Sedimentation velocity FDS studies of antibodies in pooled human serum - J. J. Correia; G. R. Bishop; P. B. Kyle; R. T. Wright; P. J. Sherwood; W. F. Stafford (European Biophysics Journal, 2023)

Hydrodynamic characterization of a vesicular stomatitis virus-based oncolytic virus using analytical ultracentrifugation - Simon Wawra; Sophia Kessler; Arina Egel; Johannes Solzin; Oliver Burkert; Daniel Hochdorfer (European Biophysics Journal, 2023)
Determination of the size, density, and mass of viral particles can provide valuable information to support process and formulation studies in clinical development. Analytical ultracentrifugation (AUC), as a first principal method, has been shown to be a beneficial tool for the characterization of the non-enveloped adeno associated virus (AAV). Here, we demonstrate the suitability of AUC for the challenging characterization of a representative for enveloped viruses, which usually are expected to exhibit higher dispersity than non-enveloped viruses. Specifically, the vesicular stomatitis virus (VSV)-based oncolytic virus VSV-GP was used to evaluate potential occurrence of non-ideal sedimentation by testing different rotor speeds and loading concentrations. The partial specific volume was determined via density gradients and density contrast experiments. Additionally, nanoparticle tracking analysis (NTA) was used to determine the hydrodynamic diameter of VSV-GP particles to calculate their molecular weight via the Svedberg equation. Overall, this study demonstrates the applicability of AUC and NTA for the characterization of size, density, and molar mass of an enveloped virus, namely VSV-GP.

Optimizing high-throughput viral vector characterization with density gradient equilibrium analytical ultracentrifugation - Shawn M. Sternisha; Abraham D. Wilson; Emilie Bouda; Akash Bhattacharya; Ross VerHeul (European Biophysics Journal, 2023)
Viral vector-based gene therapies and vaccines require accurate characterization of capsid species. The current gold standard for assessing capsid loading of adeno-associated virus (AAV) is sedimentation velocity analytical ultracentrifugation (SV-AUC). However, routine SV-AUC analysis is often size-limited, especially without the use of advanced techniques (e.g., gravitational-sweep) or when acquiring the multiwavelength data needed for assessing the loading fraction of viral vectors, and requires analysis by specialized software packages. Density gradient equilibrium AUC (DGE-AUC) is a highly simplified analytical method that provides high-resolution separation of biologics of different densities (e.g., empty and full viral capsids). The analysis required is significantly simpler than SV-AUC, and larger viral particles such as adenovirus (AdV) are amenable to characterization by DGE-AUC using cesium chloride gradients. This method provides high-resolution data with significantly less sample (estimated 56-fold improvement in sensitivity compared to SV-AUC). Multiwavelength analysis can also be used without compromising data quality. Finally, DGE-AUC is serotype-agnostic and amenable to intuitive interpretation and analysis (not requiring specialized AUC software). Here, we present suggestions for optimizing DGE-AUC methods and demonstrate a high-throughput AdV packaging analysis with the AUC, running as many as 21 samples in 80 min.

Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations - Sylvie Saleun; Caroline Mas; Aline Le Roy; Magalie Penaud-Budloo; Oumeya Adjali; Véronique Blouin; Christine Ebel (European Biophysics Journal, 2023)

Strong non-ideality effects at low protein concentrations: considerations for elongated proteins - Alexander E. Yarawsky; Vlad Dinu; Stephen E. Harding; Andrew B. Herr (European Biophysics Journal, 2023)

AAV analysis by sedimentation velocity analytical ultracentrifugation: beyond empty and full capsids - Alexander E. Yarawsky; Valeria Zai-Rose; Hazel M. Cunningham; John W. Burgner; Michael T. DeLion; Lake N. Paul (European Biophysics Journal, 2023)

A new UltraScan module for the characterization and quantification of analytical buoyant density equilibrium experiments to determine AAV capsid loading - Alexey Savelyev; Emre H. Brookes; Amy Henrickson; Borries Demeler (European Biophysics Journal, 2023)

Simulation of Gilbert theory for self-association in sedimentation velocity experiments: a guide to evaluate best fitting models - G. R. Bishop; J. J. Correia (European Biophysics Journal, 2023)

A spectral decomposition quality assessment tool for multi-wavelength AUC experiments with UltraScan - Saeed Mortezazadeh; Borries Demeler (European Biophysics Journal, 2023)

Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation - Haben Gabir; Monika Gupta; Markus Meier; Fabian Heide; Manuel Koch; Joerg Stetefeld; Borries Demeler (European Biophysics Journal, 2023)

Generation of unfolded outer membrane protein ensembles defined by hydrodynamic properties - Taylor Devlin; Patrick J. Fleming; Nicole Loza; Karen G. Fleming (European Biophysics Journal, 2023)

SViMULATE: a computer program facilitating interactive, multi-mode simulation of analytical ultracentrifugation data - Chad A. Brautigam (European Biophysics Journal, 2023)

Beyond the US-SOMO-AF database: a new website for hydrodynamic, structural, and circular dichroism calculations on user-supplied structures - Emre H. Brookes; Mattia Rocco (European Biophysics Journal, 2023)

SEDNTERP: a calculation and database utility to aid interpretation of analytical ultracentrifugation and light scattering data - John S. Philo (European Biophysics Journal, 2023)

Systematic noise removal from analytical ultracentrifugation data with UltraScan - Saeed Mortezazadeh; Borries Demeler (European Biophysics Journal, 2023)

Revisiting macromolecular hydration with HullRadSAS - Patrick J. Fleming; John J. Correia; Karen G. Fleming (European Biophysics Journal, 2023)

On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications - Serena A. J. Watkin; Rachel Z. Bennie; Jenna M. Gilkes; Volker M. Nock; F. Grant Pearce; Renwick C. J. Dobson (European Biophysics Journal, 2023)

A model for ribosome translocation based on the alternated displacement of its subunits - José S. González-García (European Biophysics Journal, 2023)

Pulling short DNA with mismatch base pairs - Navin Singh; Nehal Mathur (European Biophysics Journal, 2023)

Mechanistic insights of neuronal calcium and IP3 signaling system regulating ATP release during ischemia in progression of Alzheimer’s disease - Anand Pawar; Kamal Raj Pardasani (European Biophysics Journal, 2023)

Rethinking the protein folding problem from a new perspective - Jorge A. Vila (European Biophysics Journal, 2023)

Revealing the single-channel characteristics of OprD (OccAB1) porins from hospital strains of Acinetobacter baumannii - Aliakbar Ebrahimi; Tuğçe Ergün; Özge Kaygusuz İzgördü; Cihan Darcan; Hüseyin Avci; Barçin Öztürk; Hatice Rahmet Güner; Hamed Ghorbanpoor; Fatma Doğan Güzel (European Biophysics Journal, 2023)

Experimental extraction of Young’s modulus of MCF-7 tissue using atomic force microscopy and the spherical contact models - Mahdi Mirzaluo; Fateme Fereiduni; Moein Taheri; Mehdi Modabberifar (European Biophysics Journal, 2023)

Correction: Abstracts (European Biophysics Journal, 2023)

The effects of lithium on human red blood cells studied using optical spectroscopy and laser trap - Yashveer Singh; Aniket Chowdhury; Raktim Dasgupta; Shovan Kumar Majumder (European Biophysics Journal, 2023)

Molecular dynamics study of collective water vibrations in a DNA hydration shell - Tetiana Bubon; Oleksii Zdorevskyi; Sergiy Perepelytsya (European Biophysics Journal, 2023)

Mechanical anisotropy of hair affected by genetic diseases highlights structural information related to differential crosslinking in keratins - Steven Breakspear; Bernd Noecker; Crisan Popescu (European Biophysics Journal, 2023)

Ionizable lipids in bio-inspired nanocarriers - Vladimir P. Zhdanov (European Biophysics Journal, 2023)
In applications of bio-inspired nanoparticles (NPs), their composition is often optimised by including ionizable lipids. I use a generic statistical model to describe the charge and potential distributions in lipid nanoparticles (LNPs) containing such lipids. The LNP structure is considered to contain the biophase regions separated by narrow interphase boundaries with water. Ionizable lipids are uniformly distributed at the biophase–water boundaries. The potential is there described at the mean-filed level combining the Langmuir–Stern equation for ionizable lipids and the Poisson–Boltzmann equation for other charges in water. The latter equation is used outside a LNP as well. With physiologically reasonable parameters, the model predicts the scale of the potential in a LNP to be rather low, smaller or about $$k_BT/e$$ k B T / e , and to change primarily near the LNP-solution interface or, more precisely, inside an NP near this interface because the charge of ionizable lipids becomes rapidly neutralized along the coordinate towards the center of a LNP. The extent of dissociation-mediated neutralization of ionizable lipids along this coordinate increases but only slightly. Thus, the neutralization is primarily due to the negative and positive ions related to the ionic strength in solution and located inside a LNP.

A comparative characterisation of commercially available lipid-polymer nanoparticles formed from model membranes - Henry Sawczyc; Sabine Heit; Anthony Watts (European Biophysics Journal, 2023)
From the discovery of the first membrane-interacting polymer, styrene maleic-acid (SMA), there has been a rapid development of membrane solubilising polymers. These new polymers can solubilise membranes under a wide range of conditions and produce varied sizes of nanoparticles, yet there has been a lack of broad comparison between the common polymer types and solubilising conditions. Here, we present a comparative study on the three most common commercial polymers: SMA 3:1, SMA 2:1, and DIBMA. Additionally, this work presents, for the first time, a comparative characterisation of polymethacrylate copolymer (PMA). Absorbance and dynamic light scattering measurements were used to evaluate solubilisation across key buffer conditions in a simple, adaptable assay format that looked at pH, salinity, and divalent cation concentration. Lipid-polymer nanoparticles formed from SMA variants were found to be the most susceptible to buffer effects, with nanoparticles from either zwitterionic DMPC or POPC:POPG (3:1) bilayers only forming in low to moderate salinity (< 600 mM NaCl) and above pH 6. DIBMA-lipid nanoparticles could be formed above a pH of 5 and were stable in up to 4 M NaCl. Similarly, PMA-lipid nanoparticles were stable in all NaCl concentrations tested (up to 4 M) and a broad pH range (3–10). However, for both DIBMA and PMA nanoparticles there is a severe penalty observed for bilayer solubilisation in non-optimal conditions or when using a charged membrane. Additionally, lipid fluidity of the DMPC-polymer nanoparticles was analysed through cw-EPR, showing no cooperative gel-fluid transition as would be expected for native-like lipid membranes.

Effects of inter-radical interactions and scavenging radicals on magnetosensitivity: spin dynamics simulations of proposed radical pairs - Gongyi Hong; Ruth Pachter (European Biophysics Journal, 2023)

Analysis of residue–residue interactions in the structures of ASIC1a suggests possible gating mechanisms - Vyacheslav S. Korkosh; Denis B. Tikhonov (European Biophysics Journal, 2023)

Active wetting of epithelial tissues: modeling considerations - Ivana Pajic-Lijakovic; Milan Milivojevic (European Biophysics Journal, 2023)

Coupled folding-upon-binding of human tumor suppressor MIG6 to lung cancer EGFR kinase domain and molecular trimming/stapling of MIG6-derived β-hairpins to target the coupling event - Quan He; Shuanglan Xu; Xiaomei Ma; Ting Ling; Weiqi Feng; Xuzhi Lu; Weihua Liu; Zi Chen (European Biophysics Journal, 2023)

A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens - Jeremiah N. Sims; EJun Yun; Jonathan Chu; Mansoor A. Siddiqui; Sanjay A. Desai (European Biophysics Journal, 2023)

Numerical solution of compartment-based reaction/diffusion models with DABOSS algorithm - Dirk Gillespie (European Biophysics Journal, 2022)

Production and characterisation of modularly deuterated UBE2D1–Ub conjugate by small angle neutron and X-ray scattering - Zuzanna Pietras; Anthony P. Duff; Vivian Morad; Kathleen Wood; Cy M. Jeffries; Maria Sunnerhagen (European Biophysics Journal, 2022)
This structural study exploits the possibility to use modular protein deuteration to facilitate the study of ubiquitin signalling, transfer, and modification. A protein conjugation reaction is used to combine protonated E2 enzyme with deuterated ubiquitin for small angle X-ray and neutron scattering with neutron contrast variation. The combined biomolecules stay as a monodisperse system during data collection in both protonated and deuterated buffers indicating long stability of the E2–Ub conjugate. With multiphase ab initio shape restoration and rigid body modelling, we reconstructed the shape of a E2–Ub-conjugated complex of UBE2D1 linked to ubiquitin via an isopeptide bond. Solution X-ray and neutron scattering data for this E2–Ub conjugate in the absence of E3 jointly indicate an ensemble of open and backbent states, with a preference for the latter in solution. The approach of combining protonated and labelled proteins can be used for solution studies to assess localization and movement of ubiquitin and could be widely applied to modular Ub systems in general.

Biophysical characterization of the DNA binding motif of human phospholipid scramblase 1 - Sarika Rayala; Ulaganathan Sivagnanam; Sathyanarayana N. Gummadi (European Biophysics Journal, 2022)

Molecular and thermodynamic mechanisms for protein adaptation - Qinyi Zhao (European Biophysics Journal, 2022)

Structural heterogeneity and precision of implications drawn from cryo-electron microscopy structures: SARS-CoV-2 spike-protein mutations as a test case - Rukmankesh Mehra; Kasper P. Kepp (European Biophysics Journal, 2022)

A stochastic mathematical model for coupling the cytosolic and sarcoplasmic calcium movements in diseased cardiac myocytes - Serife Arif (European Biophysics Journal, 2022)
Several computational studies have been undertaken to explore the Ca 2+ -induced Ca 2+ release (CICR) events in cardiac myocytes and along with experimental studies it has given us invaluable insight into the mechanism of CICR from spark/blink initiation to termination and regulation, and their interplay under normal and pathological conditions. The computational modelling of this mechanism has mainly been investigated using coupled differential equations (DEs). However, there is a lack of computational investigation into (1) how the different formulation of coupled DEs capture the Ca 2+ movement in the cytosol and sarcoplasmic reticulum (SR), (2) the buffer and dye inclusion in both compartments, and (3) the effect of buffer and dye properties on the calcium behaviour. This work is set out to explore (1) the effect of different coupled formulation of DEs on spark/blink occurrence, (2) the inclusion of improved sarcoplasmic buffering properties, and (3) the effects of cytosolic and sarcoplasmic dye and buffer properties on Ca 2+ movement. The simulation results show large discrepancies between different formulations of the governing equations. Additionally, extension of the model to include sarcoplasmic buffering properties show normalised fluorescent dye profiles to be in good agreement with experimental and amongst its one- and two-dimensional representations.

Rational design of stapled helical peptides as antidiabetic PPARγ antagonists to target coactivator site by decreasing unfavorable entropy penalty instead of increasing favorable enthalpy contribution - Yang Zhang; Jie Wang; Wenchao Li; Ying Guo (European Biophysics Journal, 2022)

Structural analysis of the peptides temporin-Ra and temporin-Rb and interactions with model membranes - José L. S. Lopes; Caio C. F. Araujo; Rogério C. Neves; Jochen Bürck; Sheila G. Couto (European Biophysics Journal, 2022)

Non-linear Hamiltonian models for DNA - Marco Zoli (European Biophysics Journal, 2022)

Energetic basis of hydrogen bond formation in aqueous solution - Colyn Crane-Robinson; Peter Privalov (European Biophysics Journal, 2022)
The thermodynamic forces driving the formation of H-bonds in macromolecules have long been the subject of speculation, theory and experiment. Comparison of the energetic parameters of AT and GC base pairs in DNA duplexes has recently led to the realisation that formation of a ‘naked’ hydrogen bond, i.e. without other accompanying Van der Waals close contacts, is a non-enthalpic process driven by the entropy increase resulting from release of tightly bound water molecules from the component polar groups. This unexpected conclusion finds a parallel in the formation of ionic bonds, for example between the amino groups of DNA binding proteins and the oxygens of DNA phosphate groups that are also non-enthalpic and entropy driven. The thermodynamic correspondence between these two types of polar non-covalent bonding implies that the non-enthalpic nature of base pairing in DNA is not particular to that specific structural circumstance.

An improved platform for cultured neuronal network electrophysiology: multichannel optogenetics integrated with MEAs - F. Kemal Bayat; M. İkbal Alp; Sevginur Bostan; H. Özcan Gülçür; Gürkan Öztürk; Albert Güveniş (European Biophysics Journal, 2022)

Marangoni effect and cell spreading - Ivana Pajic-Lijakovic; Milan Milivojevic (European Biophysics Journal, 2022)

Neurotransmitter uptake of synaptic vesicles studied by X-ray diffraction - Karlo Komorowski; Julia Preobraschenski; Marcelo Ganzella; Jette Alfken; Charlotte Neuhaus; Reinhard Jahn; Tim Salditt (European Biophysics Journal, 2022)
The size, polydispersity, and electron density profile of synaptic vesicles (SVs) can be studied by small-angle X-ray scattering (SAXS), i.e. by X-ray diffraction from purified SV suspensions in solution. Here we show that size and shape transformations, as they appear in the functional context of these important synaptic organelles, can also be monitored by SAXS. In particular, we have investigated the active uptake of neurotransmitters, and find a mean vesicle radius increase of about 12% after the uptake of glutamate, which indicates an unusually large extensibility of the vesicle surface, likely to be accompanied by conformational changes of membrane proteins and rearrangements of the bilayer. Changes in the electron density profile (EDP) give first indications for such a rearrangement. Details of the protein structure are screened, however, by SVs polydispersity. To overcome the limitations of large ensemble averages and heterogeneous structures, we therefore propose serial X-ray diffraction by single free electron laser pulses. Using simulated data for realistic parameters, we show that this is in principle feasible, and that even spatial distances between vesicle proteins could be assessed by this approach.

Syndiotactic hexamer peptide nanodots - Vivek Prakash; B. Mukesh; Sajitha Sasidharan; Amay Sanjay Redkar; Abhishek Roy; R. Anandalakshmi; Vibin Ramakrishnan (European Biophysics Journal, 2022)

Self-assembly of smooth muscle myosin filaments: adaptation of filament length by telokin and Mg·ATP - Apolinary Sobieszek (European Biophysics Journal, 2022)

The role of cholesterol binding in the control of cholesterol by the Scap–Insig system - Anthony G. Lee (European Biophysics Journal, 2022)
Scap and Insig, two proteins embedded in the membrane of the endoplasmic reticulum (ER), regulate the synthesis of cholesterol in animal cells by forming a dimer in the presence of high concentrations of cholesterol. Cryo-electron microscopic structures for the Scap–Insig dimer show a sterol-binding site at the dimer interface, but none of the structures include cholesterol itself. Here, a molecular docking approach developed to characterise cholesterol binding to the transmembrane (TM) regions of membrane proteins is used to characterise cholesterol binding to sites on the TM surface of the dimer and to the interfacial binding site. Binding of cholesterol is also observed at sites on the extra-membranous luminal domains of Scap, but the properties of these sites suggest that they will be unoccupied in vivo. Comparing the structure of Scap in the dimer with that predicted by AlphaFold for monomeric Scap suggests that dimer formation could result in relocation of TM helix 7 of Scap and of the loop between TM6 and 7, and that this could be the key change on Scap that signals that there is a high concentration of cholesterol in the ER.

Effects of sugar concentration on the electroporation, size distribution and average size of charged giant unilamellar vesicles - Malay Kumar Sarkar; Mohammad Abu Sayem Karal; Victor Levadny; Marina Belaya; Marzuk Ahmed; Md. Kabir Ahamed; Shareef Ahammed (European Biophysics Journal, 2022)

NMR and dynamic light scattering give different diffusion information for short-living protein oligomers. Human serum albumin in water solutions of metal ions - A. M. Kusova; A. K. Iskhakova; Yu. F. Zuev (European Biophysics Journal, 2022)

Wave propagation through a viscous fluid-filled elastic tube under initial pressure: theoretical and biophysical model - Dejan Žikić; Katarina Žikić (European Biophysics Journal, 2022)

A fit-less approach to the elasticity of the handles in optical tweezers experiments - Alessandro Mossa; Ciro Cecconi (European Biophysics Journal, 2022)

Ultrasound-induced protein restructuring and ordered aggregation to form amyloid crystals - Rachana Pathak; Sukhvir Kaur Bhangu; Gregory J. O. Martin; Frances Separovic; Muthupandian Ashokkumar (European Biophysics Journal, 2022)
Amyloid crystals, a form of ordered protein aggregates documented relatively recently, have not been studied as extensively as amyloid fibres. This study investigates the formation of amyloid crystals with low frequency ultrasound (20 kHz) using β-lactoglobulin, as a model protein for amyloid synthesis. Acoustic cavitation generates localised zones of intense shear, with extreme heat and pressure that could potentially drive the formation of amyloid structures at ambient bulk fluid temperatures (20 ± 1 °C). Thioflavin T fluorescence and electron microscopy showed that low-frequency ultrasound at 20 W/cm 3 input power induced β-stacking to produce amyloid crystals in the mesoscopic size range, with a mean length of approximately 22 µm. FTIR spectroscopy indicated a shift towards increased intermolecular antiparallel β-sheet content. An increase in sonication time (0–60 min) and input power (4–24 W/cm 3 ) increased the mean crystal length, but this increase was not linearly proportional to sonication time and input power due to the delayed onset of crystal growth. We propose that acoustic cavitation causes protein unfolding and aggregation and imparts energy to aggregates to cross the torsion barrier, to achieve their lowest energy state as amyloid crystals. The study contributes to a further understanding of protein chemistry relating to the energy landscape of folding and aggregation. Ultrasound presents opportunities for practical applications of amyloid structures, presenting a more adaptable and scalable approach for synthesis. Graphical abstract

Cell wall channels of Rhodococcus species: identification and characterization of the cell wall channels of Rhodococcus corynebacteroides and Rhodococcus ruber - Claudio Piselli; Lorraine Benier; Cornelia Koy; Michael O. Glocker; Roland Benz (European Biophysics Journal, 2022)
The cell wall of Rhodococcus corynebacteroides formerly known as Nocardia corynebacteroides contains cell wall channels that are responsible for the cell wall permeability of this bacterium. Based on partial sequencing of the polypeptide subunits and a BLAST search, we identified one polypeptide of R. corynebacteroides (PorARc) and two polypeptides (PorARr and PorBRr) from the closely related bacterium Rhodococcus ruber. The corresponding genes, porARc (606 bp), porARr (702 bp), and porBRr (540 bp) are constituents of the known genome of R. corynebacteroides DSM-20151 and R. ruber DSM-43338, respectively. porARr and porBRr of R. ruber are possibly forming a common operon coding for the polypeptide subunits of the cell wall channel. The genes coding for PorARc and for PorARr and PorBRr without signal peptide were separately expressed in the porin-deficient Escherichia coli BL21DE3Omp8 strain and the proteins were purified to homogeneity. All proteins were checked for channel formation in lipid bilayers. PorARc formed channels with characteristics that were very similar to those of a previous study. The proteins PorARr and PorBRr expressed in E. coli could alone create channels in lipid bilayer membranes, despite the possibility that the two corresponding genes form a porin operon and that both subunits possibly form the cell wall channels in vivo. Based on amino acid sequence comparison of a variety of proteins forming cell wall channels in bacteria of the suborder Corynebacterineae, it seems very likely that PorARc, PorARr, and PorBRr are members of a huge family of proteins (PF09203) that form MspA-like cell wall channels.

Amyloid fibril length distribution from dynamic light scattering data - Petr A. Sokolov; Valeriy I. Rolich; Olga S. Vezo; Mikhail V. Belousov; Stanislav A. Bondarev; Galina A. Zhouravleva; Nina A. Kasyanenko (European Biophysics Journal, 2022)

The inhibition of glucose uptake to erythrocytes: microwave dielectric response - Cindy Galindo; Larisa Latypova; Gregory Barshtein; Leonid Livshits; Dan Arbell; Sharon Einav; Yuri Feldman (European Biophysics Journal, 2022)

NMR measurement of biomolecular translational and rotational motion for evaluating changes of protein oligomeric state in solution - Shenggen Yao; David W. Keizer; Jeffrey J. Babon; Frances Separovic (European Biophysics Journal, 2022)
Defining protein oligomeric state and/or its changes in solution is of significant interest for many biophysical studies carried out in vitro, especially when the nature of the oligomeric state is crucial in the subsequent interpretation of experimental results and their biological relevance. Nuclear magnetic resonance (NMR) is a well-established methodology for the characterization of protein structure, dynamics, and interactions at the atomic level. As a spectroscopic method, NMR also provides a compelling means for probing both molecular translational and rotational motion, two predominant measures of effective molecular size in solution, under identical conditions as employed for structural, dynamic and interaction studies. Protein translational diffusion is readily measurable by pulse gradient spin echo (PGSE) NMR, whereas its rotational correlation time, or rotational diffusion tensor when its 3D structure is known, can also be quantified from NMR relaxation parameters, such as 15 N relaxation parameters of backbone amides which are frequently employed for probing residue-specific protein backbone dynamics. In this article, we present an introductory overview to the NMR measurement of bimolecular translational and rotational motion for assessing changes of protein oligomeric state in aqueous solution, via translational diffusion coefficients measured by PGSE NMR and rotational correlation times derived from composite 15 N relaxation parameters of backbone amides, without need for the protein structure being available. Graphical abstract

Systematic analysis and molecular profiling of EGFR allosteric inhibitor cross-reactivity across the proto-oncogenic ErbB family kinases by integrating dynamics simulation, energetics calculation and biochemical assay - Yanli Ma; Bingli Qi; Meiying Ning; Lijuan Zhang; Zeyu An; Jing Zhao (European Biophysics Journal, 2022)

Interceptor potential of C60 fullerene aqueous solution: a comparative analysis using the example of the antitumor antibiotic mitoxantrone - Victoria A. Salo; Anatoly S. Buchelnikov; Maxim P. Evstigneev (European Biophysics Journal, 2022)

Molecular dynamics simulation or structure refinement of proteins: are solvent molecules required? A case study using hen lysozyme - Maria Pechlaner; Wilfred F. van Gunsteren; Niels Hansen; Lorna J. Smith (European Biophysics Journal, 2022)
In protein simulation or structure refinement based on values of observable quantities measured in (aqueous) solution, solvent (water) molecules may be explicitly treated, omitted, or represented by a potential of mean-solvation-force term, depending on protein coordinates only, in the force field used. These three approaches are compared for hen egg white lysozyme (HEWL). This 129-residue non-spherical protein contains a variety of secondary-structure elements, and ample experimental data are available: 1630 atom–atom Nuclear Overhauser Enhancement (NOE) upper distance bounds, 213 3 J -couplings and 200 S 2 order parameters. These data are used to compare the performance of the three approaches. It is found that a molecular dynamics (MD) simulation in explicit water approximates the experimental data much better than stochastic dynamics (SD) simulation in vacuo without or with a solvent-accessible-surface-area (SASA) implicit-solvation term added to the force field. This is due to the missing energetic and entropic contributions and hydrogen-bonding capacities of the water molecules and the missing dielectric screening effect of this high-permittivity solvent. Omission of explicit water molecules leads to compaction of the protein, an increased internal strain, distortion of exposed loop and turn regions and excessive intra-protein hydrogen bonding. As a consequence, the conformation and dynamics of groups on the surface of the protein, which may play a key role in protein–protein interactions or ligand or substrate binding, may be incorrectly modelled. It is thus recommended to include water molecules explicitly in structure refinement of proteins in aqueous solution based on nuclear magnetic resonance (NMR) or other experimentally measured data.

Force spectroscopy of interactions between Yersinia pseudotuberculosis and Yersinia pestis cells and monoclonal antibodies using optical tweezers - Andrey Byvalov; Ilya Konyshev; Boris Ananchenko; Vladislav Belozerov (European Biophysics Journal, 2022)

Kinetic and thermodynamic modeling of a voltage-gated sodium channel - Mara Almog; Nurit Degani-Katzav; Alon Korngreen (European Biophysics Journal, 2022)

Lipid headgroup and side chain architecture determine manganese-induced dose dependent membrane rigidification and liposome size increase - Kevin Sule; Elmar J. Prenner (European Biophysics Journal, 2022)

Flagellar rotational features of an optically confined bacterium at high frequency and temporal resolution reveal the microorganism’s response to changes in the fluid environment - Ashwini Venkateswara Bhat; Roshan Akbar Basha; Mohana Devihalli Chikkaiah; Sharath Ananthamurthy (European Biophysics Journal, 2022)

Nanoengineering for Mechanobiology “N4M-20” - Aldo Ferrari; Massimo Vassalli; Silvia Caponi (European Biophysics Journal, 2022)

Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing - Haoqing Wang; Peyman Obeidy; Zihao Wang; Yunduo Zhao; Yao Wang; Qian Peter Su; Charles D. Cox; Lining Arnold Ju (European Biophysics Journal, 2022)
Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca 2+ ) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca 2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca 2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (− 2.94 to 0 mmH 2 O) and a pneumatic high-speed pressure clamp (− 25 to 0 mmHg). To visualize Ca 2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca 2+ levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.

Micropipette-based biomechanical nanotools on living cells - Haoqing Wang; Fang Zhou; Yuze Guo; Lining Arnold Ju (European Biophysics Journal, 2022)
Mechanobiology is an emerging field at the interface of biology and mechanics, investigating the roles of mechanical forces within biomolecules, organelles, cells, and tissues. As a highlight, the recent advances of micropipette-based aspiration assays and dynamic force spectroscopies such as biomembrane force probe (BFP) provide unprecedented mechanobiological insights with excellent live-cell compatibility. In their classic applications, these assays measure force-dependent ligand–receptor-binding kinetics, protein conformational changes, and cellular mechanical properties such as cortical tension and stiffness. In recent years, when combined with advanced microscopies in high spatial and temporal resolutions, these biomechanical nanotools enable characterization of receptor-mediated cell mechanosensing and subsequent organelle behaviors at single-cellular and molecular level. In this review, we summarize the latest developments of these assays for live-cell mechanobiology studies. We also provide perspectives on their future upgrades with multimodal integration and high-throughput capability.

Acoustofluidic interferometric device for rapid single-cell physical phenotyping - J. Mejía Morales; P. Glynne-Jones; M. Vassalli; G. L. Lippi (European Biophysics Journal, 2022)

Quantifying force transmission through fibroblasts: changes of traction forces under external shearing - Steven Huth; Johannes W. Blumberg; Dimitri Probst; Jan Lammerding; Ulrich S. Schwarz; Christine Selhuber-Unkel (European Biophysics Journal, 2022)
Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

Systems of conductive skin for power transfer in clinical applications - Andreas P. Kourouklis; Julius Kaemmel; Xi Wu; Evgenij Potapov; Nikola Cesarovic; Aldo Ferrari; Christoph Starck; Volkmar Falk; Edoardo Mazza (European Biophysics Journal, 2022)
The primary aim of this article is to review the clinical challenges related to the supply of power in implanted left ventricular assist devices (LVADs) by means of transcutaneous drivelines. In effect of that, we present the preventive measures and post-operative protocols that are regularly employed to address the leading problem of driveline infections. Due to the lack of reliable wireless solutions for power transfer in LVADs, the development of new driveline configurations remains at the forefront of different strategies that aim to power LVADs in a less destructive manner. To this end, skin damage and breach formation around transcutaneous LVAD drivelines represent key challenges before improving the current standard of care. For this reason, we assess recent strategies on the surface functionalization of LVAD drivelines, which aim to limit the incidence of driveline infection by directing the responses of the skin tissue. Moreover, we propose a class of power transfer systems that could leverage the ability of skin tissue to effectively heal short diameter wounds. In this direction, we employed a novel method to generate thin conductive wires of controllable surface topography with the potential to minimize skin disruption and eliminate the problem of driveline infections. Our initial results suggest the viability of the small diameter wires for the investigation of new power transfer systems for LVADs. Overall, this review uniquely compiles a diverse number of topics with the aim to instigate new research ventures on the design of power transfer systems for IMDs, and specifically LVADs.

Non-contact elastography methods in mechanobiology: a point of view - Silvia Caponi; Alessandra Passeri; Giulio Capponi; Daniele Fioretto; Massimo Vassalli; Maurizio Mattarelli (European Biophysics Journal, 2022)
In recent decades, mechanobiology has emerged as a novel perspective in the context of basic biomedical research. It is now widely recognized that living cells respond not only to chemical stimuli (for example drugs), but they are also able to decipher mechanical cues, such as the rigidity of the underlying matrix or the presence of shear forces. Probing the viscoelastic properties of cells and their local microenvironment with sub-micrometer resolution is required to study this complex interplay and dig deeper into the mechanobiology of single cells. Current approaches to measure mechanical properties of adherent cells mainly rely on the exploitation of miniaturized indenters, to poke single cells while measuring the corresponding deformation. This method provides a neat implementation of the everyday approach to measure mechanical properties of a material, but it typically results in a very low throughput and invasive experimental protocol, poorly translatable towards three-dimensional living tissues and biological constructs. To overcome the main limitations of nanoindentation experiments, a radical paradigm change is foreseen, adopting next generation contact-less methods to measure mechanical properties of biological samples with sub-cell resolution. Here we briefly introduce the field of single cell mechanical characterization, and we concentrate on a promising high resolution optical elastography technique, Brillouin spectroscopy. This non-contact technique is rapidly emerging as a potential breakthrough innovation in biomechanics, but the application to single cells is still in its infancy.

Microfabricated cantilevers for parallelized cell-cell adhesion measurements - Michele Zanetti; Suet Nee Chen; Martina Conti; Matthew R. G. Taylor; Orfeo Sbaizero; Luisa Mestroni; Marco Lazzarino (European Biophysics Journal, 2022)

The role of physical cues in the development of stem cell-derived organoids - Ilaria Tortorella; Chiara Argentati; Carla Emiliani; Sabata Martino; Francesco Morena (European Biophysics Journal, 2022)
Organoids are a novel three-dimensional stem cells’ culture system that allows the in vitro recapitulation of organs/tissues structure complexity. Pluripotent and adult stem cells are included in a peculiar microenvironment consisting of a supporting structure (an extracellular matrix (ECM)-like component) and a cocktail of soluble bioactive molecules that, together, mimic the stem cell niche organization. It is noteworthy that the balance of all microenvironmental components is the most critical step for obtaining the successful development of an accurate organoid instead of an organoid with heterogeneous morphology, size, and cellular composition. Within this system, mechanical forces exerted on stem cells are collected by cellular proteins and transduced via mechanosensing—mechanotransduction mechanisms in biochemical signaling that dictate the stem cell specification process toward the formation of organoids. This review discusses the role of the environment in organoids formation and focuses on the effect of physical components on the developmental system. The work starts with a biological description of organoids and continues with the relevance of physical forces in the organoid environment formation. In this context, the methods used to generate organoids and some relevant published reports are discussed as examples showing the key role of mechanosensing–mechanotransduction mechanisms in stem cell-derived organoids.

Mechanical waves caused by collective cell migration: generation - Ivana Pajic-Lijakovic; Milan Milivojevic (European Biophysics Journal, 2022)

A quantitative interspecies comparison of the respiratory mucociliary clearance mechanism - Andreas Burn; Martin Schneiter; Manuel Ryser; Peter Gehr; Jaroslav Rička; Martin Frenz (European Biophysics Journal, 2022)
Collectively coordinated ciliary activity propels the airway mucus, which lines the luminal surface of the vertebrate respiratory system, in cranial direction. Our contemporary understanding on how the quantitative characteristics of the metachronal wave field determines the resulting mucociliary transport is still limited, partly due to the sparse availability of quantitative observational data. We employed high-speed video reflection microscopy to image and quantitatively characterize the metachronal wave field as well as the mucociliary transport in excised bovine, porcine, ovine, lapine, turkey and ostrich samples. Image processing techniques were used to determine the ciliary beating frequency (CBF), the velocity and wavelength of the metachronal wave and the mucociliary transport velocity. The transport direction was found to strongly correlate with the mean wave propagation direction in all six species. The CBF yielded similar values (10–15 Hz) for all six species. Birds were found to exhibit higher transport speeds (130–260 $$\upmu$$ μ m/s) than mammals (20–80 $$\upmu$$ μ m/s). While the average transport direction significantly deviates from the tracheal long axis in mammals, no significant deviation was found in birds. The metachronal waves were found to propagate at about 4–8 times the speed of mucociliary transport in mammals, whereas in birds they propagate at about the transport speed. The mucociliary transport in birds is fast and roughly follows the TLA, whereas the transport is slower and proceeds along a left-handed spiral in mammals. The longer wavelengths and the lower ratio between the metachronal wave speed and the mucociliary transport speed provide evidence that the mucociliary clearance mechanism operates differently in birds than in mammals.

Predicting optoacoustic spectral behaviour of human erythrocytes, stomatocytes and echinocytes using a modified Green’s function method - Ratan K. Saha (European Biophysics Journal, 2022)

Interactive multiple binding of oleic acid, warfarin and ibuprofen with human serum albumin revealed by thermal and fluorescence studies - Rita Guzzi; Rosa Bartucci (European Biophysics Journal, 2022)

Atomic force spectroscopy with magainin 1 functionalized tips and biomimetic supported lipid membranes - Alexandra Besleaga; Aurelia Apetrei; Lucel Sirghi (European Biophysics Journal, 2022)

Isothermal titration calorimetry in the single-injection mode with imperfect mixing - Philippe Dumas (European Biophysics Journal, 2022)

Correction to: Thermodynamics of protein folding: methodology, data analysis and interpretation of data - Seyedeh Akram Shirdel; Khosrow Khalifeh (European Biophysics Journal, 2022)

Clostridium perfringens Beta2 toxin forms highly cation-selective channels in lipid bilayers - Roland Benz; Claudio Piselli; Cezarela Hoxha; Cornelia Koy; Michael O. Glocker; Michel R. Popoff (European Biophysics Journal, 2022)
Clostridium perfringens is a potent producer of a variety of toxins. Well studied from these are five toxins (alpha, Beta (CPB), epsilon, iota and CPE) that are produced by seven toxinotype strains (A–G) of C. perfringens . Besides these toxins, C. perfringens produces also another toxin that causes necrotizing enterocolitis in piglets. This toxin termed consensus Beta2 toxin (cCPB2) has a molecular mass of 27,620 Da and shows only little homology to CPB and no one to the other toxins of C. perfringens . Its primary action on cells remained unknown to date. cCPB2 was heterogeneously expressed as fusion protein with GST in Escherichia coli and purified to homogeneity. Although cCPB2 does not exhibit the typical structure of beta-stranded pore-forming proteins and contains no indication for the presence of amphipathic alpha-helices we could demonstrate that cCPB2 is a pore-forming component with an extremely high activity in lipid bilayers. The channels have a single-channel conductance of about 700 pS in 1 M KCl and are highly cation-selective as judged from selectivity measurements in the presence of salt gradients. The high cation selectivity is caused by the presence of net negative charges in or near the channel that allowed an estimate of the channel size being about 1.4 nm wide. Our measurements suggest that the primary effect of cCPB2 is the formation of cation-selective channels followed by necrotic enteritis in humans and animals. We searched in databases for homologs of cCPB2 and constructed a cladogram representing the phylogenetic relationship to the next relatives of cCPB2.

Observations of three “re-entrant” twisted structures in double-stranded DNA dispersion particles - Yuri M. Yevdokimov; Sergey G. Skuridin; Viktor I. Salyanov; Efim I. Kats (European Biophysics Journal, 2022)

Correction to: Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study - Blanca López-Méndez; Bruno Baron; Chad A. Brautigam; Thomas A. Jowitt; Stefan H. Knauer; Stephan Uebel; Mark A. Williams; Arthur Sedivy; Olga Abian; Celeste Abreu; Malgorzata Adamczyk; Wojciech Bal; et al. (European Biophysics Journal, 2021)

Morphological, fractal, and textural features for the blood cell classification: the case of acute myeloid leukemia - Marko Dinčić; Tamara B. Popović; Milica Kojadinović; Alexander M. Trbovich; Andjelija Ž. Ilić (European Biophysics Journal, 2021)

Correction to: Lipoic acid nanoforms based on phosphatidylcholine: production and characteristics - V. A. Shchelkonogov; S. O. Alyaseva; N. Yu. Lotosh; O. A. Baranova; A. V. Chekanov; E. Yu. Solov’eva; R. A. Kamyshinskii; R. G. Vasilov; N. S. Shastina; E. A. Korepanova; A. A. Anosov; A. A. Selishcheva (European Biophysics Journal, 2021)

Partial structure, dampened mobility, and modest impact of a His tag in the SARS-CoV-2 Nsp2 C-terminal region - Miguel Mompeán; Miguel Á. Treviño; Douglas V. Laurents (European Biophysics Journal, 2021)
Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many viruses use their own IDPs to “hack” these processes to deactivate host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational preferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2, whose C-terminal region (CtR) is predicted to be disordered, interacts with human proteins that regulate translation initiation and endosome vesicle sorting. Molecules that block these interactions could be valuable leads for drug development. The 13 Cβ and backbone 13 CO, 1 HN, 13 Cα, and 15 N nuclei of Nsp2’s 45-residue CtR were assigned and used to characterize its structure and dynamics in three contexts; namely: (1) retaining an N-terminal His tag, (2) without the His tag and with an adventitious internal cleavage, and (3) lacking both the His tag and the internal cleavage. Two five-residue segments adopting a minor extended population were identified. Overall, the dynamic behavior is midway between a completely rigid and a fully flexible chain. Whereas the presence of an N-terminal His tag and internal cleavage stiffen and loosen, respectively, neighboring residues, they do not affect the tendency of two regions to populate extended conformations.

Thermodynamics of semi-specific ligand recognition: the binding of dipeptides to the E.coli dipeptide binding protein DppA - Mohamad K. M. Zainol; Robert J. C. Linforth; Donald J. Winzor; David J. Scott (European Biophysics Journal, 2021)
This investigation of the temperature dependence of DppA interactions with a subset of three dipeptides (AA. AF and FA) by isothermal titration calorimetry has revealed the negative heat capacity ( $$Δ C_{p}^{o}$$ Δ C p o ) that is a characteristic of hydrophobic interactions. The observation of enthalpy–entropy compensation is interpreted in terms of the increased structuring of water molecules trapped in a hydrophobic environment, the enthalpic energy gain from which is automatically countered by the entropy decrease associated with consequent loss of water structure flexibility. Specificity for dipeptides stems from appropriate spacing of designated DppA aspartate and arginine residues for electrostatic interaction with the terminal amino and carboxyl groups of a dipeptide, after which the binding pocket closes to become completely isolated from the aqueous environment. Any differences in chemical reactivity of the dipeptide sidechains are thereby modulated by their occurrence in a hydrophobic environment where changes in the structural state of entrapped water molecules give rise to the phenomenon of enthalpy–entropy compensation. The consequent minimization of differences in the value of Δ G 0 for all DppA–dipeptide interactions thus provides thermodynamic insight into the biological role of DppA as a transporter of all dipeptides across the periplasmic membrane.

Insight into dual fluorescence effects induced by molecular aggregation occurring in membrane model systems containing 1,3,4-thiadiazole derivatives - Melinda David; Iwona Budziak-Wieczorek; Dariusz Karcz; Monica Florescu; Arkadiusz Matwijczuk (European Biophysics Journal, 2021)
This work reports on biophysical insights into the excited state intramolecular proton transfer (ESIPT) processes taking place in three 1,3,4-thiadiazole derivatives that served as model compounds, on which electronic absorption, fluorescence, Fourier-transform infrared spectroscopy (FTIR), surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) studies were performed. The fluorescence spectra recorded in various solvents revealed an interesting dual fluorescence effect. In molecules in their monomeric form, the effect is associated with the ESIPT phenomenon, and may be further enhanced by aggregation-related effects, such as aggregation-induced emissions. Other spectroscopic studies on the selected molecules in a liposomal medium as a model revealed that, in a biomimetic environment, they can exist in both monomeric and aggregated forms. In both cases, however, the effects observed are closely related to the lipid’s main phase transition temperature and the structure of the molecule. Introduction of specific substituents to the phenyl moiety either allows or prevents proton transfer from occurring in the excited state. The hydrophobicity changes in a lipid environment may result in an emergence of specific molecular forms and therefore either facilitate or hinder ESIPT processes. SPR and EIS confirmed the significant hydrophobicity changes in the model lipid systems, while FTIR measurements revealed a notable influence of 1,3,4 - thiadiazoles on the fluidity of liposomal membranes. The results obtained clearly show that the thiadiazole derivatives are very good model molecules for studying hydrophobic-hydrophilic environments, and particularly with polymers or liposomes used as drug delivery systems.

Direct visualization of local activities of long DNA strands via image–time correlation - Kyongok Kang; Yue Ma; Koichiro Sadakane (European Biophysics Journal, 2021)
Bacteriophages with long DNA genomes are of interest due to their diverse mutations dependent on environmental factors. By lowering the ionic strength of a hydrophobic (PPh4Cl) antagonistic salt (at 1 mM), single long T4 DNA strand fluctuations were clearly observed, while condensed states of T4 DNA globules were formed above 5–10 mM salt. These long DNA strands were treated with fluorescently labeled probes, for which photo bleaching is often unavoidable over a short time of measurement. In addition, long (few tens of $$\upmu m$$ μ m ) length scales are required to have larger fields of view for better sampling, with shorter temporal resolutions. Thus, an optimization between length and time is crucial to obtain useful information. To facilitate the challenge of detecting large biomacromolecules, we here introduce an effective method of live image data analysis for direct visualization and quantification of local thermal fluctuations. The motions of various conformations for the motile long DNA strands were examined for the single- and multi-T4 DNA strands. We find that the unique correlation functions exhibit a relatively high-frequency oscillatory behavior superimposed on the overall slower decay of the correlation function with a splitting of amplitudes deriving from local activities of the long DNA strands. This work shows not only the usefulness of an image–time correlation for analyzing large biomacromolecules, but also provides insight into the effects of a hydrophobic antagonistic salt on active T4 bacteriophage long DNA strands, including thermal translocations in their electrostatic interactions.

Effective inhibition of insulin amyloid fibril aggregation by nickel(II) complexes containing heterocyclic thiosemicarbazones - Kannayiram Gomathi; Jebiti Haribabu; Sivaraj Saranya; Dasararaju Gayathri; Kumaramangalam Jeyalakshmi; Subramanian Sendilvelan; Cesar Echeverria; Ramasamy Karvembu (European Biophysics Journal, 2021)

DNA–MBF1 study using molecular dynamics simulations - Daniel Salgado-Blanco; Florentino López-Urías; Cesaré Ovando-Vázquez; Fabiola Jaimes-Miranda (European Biophysics Journal, 2021)

Reflectometry and molecular dynamics study of the impact of cholesterol and melatonin on model lipid membranes - Pavol Hrubovčák; Ermuhammad Dushanov; Tomáš Kondela; Oleksandr Tomchuk; Kholmirzo Kholmurodov; Norbert Kučerka (European Biophysics Journal, 2021)

Structure and mechanism of secondary sulfonamide binding to carbonic anhydrases - Denis Baronas; Virginija Dudutienė; Vaida Paketurytė; Visvaldas Kairys; Alexey Smirnov; Vaida Juozapaitienė; Aivaras Vaškevičius; Elena Manakova; Saulius Gražulis; Asta Zubrienė; Daumantas Matulis (European Biophysics Journal, 2021)

‘Chain and running’ induced by mechanical interactions among cells of different phenotypes in the Bacillus subtilis biofilm - Shuaishuai Hu; Xiaoling Wang; Yifan Tan; Jiali Liu; Hui Zhao (European Biophysics Journal, 2021)

KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements - Diana Spiegelberg; Jonas Stenberg; Pascale Richalet; Marc Vanhove (European Biophysics Journal, 2021)
Design of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point K D determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

Structure and dynamics of sodium alginate as elucidated by chemical shift anisotropy and site-specific spin–lattice relaxation time measurements - Krishna Kishor Dey; Shovanlal Gayen; Manasi Ghosh (European Biophysics Journal, 2021)

Atomistic molecular dynamics simulations of tubulin heterodimers explain the motion of a microtubule - Alexandr Nasedkin; Inna Ermilova; Jan Swenson (European Biophysics Journal, 2021)
Microtubules are essential parts of the cytoskeleton that are built by polymerization of tubulin heterodimers into a hollow tube. Regardless that their structures and functions have been comprehensively investigated in a modern soft matter, it is unclear how properties of tubulin heterodimer influence and promote the self-assembly. A detailed knowledge of such structural mechanisms would be helpful in drug design against neurodegenerative diseases, cancer, diabetes etc. In this work atomistic molecular dynamics simulations were used to investigate the fundamental dynamics of tubulin heterodimers in a sheet and a short microtubule utilizing well-equilibrated structures. The breathing motions of the tubulin heterodimers during assembly show that the movement at the lateral interface between heterodimers (wobbling) dominates in the lattice. The simulations of the protofilament curvature agrees well with recently published experimental data, showing curved protofilaments at polymerization of the microtubule plus end. The tubulin heterodimers exposed at the microtubule minus end were less curved and displayed altered interactions at the site of sheet closure around the outmost heterodimers, which may slow heterodimer binding and polymerization, providing a potential explanation for the limited dynamics observed at the minus end.

A novel assay based on pre-equilibrium titration curves for the determination of enzyme inhibitor binding kinetics - Bernard Noppen; Anouk Vanbelle; Alan W. Stitt; Marc Vanhove (European Biophysics Journal, 2021)
Selection of pharmacological agents based on potency measurements performed at equilibrium fail to incorporate the kinetic aspects of the drug–target interaction. Here we describe a method for screening or characterization of enzyme inhibitors that allows the concomitant determination of the equilibrium inhibition constant in unison with rates of complex formation and dissociation. The assay is distinct from conventional enzymatic assays and is based on the analysis of inhibition curves recorded prior to full equilibration of the system. The methodology is illustrated using bicyclic peptide inhibitors of the serine protease plasma kallikrein.

Single-molecule localisation microscopy: accounting for chance co-localisation between foci in bacterial cells - Christoffer Åberg; Andrew Robinson (European Biophysics Journal, 2021)
Using single-molecule fluorescence microscopes, individual biomolecules can be observed within live bacterial cells. Using differently coloured probes, physical associations between two different molecular species can be assessed through co-localisation measurements. However, bacterial cells are finite and small (~ 1 μm) relative to the resolution limit of optical microscopes (~ 0.25 μm). Furthermore, the images produced by optical microscopes are typically two-dimensional projections of three-dimensional objects. These limitations mean that a certain proportion of object pairs (molecules) will inevitably be assigned as being co-localised, even when they are distant at molecular distance scales (nm). What is this proportion? Here, we attack this problem, theoretically and computationally, by creating a model of the co-localisation expected purely due to chance. We thus consider a bacterial cell wherein objects are distributed at random and evaluate the co-localisation in a fashion that emulates an experimental analysis. We consider simplified geometries where we can most transparently investigate the effect of a finite size of the cell and the effect of probing a three-dimensional cell in only two dimensions. Coupling theory to simulations, we also study the co-localisation expected due to chance using parameters relevant to bacterial cells. Overall, we show that the co-localisation expected purely due to chance can be quite substantial and describe the parameters that it depends upon.

A method to assess the robustness of complex mathematical models used for quantitative interpretation of experimental data by nonlinear regression analysis: application to high-affinity binding models - Marc Vanhove (European Biophysics Journal, 2021)

The importance of hydrophobic interactions in the structure of transcription systems - Angel Mozo-Villarías; Juan Cedano; Enrique Querol (European Biophysics Journal, 2021)

Improved Hodgkin–Huxley type model for neural action potentials - P. J. Stiles; C. G. Gray (European Biophysics Journal, 2021)

DNA translocation through pH-dependent soft nanopores - Alireza Yousefi; Ardalan Ganjizade; Seyed Nezameddin Ashrafizadeh (European Biophysics Journal, 2021)

The effects of short term hyperglycemia on human red blood cells studied using Raman spectroscopy and optical trap - Yashveer Singh; Aniket Chowdhury; Raktim Dasgupta; Shovan Kumar Majumder (European Biophysics Journal, 2021)

Molecular dynamics simulation study of the positioning and dynamics of α-tocopherol in phospholipid bilayers - Sepideh Kavousi; Brian R. Novak; Xinjie Tong; Dorel Moldovan (European Biophysics Journal, 2021)

Influence of temperature and salt on coacervation in an aqueous mixture of poly-L-lysine (PLL) and poly-(sodium styrene sulfonate) (PSSNa) - Imen Naassaoui; Adel Aschi (European Biophysics Journal, 2021)

Extending the double-dispersion Cole–Cole, Cole–Davidson and Havriliak–Negami electrochemical impedance spectroscopy models - Ahmed S. Elwakil; Abdulwadood A. Al–Ali; Brent J. Maundy (European Biophysics Journal, 2021)

The need for reconsideration of a mechanism of membrane potential generation using Ling’s adsorption theory - Hirohisa Tamagawa; Titus Mulembo; Bernard Delalande (European Biophysics Journal, 2021)

A quantitative calcium phosphate nanocluster model of the casein micelle: the average size, size distribution and surface properties - Carl Holt (European Biophysics Journal, 2021)

What can S-shaped potential profiles tell us about the mechanism of membrane potential generation? - Hirohisa Tamagawa; Titus Mulembo; Bernard Delalande (European Biophysics Journal, 2021)

The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method - Ivana Drvenica; Slavko Mojsilović; Ana Stančić; Dragana Marković; Marijana Kovačić; Irina Maslovarić; Ivana Rapajić; Dušan Vučetić; Vesna Ilić (European Biophysics Journal, 2021)

13th EBSA congress, July 24–28, 2021, Vienna, Austria (European Biophysics Journal, 2021)

Cu(ii) complex with auxin (3-indoleacetic acid) and an aromatic planar ligand: synthesis, crystal structure, biomolecular interactions and radical scavenging activity - Duygu İnci; Rahmiye Aydın; Yunus Zorlu (European Biophysics Journal, 2021)

Thermodynamic basis of the α-helix and DNA duplex - A. I. Dragan; C. Crane-Robinson; P. L. Privalov (European Biophysics Journal, 2021)
Analysis of calorimetric and crystallographic information shows that the α-helix is maintained not only by the hydrogen bonds between its polar peptide groups, as originally supposed, but also by van der Waals interactions between tightly packed apolar groups in the interior of the helix. These apolar contacts are responsible for about 60% of the forces stabilizing the folded conformation of the α-helix and their exposure to water on unfolding results in the observed heat capacity increment, i.e. the temperature dependence of the melting enthalpy. The folding process is also favoured by an entropy increase resulting from the release of water from the peptide groups. A similar situation holds for the DNA double helix: calorimetry shows that the hydrogen bonding between conjugate base pairs provides a purely entropic contribution of about 40% to the Gibbs energy while the enthalpic van der Waals interactions between the tightly packed apolar parts of the base pairs provide the remaining 60%. Despite very different structures, the thermodynamic basis of α-helix and B-form duplex stability are strikingly similar. The general conclusion follows that the stability of protein folds is primarily dependent on internal atomic close contacts rather than the hydrogen bonds they contain.

Molecular dynamics study of the competitive binding of hydrogen peroxide and water molecules with DNA phosphate groups - Sergiy Perepelytsya; Jozef Uličný; Sergey N. Volkov (European Biophysics Journal, 2021)

Are cysteine residues of human phospholipid scramblase 1 essential for Pb2+ and Hg2+ binding-induced scrambling of phospholipids? - Ashok Kumar Shettihalli; Santosh Kumar Palanirajan; Sathyanarayana N. Gummadi (European Biophysics Journal, 2021)

The effects of the chemical environment of menaquinones in lipid monolayers on mercury electrodes on the thermodynamics and kinetics of their electrochemistry - Karuppasamy Dharmaraj; Dirk Dattler; Heike Kahlert; Uwe Lendeckel; Felix Nagel; Mihaela Delcea; Fritz Scholz (European Biophysics Journal, 2021)
The effects of the chemical environment of menaquinones (all- trans MK-4 and all -trans MK-7) incorporated in lipid monolayers on mercury electrodes have been studied with respect to the thermodynamics and kinetics of their electrochemistry. The chemical environment relates to the composition of lipid films as well as the adjacent aqueous phase. It could be shown that the addition of all- trans MK-4 to TMCL does not change the phase transition temperatures of TMCL. In case of DMPC monolayers, the presence of cholesterol has no effect on the thermodynamics (formal redox potentials) of all- trans MK-7, but the kinetics are affected. Addition of an inert electrolyte (sodium perchlorate; change of ionic strength) to the aqueous phase shifts the redox potentials of all- trans MK-7 only slightly. The formal redox potentials of all- trans MK-4 were determined in TMCL and nCL monolayers and found to be higher in nCL monolayers than in TMCL monolayers. The apparent electron transfer rate constants, transfer coefficients and activation energies of all- trans MK-4 in cardiolipins have been also determined. Most surprisingly, the apparent electron transfer rate constants of all- trans MK-4 exhibit an opposite pH dependence for TMCL and nCL films: the rate constants increase in TMCL films with increasing pH, but in nCL films they increase with decreasing pH. This study is a contribution to understand environmental effects on the redox properties of membrane bond redox systems. Graphical abstract

Structural and biochemical analyses of the tetrameric cell binding domain of Lys170 from enterococcal phage F170/08 - Xiaolong Xu; Dandan Zhang; Biao Zhou; Xiangkai Zhen; Songying Ouyang (European Biophysics Journal, 2021)

Evolution of frustrated and stabilising contacts in reconstructed ancient proteins - Martina Crippa; Damiano Andreghetti; Riccardo Capelli; Guido Tiana (European Biophysics Journal, 2021)
Energetic properties of a protein are a major determinant of its evolutionary fitness. Using a reconstruction algorithm, dating the reconstructed proteins and calculating the interaction network between their amino acids through a coevolutionary approach, we studied how the interactions that stabilise 890 proteins, belonging to five families, evolved for billions of years. In particular, we focused our attention on the network of most strongly attractive contacts and on that of poorly optimised, frustrated contacts. Our results support the idea that the cluster of most attractive interactions extends its size along evolutionary time, but from the data, we cannot conclude that protein stability or that the degree of frustration tends always to decrease.

Fluorescence-based techniques for the detection of the oligomeric status of proteins: implication in amyloidogenic diseases - Lipika Mirdha; Hirak Chakraborty (European Biophysics Journal, 2021)

Effect of setting data collection parameters on the reliability of a circular dichroism spectrum - Victor K. Sousa; Jéssica A. F. Pedro; Patricia S. Kumagai; Jose L. S. Lopes (European Biophysics Journal, 2021)

Proximity-induced electrodeformation and membrane capacitance coupling between cells - E. Sabri; C. Brosseau (European Biophysics Journal, 2021)

Community-building and promotion of technological excellence in molecular biophysics: the ARBRE–MOBIEU network - Patrick England; Thomas A. Jowitt (European Biophysics Journal, 2021)

Correction to: Standard operation procedure for switchSENSE DRX systems - Hanna Müller-Landau; Paloma Fernández Varela (European Biophysics Journal, 2021)

Substitution of histidine 30 by asparagine in manganese superoxide dismutase alters biophysical properties and supports proliferation in a K562 leukemia cell line - Rosalin Bonetta; Gary J. Hunter; Chi H. Trinh; Tomasz Borowski; Anthony G. Fenech; Maria Kulp; Leandro C. Tabares; Sun Un; Thérèse Hunter (European Biophysics Journal, 2021)
We have generated a mutant of C. elegans manganese superoxide dismutase at histidine 30 by site-directed mutagenesis. The structure was solved at a resolution of 1.52 Å by X-ray crystallography (pdb: 6S0D). His30 was targeted, as it forms as a gateway residue at the top of the solvent access funnel to the active site, together with Tyr34. In the wild-type protein, these gateway residues are involved in the hydrogen-bonding network providing the protons necessary for the catalytic reaction at the metal center. However, biophysical characterization and cell viability experiments reveal that a mutation from histidine to asparagine in the H30N mutant modifies metal selectivity in the protein, favoring the uptake of iron over manganese in minimal media conditions, alters active-site coordination from the characteristic trigonal bipyramidal to octahedral geometry, and encourages cellular proliferation in K562 cells, when added exogenously to the cells.

Structural features and stability of apo- and holo-forms of a simple iron–sulfur protein - Ana V. Almeida; João P. Jacinto; João P. L. Guerra; Bruno J. C. Vieira; João C. Waerenborgh; Nykola C. Jones; Søren V. Hoffmann; Alice S. Pereira; Pedro Tavares (European Biophysics Journal, 2021)

Probing the oligomeric re-assembling of bacterial fimbriae in vitro: a small-angle X-ray scattering and analytical ultracentrifugation study - Alexandra S. Solovyova; Daniel T. Peters; Gema Dura; Helen Waller; Jeremy H. Lakey; David A. Fulton (European Biophysics Journal, 2021)
Capsular antigen fragment 1 (Caf1) is an oligomeric protein consisting of 15 kDa monomeric subunits that are non-covalently linked through exceptionally strong and kinetically inert interactions into a linear polymer chain. It has been shown that after its thermal depolymerisation into unfolded monomeric subunits, Caf1 is able to efficiently repolymerise in vitro to reform its polymeric structure. However, little is known about the nature of the repolymerisation process. An improved understanding of this process will lead to the development of methods to better control the lengths of the repolymerised species, and ultimately, to better design of the properties of Caf1-based materials. Here we utilize small-angle X-ray scattering to estimate the size of Caf1 polymers during the first 24 h of the re-polymerisation process. Analytical ultracentrifugation measurements were also used to investigate the process post-24 h, where the rate of repolymerisation becomes considerably slower. Results show that in vitro polymerisation proceeds in a linear manner with no evidence observed for the formation of a lateral polymer network or uncontrolled aggregates. The rate of Caf1 in vitro repolymerisation was found to be concentration-dependent. Importantly, the rate of polymer growth was found to be relatively fast over the first few hours, before continuing at a dramatically slower rate. This observation is not consistent with the previously proposed step-growth mechanism of in vitro polymerisation of Caf1, where a linear increase in polymer length would be expected with time. We speculate how our observations may support the idea that the polymerisation process may be occurring at the ends of the chains with monomers adding sequentially. Our findings will contribute towards the development of new biomaterials for 3D cell culture and bio-printing.

Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein–ligand binding and protein stability - Egidijus Kazlauskas; Vytautas Petrauskas; Vaida Paketurytė; Daumantas Matulis (European Biophysics Journal, 2021)

A study on the secondary structure of the metalloregulatory protein CueR: effect of pH, metal ions and DNA - Ria K. Balogh; Eszter Németh; Nykola C. Jones; Søren Vrønning Hoffmann; Attila Jancsó; Béla Gyurcsik (European Biophysics Journal, 2021)

Correction to: Macromolecular interactions in vitro, comparing classical and novel approaches - Christophe Velours; Magali Aumont-Nicaise; Stephan Uebel; Patrick England; Adrian Velazquez-Campoy; David Stroebel; Guillaume Bec; Pierre Soule; Christophe Quétard; Christine Ebel; Alain Roussel; Jean-Baptiste Charbonnier; et al. (European Biophysics Journal, 2021)

Dps–DNA interaction in Marinobacter hydrocarbonoclasticus protein: effect of a single-charge alteration - João P. Jacinto; Daniela Penas; João P. L. Guerra; Ana V. Almeida; Nykola C. Jones; Søren V. Hoffmann; Pedro Tavares; Alice S. Pereira (European Biophysics Journal, 2021)

Quality control of purified proteins to improve data quality and reproducibility: results from a large-scale survey - Nick Berrow; Ario de Marco; Mario Lebendiker; Maria Garcia-Alai; Stefan H. Knauer; Blanca Lopez-Mendez; André Matagne; Annabel Parret; Kim Remans; Stephan Uebel; Bertrand Raynal (European Biophysics Journal, 2021)

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study - Blanca López-Méndez; Bruno Baron; Chad A. Brautigam; Thomas A. Jowitt; Stefan H. Knauer; Stephan Uebel; Mark A. Williams; Arthur Sedivy; Olga Abian; Celeste Abreu; Malgorzata Adamczyk; Wojciech Bal; et al. (European Biophysics Journal, 2021)
Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments , employing a standard operation procedure and centrally prepared samples. A protein–small molecule interaction, a newly developed protein–protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM-based single-cell force spectroscopy - Prem Kumar Viji Babu; Ursula Mirastschijski; Ganzanfer Belge; Manfred Radmacher (European Biophysics Journal, 2021)
Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin–cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins’ native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins’ homophilic bonds.

Standard operating procedure for NanoTemper Monolith measurements - Arthur Sedivy (European Biophysics Journal, 2021)

Microscale Thermophoresis and additional effects measured in NanoTemper Monolith instruments - Blanca López-Méndez; Stephan Uebel; Li Peng Lundgren; Arthur Sedivy (European Biophysics Journal, 2021)

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA - Adrian Velazquez-Campoy; Bárbara Claro; Olga Abian; Jonas Höring; Louis Bourlon; Rafael Claveria-Gimeno; Eric Ennifar; Patrick England; Jonathan Brad Chaires; Di Wu; Grzegorz Piszczek; Chad Brautigam; et al. (European Biophysics Journal, 2021)

A standard operating procedure for an enzymatic activity inhibition assay - Joana Smirnovienė; Lina Baranauskienė; Asta Zubrienė; Daumantas Matulis (European Biophysics Journal, 2021)

Interaction standards for biophysics: anti-lysozyme nanobodies - Holly L. Birchenough; Hilda D. Ruiz Nivia; Thomas A. Jowitt (European Biophysics Journal, 2021)
There is a significant demand in the molecular biophysics community for robust standard samples. They are required by researchers, instrument developers and pharmaceutical companies for instrumental quality control, methodological development and in the design and validation of devices, diagnostics and instrumentation. To-date there has been no clear consensus on the need and type of standards that should be available and different research groups and instrument manufacturers use different standard systems which significantly hinders comparative analysis. One of the major objectives of the Association of Resources for Biophysical Research in Europe (ARBRE) is to establish a common set of standard samples that can be used throughout the biophysics community and instrument developers. A survey was circulated among ARBRE members to ascertain the requirements of laboratories when using standard systems and the results are documented in this article. In summary, the major requirements are protein samples which are cheap, relatively small, stable and have different binding strengths. We have developed a panel of sdAb’s or ‘nanobodies’ against hen-egg white lysozyme with different binding strengths and suitable stability characteristics. Here we show the results of the survey, the selection procedure, validation and final selection of a panel of nanobody interaction standards.

Uncertainty in protein–ligand binding constants: asymmetric confidence intervals versus standard errors - Vaida Paketurytė; Vytautas Petrauskas; Asta Zubrienė; Olga Abian; Margarida Bastos; Wen-Yih Chen; Maria João Moreno; Georg Krainer; Vaida Linkuvienė; Arthur Sedivy; Adrian Velazquez-Campoy; Mark A. Williams; et al. (European Biophysics Journal, 2021)

Macromolecular interactions in vitro, comparing classical and novel approaches - Christophe Velours; Magali Aumont-Nicaise; Stephan Uebel; Patrick England; Adrian Velazquez-Campoy; David Stroebel; Guillaume Bec; Pierre Soule; Christophe Quétard; Christine Ebel; Alain Roussel; Jean-Baptiste Charbonnier; et al. (European Biophysics Journal, 2021)

Standard operation procedure for switchSENSE DRX systems - Hanna Müller-Landau; Paloma Fernández Varela (European Biophysics Journal, 2021)

Attenuated total reflection-Fourier transform infrared spectroscopy: a tool to characterize antimicrobial cyclic peptide–membrane interactions - Bárbara Claro; Erik Goormaghtigh; Margarida Bastos (European Biophysics Journal, 2021)

Biophysical characterization of melanoma cell phenotype markers during metastatic progression - Anna Sobiepanek; Alessio Paone; Francesca Cutruzzolà; Tomasz Kobiela (European Biophysics Journal, 2021)
Melanoma is the most fatal form of skin cancer, with increasing prevalence worldwide. The most common melanoma genetic driver is mutation of the proto-oncogene serine/threonine kinase BRAF ; thus, the inhibition of its MAP kinase pathway by specific inhibitors is a commonly applied therapy. However, many patients are resistant, or develop resistance to this type of monotherapy, and therefore combined therapies which target other signaling pathways through various molecular mechanisms are required. A possible strategy may involve targeting cellular energy metabolism, which has been recognized as crucial for cancer development and progression and which connects through glycolysis to cell surface glycan biosynthetic pathways. Protein glycosylation is a hallmark of more than 50% of the human proteome and it has been recognized that altered glycosylation occurs during the metastatic progression of melanoma cells which, in turn facilitates their migration. This review provides a description of recent advances in the search for factors able to remodel cell metabolism between glycolysis and oxidative phosphorylation, and of changes in specific markers and in the biophysical properties of cells during melanoma development from a nevus to metastasis. This development is accompanied by changes in the expression of surface glycans, with corresponding changes in ligand-receptor affinity, giving rise to structural features and viscoelastic parameters particularly well suited to study by label-free biophysical methods.

Isothermal titration calorimetry (ITC): a standard operating procedure (SOP) - Margarida Bastos; Adrian Velazquez-Campoy (European Biophysics Journal, 2021)

Standard protocol for mass photometry experiments - Di Wu; Grzegorz Piszczek (European Biophysics Journal, 2021)

Protein formulation through automated screening of pH and buffer conditions, using the Robotein® high throughput facility - Ruth Kellner; Romain Malempré; Julie Vandenameele; Alain Brans; Anne-Françoise Hennen; Noémie Rochus; Alexandre Di Paolo; Marylène Vandevenne; André Matagne (European Biophysics Journal, 2021)

Amino acid side chain contribution to protein FTIR spectra: impact on secondary structure evaluation - Joëlle De Meutter; Erik Goormaghtigh (European Biophysics Journal, 2021)
Prediction of protein secondary structure from FTIR spectra usually relies on the absorbance in the amide I–amide II region of the spectrum. It assumes that the absorbance in this spectral region, i.e., roughly 1700–1500 cm −1 is solely arising from amide contributions. Yet, it is accepted that, on the average, about 20% of the absorbance is due to amino acid side chains. The present paper evaluates the contribution of amino acid side chains in this spectral region and the potential to improve secondary structure prediction after correcting for their contribution. We show that the β-sheet content prediction is improved upon subtraction of amino acid side chain contributions in the amide I–amide II spectral range. Improvement is relatively important, for instance, the error of prediction of β-sheet content decreases from 5.42 to 4.97% when evaluated by ascending stepwise regression. Other methods tested such as partial least square regression and support vector machine have also improved accuracy for β-sheet content evaluation. The other structures such as α-helix do not significantly benefit from side chain contribution subtraction, in some cases prediction is even degraded. We show that co-linearity between secondary structure content and amino acid composition is not a main limitation for improving secondary structure prediction. We also show that, even though based on different criteria, secondary structures defined by DSSP and XTLSSTR both arrive at the same conclusion: only the β-sheet structure clearly benefits from side chain subtraction. It must be concluded that side chain contribution subtraction benefit for the evaluation of other secondary structure contents is limited by the very rough description of side chain absorbance which does not take into account the variations related to their environment. The study was performed on a large protein set. To deal with the large number of proteins present, we worked on protein microarrays deposited on BaF 2 slides and FTIR spectra were acquired with an imaging system.

Development of 48-condition buffer screen for protein stability assessment - Josef Houser; Jana Kosourova; Monika Kubickova; Michaela Wimmerova (European Biophysics Journal, 2021)

Evaluation of protein secondary structure from FTIR spectra improved after partial deuteration - Joëlle De Meutter; Erik Goormaghtigh (European Biophysics Journal, 2021)
FTIR spectroscopy has become a major tool to determine protein secondary structure. One of the identified obstacle for reaching better predictions is the strong overlap of bands assigned to different secondary structures. Yet, while for instance disordered structures and α-helical structures absorb almost at the same wavenumber, the absorbance bands are differentially shifted upon deuteration, in part because exchange is much faster for disordered structures. We recorded the FTIR spectra of 85 proteins at different stages of hydrogen/deuterium exchange process using protein microarrays and infrared imaging for high throughput measurements. Several methods were used to relate spectral shape to secondary structure content. While in absolute terms, β-sheet is always better predicted than α-helix content, results consistently indicate an improvement of secondary structure predictions essentially for the α-helix and the category called “Others” (grouping random, turns, bends, etc.) after 15 min of exchange. On the contrary, the β-sheet fraction is better predicted in non-deuterated conditions. Using partial least square regression, the error of prediction for the α-helix content is reduced after 15-min deuteration. Further deuteration degrades the prediction. Error on the prediction for the “Others” structures also decreases after 15-min deuteration. Cross-validation or a single 25-protein test set result in the same overall conclusions.

Protein intrinsic viscosity determination with the Viscosizer TD instrument: reaching beyond the initially expected applications - Sébastien Brûlé; Raffaele Leroux; Patrick England; Bertrand Raynal (European Biophysics Journal, 2021)

Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA - Adriana E. Miele; Laura Cervoni; Aline Le Roy; Antimo Cutone; Giovanni Musci; Christine Ebel; Maria Carmela Bonaccorsi di Patti (European Biophysics Journal, 2021)

Calibrating analytical ultracentrifuges - Huaying Zhao; Ai Nguyen; Samuel C. To; Peter Schuck (European Biophysics Journal, 2021)

Special issue: Multicomponent lipid membranes—how molecular organisation leads to function - Bert de Groot; Andreas Janshoff; Claudia Steinem; Markus Zweckstetter (European Biophysics Journal, 2021)

Analysis of patchclamp recordings: model-free multiscale methods and software - Florian Pein; Benjamin Eltzner; Axel Munk (European Biophysics Journal, 2021)
Analysis of patchclamp recordings is often a challenging issue. We give practical guidance how such recordings can be analyzed using the model-free multiscale idealization methodology JSMURF, JULES, and HILDE. We provide an operational manual how to use the accompanying software available as an R-package and as a graphical user interface. This includes selection of the right approach and tuning of parameters. We also discuss advantages and disadvantages of model-free approaches in comparison to hidden Markov model approaches and explain how they complement each other.

Selected tools to visualize membrane interactions - Tobias Grothe; Julia Nowak; Reinhard Jahn; Peter Jomo Walla (European Biophysics Journal, 2021)
In the past decade, we developed various fluorescence-based methods for monitoring membrane fusion, membrane docking, distances between membranes, and membrane curvature. These tools were mainly developed using liposomes as model systems, which allows for the dissection of specific interactions mediated by, for example, fusion proteins. Here, we provide an overview of these methods, including two-photon fluorescence cross-correlation spectroscopy and intramembrane Förster energy transfer, with asymmetric labelling of inner and outer membrane leaflets and the calibrated use of transmembrane energy transfer to determine membrane distances below 10 nm. We discuss their application range and their limitations using examples from our work on protein-mediated vesicle docking and fusion.

Structure, gating and interactions of the voltage-dependent anion channel - Eszter E. Najbauer; Stefan Becker; Karin Giller; Markus Zweckstetter; Adam Lange; Claudia Steinem; Bert L. de Groot; Christian Griesinger; Loren B. Andreas (European Biophysics Journal, 2021)
The voltage-dependent anion channel (VDAC) is one of the most highly abundant proteins found in the outer mitochondrial membrane, and was one of the earliest discovered. Here we review progress in understanding VDAC function with a focus on its structure, discussing various models proposed for voltage gating as well as potential drug targets to modulate the channel’s function. In addition, we explore the sensitivity of VDAC structure to variations in the membrane environment, comparing DMPC-only, DMPC with cholesterol, and near-native lipid compositions, and use magic-angle spinning NMR spectroscopy to locate cholesterol on the outside of the β-barrel. We find that the VDAC protein structure remains unchanged in different membrane compositions, including conditions with cholesterol.

How arginine derivatives alter the stability of lipid membranes: dissecting the roles of side chains, backbone and termini - Sarah F. Verbeek; Neha Awasthi; Nikolas K. Teiwes; Ingo Mey; Jochen S. Hub; Andreas Janshoff (European Biophysics Journal, 2021)
Arginine (R)-rich peptides constitute the most relevant class of cell-penetrating peptides and other membrane-active peptides that can translocate across the cell membrane or generate defects in lipid bilayers such as water-filled pores. The mode of action of R-rich peptides remains a topic of controversy, mainly because a quantitative and energetic understanding of arginine effects on membrane stability is lacking. Here, we explore the ability of several oligo-arginines R $$_n$$ n and of an arginine side chain mimic R $$_Side$$ Side to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R $$_Side$$ Side but not R $$_n$$ n reduces the line tension of a membrane with anionic lipids. While R $$_n$$ n peptides form a layer on top of a partly negatively charged lipid bilayer, R $$_Side$$ Side leads to its disintegration. Complementary, our simulations show R $$_Side$$ Side causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R $$_8$$ 8 similarly promoted pore formation in simulations, but without overall bilayer destabilization. We conclude that while the guanidine moiety is intrinsically membrane-disruptive, poly-arginines favor pore formation in negatively charged membranes via a different mechanism. Pore formation by R-rich peptides seems to be counteracted by lipids with PC headgroups. We found that long R $$_n$$ n and R $$_Side$$ Side but not short R $$_n$$ n reduce the free energy of nucleating a pore. In short R $$_n$$ n , the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer $$R_{n}$$ R n are membrane-active.

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels - Igor Tkach; Ulf Diederichsen; Marina Bennati (European Biophysics Journal, 2021)
Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5–10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes. Graphical Abstract

Membrane fusion studied by colloidal probes - Hannes Witt; Filip Savić; Sarah Verbeek; Jörn Dietz; Gesa Tarantola; Marieelen Oelkers; Burkhard Geil; Andreas Janshoff (European Biophysics Journal, 2021)
Membrane-coated colloidal probes combine the benefits of solid-supported membranes with a more complex three-dimensional geometry. This combination makes them a powerful model system that enables the visualization of dynamic biological processes with high throughput and minimal reliance on fluorescent labels. Here, we want to review recent applications of colloidal probes for the study of membrane fusion. After discussing the advantages and disadvantages of some classical vesicle-based fusion assays, we introduce an assay using optical detection of fusion between membrane-coated glass microspheres in a quasi two-dimensional assembly. Then, we discuss free energy considerations of membrane fusion between supported bilayers, and show how colloidal probes can be combined with atomic force microscopy or optical tweezers to access the fusion process with even greater detail.

A stalk fluid forming above the transition from the lamellar to the rhombohedral phase of lipid membranes - Max Scheu; Karlo Komorowski; Chen Shen; Tim Salditt (European Biophysics Journal, 2021)
In this work, we present evidence for the formation of transient stalks in aligned multilamellar stacks of lipid membranes. Just above the phase transition from the fluid ( $$L_α$$ L α ) lamellar phase to the rhombohedral phase (R), where lipid stalks crystallize on a super-lattice within the lipid bilayer stack, we observe a characteristic scattering pattern, which can be attributed to a correlated fluid of transient stalks. Excess (off-axis) diffuse scattering with a broad modulation around the position which later transforms to a sharp peak of the rhombohedral lattice, gives evidence for the stalk fluid forming as a pre-critical effect, reminiscent of critical phenomena in the vicinity of second-order phase transitions. Using high-resolution off-specular X-ray scattering and lineshape analysis we show that this pre-critical regime is accompanied by an anomalous elasticity behavior of the membrane stack, in particular an increase in inter-bilayer compressibility, i.e., a decrease in the compression modulus.

How does curvature affect the free-energy barrier of stalk formation? Small vesicles vs apposing, planar membranes - Y. G. Smirnova; M. Müller (European Biophysics Journal, 2021)
Using molecular simulations of POPC lipids in conjunction with the calculation of the Minimum Free-Energy Path (MFEP), we study the effect of strong membrane curvature on the formation of the first fusion intermediate—the stalk between a vesicle and its periodic image. We find that the thermodynamic stability of this hourglass-shaped, hydrophobic connection between two vesicles is largely increased by the strong curvature of small vesicles, whereas the intrinsic barrier to form a stalk, i.e., associated with dimple formation and lipid tails protrusions, is similar to the case of two, apposing, planar membranes. A significant reduction of the barrier of stalk formation, however, stems from the lower dehydration free energy that is required to bring highly curved vesicle into a distance, at which stalk formation may occur, compared to the case of apposing, planar membranes.

Protein-dependent membrane remodeling in mitochondrial morphology and clathrin-mediated endocytosis - Daryna Tarasenko; Michael Meinecke (European Biophysics Journal, 2021)
Cellular membranes can adopt a plethora of complex and beautiful shapes, most of which are believed to have evolved for a particular physiological reason. The closely entangled relationship between membrane morphology and cellular physiology is strikingly seen in membrane trafficking pathways. During clathrin-mediated endocytosis, for example, over the course of a minute, a patch of the more or less flat plasma membrane is remodeled into a highly curved clathrin-coated vesicle. Such vesicles are internalized by the cell to degrade or recycle plasma membrane receptors or to take up extracellular ligands. Other, steadier, membrane morphologies can be observed in organellar membranes like the endoplasmic reticulum or mitochondria. In the case of mitochondria, which are double membrane-bound, ubiquitous organelles of eukaryotic cells, especially the mitochondrial inner membrane displays an intricated ultrastructure. It is highly folded and consequently has a much larger surface than the mitochondrial outer membrane. It can adopt different shapes in response to cellular demands and changes of the inner membrane morphology often accompany severe diseases, including neurodegenerative- and metabolic diseases and cancer. In recent years, progress was made in the identification of molecules that are important for the aforementioned membrane remodeling events. In this review, we will sum up recent results and discuss the main players of membrane remodeling processes that lead to the mitochondrial inner membrane ultrastructure and in clathrin-mediated endocytosis. We will compare differences and similarities between the molecular mechanisms that peripheral and integral membrane proteins use to deform membranes.

Lipid–protein forces predict conformational changes in a mechanosensitive channel - Csaba Daday; Bert L. de Groot (European Biophysics Journal, 2021)
The mechanosensitive TREK-2 potassium channel, a member of the K2P family, has essential physiological roles and is, therefore, a pharmaceutical target. A combination of experimental and computational studies have established that of the two known conformations, “up” and “down”, membrane tension directly favors the “up” state, which displays a higher conductance. However, these studies did not reveal the exact mechanism by which the membrane affects the channel conformation. In this work, we show that changes in protein–lipid interaction patterns suffice in predicting this conformational change, and pinpoint potentially important residues involved in this phenomenon.

Membrane-embedded TSPO: an NMR view - Gwladys Rivière; Garima Jaipuria; Loren B. Andreas; Andrei Leonov; Karin Giller; Stefan Becker; Markus Zweckstetter (European Biophysics Journal, 2021)
Translocator Protein (18 kDa) (TSPO) is a mitochondrial transmembrane protein commonly used as a biomarker for neuroinflammation and is also a potential therapeutic target in neurodegenerative diseases. Despite intensive research efforts, the function of TSPO is still largely enigmatic. Deciphering TSPO structure in the native lipid environment is essential to gain insight into its cellular activities and to design improved diagnostic and therapeutic ligands. Here, we discuss the influence of lipid composition on the structure of mammalian TSPO embedded into lipid bilayers on the basis of solid-state NMR experiments. We further highlight that cholesterol can influence both the tertiary and quaternary TSPO structure and also influence TSPO localization in mitochondria-associated endoplasmic reticulum membranes.

How proteins open fusion pores: insights from molecular simulations - H. Jelger Risselada; Helmut Grubmüller (European Biophysics Journal, 2021)
Fusion proteins can play a versatile and involved role during all stages of the fusion reaction. Their roles go far beyond forcing the opposing membranes into close proximity to drive stalk formation and fusion. Molecular simulations have played a central role in providing a molecular understanding of how fusion proteins actively overcome the free energy barriers of the fusion reaction up to the expansion of the fusion pore. Unexpectedly, molecular simulations have revealed a preference of the biological fusion reaction to proceed through asymmetric pathways resulting in the formation of, e.g., a stalk-hole complex, rim-pore, or vertex pore. Force-field based molecular simulations are now able to directly resolve the minimum free-energy path in protein-mediated fusion as well as quantifying the free energies of formed reaction intermediates. Ongoing developments in Graphics Processing Units (GPUs), free energy calculations, and coarse-grained force-fields will soon gain additional insights into the diverse roles of fusion proteins.

In vitro single vesicle fusion assays based on pore-spanning membranes: merits and drawbacks - Peter Mühlenbrock; Merve Sari; Claudia Steinem (European Biophysics Journal, 2021)
Neuronal fusion mediated by soluble N -ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) is a fundamental cellular process by which two initially distinct membranes merge resulting in one interconnected structure to release neurotransmitters into the presynaptic cleft. To get access to the different stages of the fusion process, several in vitro assays have been developed. In this review, we provide a short overview of the current in vitro single vesicle fusion assays. Among those assays, we developed a single vesicle assay based on pore-spanning membranes (PSMs) on micrometre-sized pores in silicon, which might overcome some of the drawbacks associated with the other membrane architectures used for investigating fusion processes. Prepared by spreading of giant unilamellar vesicles with reconstituted t-SNAREs, PSMs provide an alternative tool to supported lipid bilayers to measure single vesicle fusion events by means of fluorescence microscopy. Here, we discuss the diffusive behaviour of the reconstituted membrane components as well as that of the fusing synthetic vesicles with reconstituted synaptobrevin 2 (v-SNARE). We compare our results with those obtained if the synthetic vesicles are replaced by natural chromaffin granules under otherwise identical conditions. The fusion efficiency as well as the different fusion states observable in this assay by means of both lipid mixing and content release are illuminated.

Chemically synthesized Gb3 glycosphingolipids: tools to access their function in lipid membranes - Jeremias Sibold; Somayeh Ahadi; Daniel B. Werz; Claudia Steinem (European Biophysics Journal, 2021)
Gb 3 glycosphingolipids are the specific receptors for bacterial Shiga toxin. Whereas the trisaccharidic head group of Gb 3 defines the specificity of Shiga toxin binding, the lipophilic part composed of sphingosine and different fatty acids is suggested to determine its localization within membranes impacting membrane organisation and protein binding eventually leading to protein internalisation. While most studies use Gb 3 extracts, chemical synthesis provides a unique tool to access different tailor-made Gb 3 glycosphingolipids. In this review, strategies to synthesize these complex glycosphingolipids are presented. Special emphasis is put on the preparation of Gb 3 molecules differing only in their fatty acid part (saturated, unsaturated, α-hydroxylated and both, unsaturated and α-hydroxylated). With these molecules in hand, it became possible to investigate the phase behaviour of liquid ordered/liquid disordered supported membranes doped with the Gb 3 species by means of fluorescence and atomic force microscopy. The results clearly highlight the influence of the different fatty acids of the Gb 3 sphingolipids on the phase behaviour and the binding properties of Shiga toxin B subunits, even though the membranes were only doped with 5 mol% of the receptor lipid. To obtain fluorescent Gb 3 derivatives, either fatty acid labelled Gb 3 molecules or head group labelled ones were synthesized. These molecules enabled us to address the question, where the Gb 3 sphingolipids are localized prior protein binding by means of fluorescence microscopy on giant unilamellar vesicles. The results again demonstrate that the fatty acid of Gb 3 plays a pivotal role for the overall membrane organisation.

Inferring functional units in ion channel pores via relative entropy - Michael Schmidt; Indra Schroeder; Daniel Bauer; Gerhard Thiel; Kay Hamacher (European Biophysics Journal, 2021)
Coarse-grained protein models approximate the first-principle physical potentials. Among those modeling approaches, the relative entropy framework yields promising and physically sound results, in which a mapping from the target protein structure and dynamics to a model is defined and subsequently adjusted by an entropy minimization of the model parameters. Minimization of the relative entropy is equivalent to maximization of the likelihood of reproduction of (configurational ensemble) observations by the model. In this study, we extend the relative entropy minimization procedure beyond parameter fitting by a second optimization level, which identifies the optimal mapping to a (dimension-reduced) topology. We consider anisotropic network models of a diverse set of ion channels and assess our findings by comparison to experimental results.

Multiscale nature of cell rearrangement caused by collective cell migration - Ivana Pajic-Lijakovic; Milan Milivojevic (European Biophysics Journal, 2021)

Towards understanding single-channel characteristics of OccK8 purified from Pseudomonas aeruginosa - Fatma Dogan Guzel; Daniel Pletzer; Araz Norouz Dizaji; Kareem Al-Nahas; Mawadah Bajrai; Mathias Winterhalter (European Biophysics Journal, 2021)

Differential dielectroscopic data on the relation of erythrocyte membrane skeleton to erythrocyte deformability and flicker - Ivan T. Ivanov; Boyana K. Paarvanova (European Biophysics Journal, 2021)

Molecular crowding accelerates aggregation of α-synuclein by altering its folding pathway - Soumojit Biswas; Antara Bhadra; Sunidhi Lakhera; Monika Soni; Venkataharsha Panuganti; Swati Jain; Ipsita Roy (European Biophysics Journal, 2021)

An investigation into the critical tension of electroporation in anionic lipid vesicles - Mohammad Abu Sayem Karal; Md. Kabir Ahamed; Urbi Shyamolima Orchi; Md. Towhiduzzaman; Marzuk Ahmed; Shareef Ahammed; Nadia Akter Mokta; Muhammad Samir Ullah (European Biophysics Journal, 2021)

Electrostatic effects in saturation of membrane binding of cationic cell-penetrating peptide - Anna Svirina; Ivan Terterov (European Biophysics Journal, 2021)

Exploring the diversity of mechanosensitive channels in bacterial genomes - Sarah C. Johnson; Jordyn Veres; Hannah R. Malcolm (European Biophysics Journal, 2021)

Measuring compressibility in the optima AUC™ analytical ultracentrifuge - Marielle Stoutjesdyk; Emre Brookes; Amy Henrickson; Borries Demeler (European Biophysics Journal, 2020)

Multi-wavelength analytical ultracentrifugation as a tool to characterise protein–DNA interactions in solution - Christopher R. Horne; Amy Henrickson; Borries Demeler; Renwick C. J. Dobson (European Biophysics Journal, 2020)

Analytical ultracentrifugation: still the gold standard that offers multiple solutions - Renwick C. J. Dobson; Trushar R. Patel (European Biophysics Journal, 2020)

Probing the effect of aroma compounds on the hydrodynamic properties of mucin glycoproteins - Vlad Dinu; Thomas MacCalman; Ni Yang; Gary G. Adams; Gleb E. Yakubov; Stephen E. Harding; Ian D. Fisk (European Biophysics Journal, 2020)
Aroma compounds are diverse low molecular weight organic molecules responsible for the flavour of food, medicines or cosmetics. Natural and artificial aroma compounds are manufactured and used by the industry to enhance the flavour and fragrance of products. While the low concentrations of aroma compounds present in food may leave no effect on the structural integrity of the mucosa, the effect of concentrated aroma volatiles is not well understood. At high concentrations, like those found in some flavoured products such as e-cigarettes, some aroma compounds are suggested to elicit a certain degree of change in the mucin glycoprotein network, depending on their functional group. These effects are particularly associated with carbonyl compounds such as aldehydes and ketones, but also phenols which may interact with mucin and other glycoproteins through other interaction mechanisms. This study demonstrates the formation of such interactions in vitro through the use of molecular hydrodynamics. Sedimentation velocity studies reveal that the strength of the carbonyl compound interaction is influenced by compound hydrophobicity, in which the more reactive short chain compounds show the largest increase in mucin-aroma sedimentation coefficients. By contrast, the presence of groups that increases the steric hindrance of the carbonyl group, such as ketones, produced a milder effect. The interaction effects were further demonstrated for hexanal using size exclusion chromatography light scattering (SEC-MALS) and intrinsic viscosity. In addition, phenolic aroma compounds were identified to reduce the sedimentation coefficient of mucin, which is consistent with interactions in the non-glycosylated mucin region.

Analysis of nonideality: insights from high concentration simulations of sedimentation velocity data - J. J. Correia; R. T. Wright; P. J. Sherwood; W. F. Stafford (European Biophysics Journal, 2020)
The Aviv fluorescence detection system (Aviv-FDS) has allowed the performance of sedimentation velocity experiments on therapeutic antibodies in highly concentrated environments like formulation buffers and serum. Methods were implemented in the software package SEDANAL for the analysis of nonideal, weakly associating AUC data acquired on therapeutic antibodies and proteins (Wright et al. Eur Biophys J 47:709–722, 2018, Anal Biochem 550:72–83, 2018). This involved fitting both hydrodynamic, k s , and thermodynamic, BM 1 , nonideality where concentration dependence is expressed as s = s o /(1 + k s c ) and D = D o (1 + 2BM 1 c )/(1 + k s c ) and s o and D o are values extrapolated to c = 0 (mg/ml). To gain insight into the consequences of these phenomenological parameters, we performed simulations with SEDANAL of a monoclonal antibody as a function of k s (0–100 ml/g) and BM 1 (0–100 ml/g). This provides a visual understanding of the separate and joint impact of k s and BM 1 on the shape of high-concentration sedimentation velocity boundaries and the challenge of their unique determination by finite element methods. In addition, mAbs undergo weak self- and hetero-association (Yang et al. Prot Sci 27:1334–1348, 2018) and thus we have simulated examples of nonideal weak association over a wide range of concentrations (1–120 mg/ml). Here we demonstrate these data are best analyzed by direct boundary global fitting to models that account for k s , BM 1 and weak association. Because a typical clinical dose of mAb is 50–200 mg/ml, these results have relevance for biophysical understanding of concentrated therapeutic proteins.

Analytical ultracentrifuge: an ideal tool for characterization of non-coding RNAs - Maulik D. Badmalia; M. Quadir Siddiqui; Tyler Mrozowich; Darren L. Gemmill; Trushar R. Patel (European Biophysics Journal, 2020)

Purification of a heterodimeric Reelin construct to investigate binding stoichiometry - Liam S. Turk; Daniel Mitchell; Davide Comoletti (European Biophysics Journal, 2020)
Reelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein’s function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin’s central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin’s known receptors, calculating the K D of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.

On the utility of fluorescence-detection analytical ultracentrifugation in probing biomolecular interactions in complex solutions: a case study in milk - Jennifer M. Crowther; Marita Broadhurst; Thomas M. Laue; Geoffrey B. Jameson; Alison J. Hodgkinson; Renwick C. J. Dobson (European Biophysics Journal, 2020)

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase - Elena Sugrue; David Coombes; David Wood; Tong Zhu; Katherine A. Donovan; Renwick C. J. Dobson (European Biophysics Journal, 2020)

A comparison of data quality using quartz vs. sapphire cell windows in analytical ultracentrifugation - Akash Bhattacharya; Eric Von Seggern (European Biophysics Journal, 2020)

Tert-butyldimethylsilyl chitosan synthesis and characterization by analytical ultracentrifugation, for archaeological wood conservation - Jennifer M. K. Wakefield; Susan Braovac; Hartmut Kutzke; Robert A. Stockman; Stephen E. Harding (European Biophysics Journal, 2020)
The Oseberg ship is one of the most important archaeological testimonies of the Vikings. After excavation in 1904, the wooden gravegoods were conserved using alum salts. This resulted in extreme degradation of a number of the objects a hundred years later through acid depolymerisation of cellulose and lignin. The fragile condition of the artefacts requires a reconsolidation which has to be done avoiding water as solvent. We synthesized tert- butyldimethylsilyl (TBDMS) chitosan which is soluble in a 50:50 solution of ethyl acetate and toluene. Measurement of its molecular weight, to anticipate its penetration, provided a challenge as the density difference of the polymer and solvent was too small to provide adequate solute redistribution under a centrifugal field, so a two-stage process was implemented (i) determination of the weight-average molar mass of the aqueous soluble activated precursor, chitosan mesylate, M w,mc using sedimentation equilibrium with the SEDFIT-MSTAR algorithm, and determination of the degree of polymerisation DP ; (ii) measurement of the average degree of substitution DS TBDMS of the TBDMS group on each chitosan monosaccharide monomer unit using NMR, to augment the M w,mc value to give the molar mass of the TBDMS-chitosan. For the preparation, we find M w = 9.8 kg·mol −1 , which is within the acceptable limit for penetration and consolidation of degraded wood. Future work will test this on archaeological wood from different sources.

Aminoethyl substitution enhances the self-assembly properties of an aminocellulose as a potential archaeological wood consolidant - Jennifer M. K. Wakefield; Robert Hampe; Richard B. Gillis; Agnes Sitterli; Gary G. Adams; Hartmut Kutzke; Thomas Heinze; Stephen E. Harding (European Biophysics Journal, 2020)
The 6-deoxy-6-aminocelluloses—or “aminocelluloses”—are a class of synthetic natural cellulose derivatives which are mostly aqueous soluble and have excellent film-forming properties. Recent studies have connected these properties at the molecular level with protein-like self-associative behaviour for a range of aminocelluloses including a 6-deoxy-6-(ω-aminoethyl) aminocellulose AEA-1 with the association being a two-stage process—a reversible oligomerisation followed by further (semi-reversible) aggregation into larger structures. Here, we synthesise and compare a new 6-deoxy-6-(ω-aminoethyl) aminocellulose AEA-1′ with different degree of substitution with one with further alkyl derivatisation, namely 6-deoxy-6-(ω-hydroxyethyl) aminocellulose HEA-1′. As with AEA-1, sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge still show a two-stage process for both AEA-1′ and HEA-1′, with the latter giving higher molar masses. The consequences of these properties for use as consolidants for archaeological wood are considered.

Using modern approaches to sedimentation velocity to detect conformational changes in proteins - Chad A. Brautigam; Shih-Chia Tso; Ranjit K. Deka; Wei Z. Liu; Michael V. Norgard (European Biophysics Journal, 2020)

A calibration disk for the correction of radial errors from chromatic aberration and rotor stretch in the Optima AUC™ analytical ultracentrifuge - Marielle Stoutjesdyk; Amy Henrickson; Geoff Minors; Borries Demeler (European Biophysics Journal, 2020)

Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation - Maximilian J. Uttinger; Timon R. Heyn; Uwe Jandt; Simon E. Wawra; Bettina Winzer; Julia K. Keppler; Wolfgang Peukert (European Biophysics Journal, 2020)
The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils. In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.

Following the footprints of variability during filopodial growth - Daniela Senra; Alejandra Páez; Geraldine Gueron; Luciana Bruno; Nara Guisoni (European Biophysics Journal, 2020)

Calcium signaling modulates the dynamics of cilia and flagella - M. V. Satarić; S. Zdravković; T. Nemeš; B. M. Satarić (European Biophysics Journal, 2020)

Modulation of the mitochondrial voltage-dependent anion channel (VDAC) by hydrogen peroxide and its recovery by curcumin - Chetan Malik; Subhendu Ghosh (European Biophysics Journal, 2020)

U-turn time and velocity dependence on the wavelength of light: multicellular magnetotactic prokaryotes of different sizes behave differently - Marcos Venicio Gonçalves Sales; Beatriz Silva Lima; Daniel Acosta-Avalos (European Biophysics Journal, 2020)

Magnetoreception in multicellular magnetotactic prokaryotes: a new analysis of escape motility trajectories in different magnetic fields - Ana Gabriela Veiga Sepulchro; Henrique Lins de Barros; Henrique Oliveira Leiras de Mota; Karen Shiroiva Berbereia; Katterine Patricia Taipe Huamani; Lis Carneiro da Silva Lopes; Vitor Sudbrack; Daniel Acosta-Avalos (European Biophysics Journal, 2020)

The general anesthetic etomidate and fenamate mefenamic acid oppositely affect GABAAR and GlyR: a structural explanation - Alexey Rossokhin (European Biophysics Journal, 2020)

Energetic and thermodynamical aspects of the cyclodextrins-cannabidiol complex in aqueous solution: a molecular-dynamics study - A. J. da Silva; E. S. dos Santos (European Biophysics Journal, 2020)