HSAFM-MIREBA - Methodology for Inferring REsolution in biological applications.

Due to a lack of requirement for any direct labelling of the target molecule, high speed atomic force microscopy (HS-AFM) is a potentially powerful procedure for the assessment of biological processes involving macromolecules. When the sample is static the AFM device can be purposefully setup to recover high-resolution information about the feature in question. However, when the feature to be studied moves an appreciable amount during the course of the measurement, the obtained image will be blurred. Encountering such blurred observations prompts the experimenter to sacrifice higher resolution images for higher scanning speeds by tuning available experimental parameters (such as the scanned image area, the image pixel size, the resonance frequency of the cantilever and/or the diameter of the AFM tip). The present work describes a software tool, HSAFM-MIREBA (High Speed Atomic Force Microscopy - Methodology for Inferring REsolution in Biological Applications) that allows for pre-experimental optimization of such parameters through iterative rounds of simulation of both the dynamic surface process and the HS-AFM measurement (based on the particular set of governing parameters). A representative set of five dynamic biological processes that describe a range of diffusive and directed motions (which can themselves be tuned by altering characteristic governing parameter sets) are provided.

MIL-CELL: a tool for multi-scale simulation of yeast replication and prion transmission.

The single-celled baker's yeast, Saccharomyces cerevisiae, can sustain a number of amyloid-based prions, the three most prominent examples being [URE3], [PSI+], and [PIN+]. In the laboratory, haploid S. cerevisiae cells of a single mating type can acquire an amyloid prion in one of two ways (i) spontaneous nucleation of the prion within the yeast cell, and (ii) receipt via mother-to-daughter transmission during the cell division cycle. Similarly, prions can be lost due to (i) dissolution of the prion amyloid by its breakage into non-amyloid monomeric units, or (ii) preferential donation/retention of prions between the mother and daughter during cell division. Here we present a computational tool (Monitoring Induction and Loss of prions in Cells; MIL-CELL) for modelling these four general processes using a multiscale approach describing both spatial and kinetic aspects of the yeast life cycle and the amyloid-prion behavior. We describe the workings of the model, assumptions upon which it is based and some interesting simulation results pertaining to the wave-like spread of the epigenetic prion elements through the yeast population. MIL-CELL is provided as a stand-alone GUI executable program for free download with the paper. MIL-CELL is equipped with a relational database allowing all simulated properties to be searched, collated and graphed. Its ability to incorporate variation in heritable properties means MIL-CELL is also capable of simulating loss of the isogenic nature of a cell population over time. The capability to monitor both chronological and reproductive age also makes MIL-CELL potentially useful in studies of cell aging.

Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation.

Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 °C. At concentrations lower than 20 µg/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.

A new look at an old view of denaturant induced protein unfolding.

We re-examine a site-binding approach independently proposed by Schellman (Schellman, J.A. (1958) Compt. rend. Lab. Carlsberg Ser. Chim. 30, 439-449) and Aune and Tanford (Aune, K.C. and Tanford, D. (1969) Biochemistry, 8, 4586-4590) for explicitly including the denaturant concentration within the protein unfolding equilibrium. We extend and formalize the approach through development of a multi-dimensional analytical model in which the folding reaction coordinate is defined by the number of denaturant molecules bound to sites located on either the initially folded, or unfolded, states of the protein. We use the developed method to re-examine the mechanistic determinants underlying the sigmoidal shape of the unfolding transition. A natural feature of our method is that it presents a landscape picture of the denaturant induced protein unfolding reaction.

Deamidation of N76 in human γS-crystallin promotes dimer formation.

BACKGROUND: Cataract formation is often attributed to the build-up of post-translational modifications in the crystallin proteins of the eye lens. One such modification, the deamidation of N76 in human γS-crystallin to D76, is highly correlated with age-related cataract (Hooi et al. Invest. Ophthalmol. Vis. Sci. 53 (2012) 3554-3561). In the current work, this modification has been extensively characterised in vitro. METHODS: Biophysical characterisation was performed on wild type and N76D γS-crystallins using turbidity measurements to monitor aggregation, intrinsic fluorescence and circular dichroism spectroscopy to determine the folded state and NMR spectroscopy for identifying local changes in structure. Protein mass was determined using SEC-MALLS and analytical ultracentrifugation methods. RESULTS: Relative to the wild type protein, deamidation at N76 in γS-crystallin causes an increase in the thermal stability and resistance to thermally induced aggregation alongside a decrease in stability to denaturants, a propensity to aggregate rapidly once destabilised and a tendency to form a dimer. We ascribe the apparent increase in thermal stability upon deamidation to the formation of dimer which prevents the unfolding of the inherently less stable monomer. CONCLUSIONS: Deamidation causes a decrease in stability of γS-crystallin but this is offset by an increased tendency for dimer formation. GENERAL SIGNIFICANCE: Deamidation at N76 in human γS-crystallin likely has a combinatorial effect with other post-translational crystallin modifications to induce age-related cataract. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

A structural and functional study of Gln147 deamidation in αA-crystallin, a site of modification in human cataract.

Deamidation of Glu147 in human αA-crystallin is common in aged cataractous lenses (Hains and Truscott, Invest. Ophthalmol. Vis. Sci. 2010, 51, 3107). Accordingly, this modification may have a causative effect in cataract. αA-crystallin is a small heat-shock molecular chaperone protein that prevents aggregation of proteins and is the principal defence against crystallin unfolding and aggregation in the ageing lens. Deamidated Q147E αA-crystallin was structurally characterised using a variety of spectroscopic and biophysical methods, including NMR, circular dichroism and fluorescence spectroscopy and dynamic light scattering. The effect of Glu147 deamidation on αA-crystallin in vitro chaperone ability was determined for a variety of aggregating proteins. Compared to the wild type protein, Q147E αA-crystallin generally exhibited slightly reduced chaperone ability and a small loss of overall structure in its central α-crystallin domain while also showing significantly enhanced thermal stability and a tendency to form slightly larger oligomers. As αA-crystallin is the major lens protein, even a small loss of function could combine with other sources of age-related damage to the crystallins to contribute to lens opacification.

Real-time monitoring of amyloid growth in a rigid gel matrix.

We demonstrate the real-time monitoring of the growth of amyloid-protein aggregates in a semi-rigid gel environment constructed from a 5% w/v gelatin solution. The kinetics of amyloid fibril growth from reduced and carboxy-methylated κ-casein occurring in the gel medium was contrasted against that obtained in a regular solution assay. Aggregation kinetics were recorded using Thioflavin T fluorescence. Transmission electron microscopy was used to confirm the aggregates' existence and morphology. The current demonstration of controlled amyloid growth in a gel environment represents the first step towards development of an experimental model for investigating the role of spatial and medium factors in the kinetics of aggregation-based proteopathies.

Recognizing and analyzing variability in amyloid formation kinetics: Simulation and statistical methods.

We examine the phenomenon of variability in the kinetics of amyloid formation and detail methods for its simulation, identification and analysis. Simulated data, reflecting intrinsic variability, were produced using rate constants, randomly sampled from a pre-defined distribution, as parameters in an irreversible nucleation-growth kinetic model. Simulated kinetic traces were reduced in complexity through description in terms of three characteristic parameters. Practical methods for assessing convergence of the reduced parameter distributions were introduced and a bootstrap procedure was applied to determine convergence for different levels of intrinsic variation. Statistical methods for assessing the significance of shifts in parameter distributions, relating to either change in parameter mean or distribution shape, were tested. Robust methods for analyzing and interpreting kinetic data possessing significant intrinsic variance will allow greater scrutiny of the effects of anti-amyloid compounds in drug trials.

Protein aggregate turbidity: Simulation of turbidity profiles for mixed-aggregation reactions.

Due to their colloidal nature, all protein aggregates scatter light in the visible wavelength region when formed in aqueous solution. This phenomenon makes solution turbidity, a quantity proportional to the relative loss in forward intensity of scattered light, a convenient method for monitoring protein aggregation in biochemical assays. Although turbidity is often taken to be a linear descriptor of the progress of aggregation reactions, this assumption is usually made without performing the necessary checks to provide it with a firm underlying basis. In this article, we outline utilitarian methods for simulating the turbidity generated by homogeneous and mixed-protein aggregation reactions containing fibrous, amorphous, and crystalline structures. The approach is based on a combination of Rayleigh-Gans-Debye theory and approximate forms of the Mie scattering equations.

RNA-LIM: a novel procedure for analyzing protein/single-stranded RNA propensity data with concomitant estimation of interface structure.

RNA-LIM is a procedure that can analyze various pseudo-potentials describing the affinity between single-stranded RNA (ssRNA) ribonucleotides and surface amino acids to produce a coarse-grained estimate of the structure of the ssRNA at the protein interface. The search algorithm works by evolving an ssRNA chain, of known sequence, as a series of walks between fixed sites on a protein surface. Optimal routes are found by application of a set of minimal "limiting" restraints derived jointly from (i) selective sampling of the ribonucleotide amino acid affinity pseudo-potential data, (ii) limited surface path exploration by prior determination of surface arc lengths, and (iii) RNA structural specification obtained from a statistical potential gathered from a library of experimentally determined ssRNA structures. We describe the general approach using a NAST (Nucleic Acid Simulation Tool)-like approximation of the ssRNA chain and a generalized pseudo-potential reflecting the location of nucleic acid binding residues. Minimum and maximum performance indicators of the methodology are established using both synthetic data, for which the pseudo-potential defining nucleic acid binding affinity is systematically degraded, and a representative real case, where the RNA binding sites are predicted by the amplified antisense RNA (aaRNA) method. Some potential uses and extensions of the routine are discussed. RNA-LIM analysis programs along with detailed instructions for their use are available on request from the authors.

Equations describing semi-confluent cell growth (I) Analytical approximations.

A set of differential equations with analytical solutions are presented that can quantitatively account for variable degrees of contact inhibition on cell growth in two- and three-dimensional cultures. The developed equations can be used for comparative purposes when assessing contribution of higher-order effects, such as culture geometry and nutrient depletion, on mean cell growth rate. These equations also offer experimentalists the opportunity to characterize cell culture experiments using a single reductive parameter.

Biophysical Reviews: peering into 2024.

After introducing the winner of this year's Michèle Auger Award for Young Scientists' Independent Research, this Editorial for Volume 16 Issue 1 then describes the Issue contents. The Editorial concludes by providing a look into what lies ahead for 2024.

Biophysical reviews Special issue call: The 21st IUPAB Congress 2024 Kyoto Japan.

This commentary describes an open call for submissions to the upcoming Biophysical Reviews' Special Issue: The 21st IUPAB Congress 2024 Kyoto Japan. The submission deadline is July 1st of 2024. Interested parties are requested to make contact with the Special Issue editors prior to submission.

Biophysical Reviews: a transition in the journal.

This Editorial for Volume 16 Issue 2 first describes the issue contents before describing some upcoming events within Biophysical Reviews and concludies with an announcement on the transition of Chief Editors thanks to the outgoing Chief Editor.

Biophysical Reviews: a call for nominations to the 2024 Michéle Auger Award.

This editorial for volume 15 issue 3 first provides a brief introduction to the issue contents before then going on to open the call for nominations to the 2024 Michéle Auger Award for Young Scientists' Independent Research-the journal's single award.

Correction to: Biophysical Reviews: Publishing short and critical reviews written by key figures in the field.

[This corrects the article DOI: 10.1007/s12551-022-01009-6.].

Biophysical Reviews: Turning the page from 2022 to 2023.

This Editorial (vol. 15 issue 1-Regular Issue featuring an Issue Focus on the "100th Anniversary of Har Gobind Khorana") first describes the issue contents before providing both, a look back at some journal highlights from 2022, and a look forward to what we can expect from 2023. The Editorial closes with a roundup of new journal access features and an acknowledgement of those supporting the journal.

Biophysical Reviews: And the winner is .

This Editorial (Vol. 15 Issue 2-Regular Issue) first announces the winner of the 2023 Michèle Auger Award for Young Scientists' Independent Research before then going on to describe the contents of the current Issue. The Editorial closes with a discussion of the pros and cons of writing in the formulation of scientific ideas.

Biophysical Reviews: A goodbye to 2023.

This Editorial for the IUPAB Biophysical Reviews journal (2023 volume 15 issue 6) first provides an overview of the contents of this "Regular Issue featuring an Issue Focus on the Computational Biophysics of Atomic Force Microscopy" before going on to highlight some of the notable work published in the journal throughout 2023. Highlights of the current Issue include the contributed review article by Antonio Benedetto, winner of the 2023 Michèle Auger Award for Young Scientists' Independent Research, the latest installment of the "Biophysical Reviews Top 5 Series" authored by Angela Dulhunty, and an Issue Focus on the topic of computational aspects of atomic force microscopy generated from an IUPAB-sponsored workshop held in 2022.

Biophysical Reviews' "Meet the Editors Series": a profile of Damien Hall.

This piece introduces Damien Hall, Chief Editor of the Biophysical Reviews journal since 2019. Currently working as an Assistant Professor at Kanazawa University, the author describes his association with the journal along with some parts of his family history and academic journey.