Biology subjects

Best, R. B.

Publications and source records attributed to Best, R. B..

12 recordsLinked to original sources

bioRxiv2026

Beyond Equilibrium Ensembles: Time Rescaling in Coarse-Grained Simulations across Single-Molecule and Condensate Regimes

Residue-level coarse-grained simulations provide a powerful route for modeling biomolecular condensates over length and time scales that are difficult to access with atomistic molecular dynamics. Coarse-grained models have been shown to reproduce many aspects of equilibrium phase behavior. However, it remains unclear to what extent such models can reproduce the relative timescales of molecular dynamics. Here, we examine this question for complex coacervates with markedly different dynamics, formed by the highly acidic intrinsically disordered protein prothymosin with four cationic partners: linker histone H1, protamine, polylysine, and polyarginine. Coexistence simulations using a residue-level coarse-grained model reproduce key equilibrium observables from experiments, including dense-phase concentrations, ionic-strength-dependent phase behavior, and chain dimensions in the dense and dilute phases. Dynamics are accelerated in these simulations, but a composition-specific time-rescaling factor captures the ionic-strength dependence of chain reconfiguration times within a given complex coacervate. In contrast, time rescaling is not transferable between dense and dilute phases or across condensate compositions and can depend on the chosen observable. These results show that agreement with measured equilibrium observables does not imply a universally transferable timescale for conformational dynamics in residue-level coarse-grained simulations. However, we find that the required time rescaling strongly correlates with the interaction energy of the protein chains, suggesting that the missing frictional effects arise from protein-protein interactions rather than solely from protein-solvent interactions, reminiscent of internal friction. Our findings highlight the need to combine thermodynamic validation with kinetic calibration when interpreting chain relaxation, molecular diffusion, and material properties from residue-level coarse-grained simulations of biomolecular condensates.

biophysics↗

bioRxiv2026

Predictive all-atom simulations of disordered proteins and biomolecular condensates through osmometry-guided force-field optimization

All-atom simulations with explicit solvent can provide a detailed and accurate description of dynamics and mechanisms in biomolecular systems, including intrinsically disordered proteins (IDPs) and their condensates. However, interactions involving charged residues and ions remain a persistent source of systematic error. Here we introduce an osmometry-guided optimization strategy that directly targets residue-residue, residue-ion and ion-ion interactions. Osmotic pressure provides key experimental information on molecular interactions and can be calculated directly and rapidly from simulations, enabling iterative force-field optimization. The resulting parameters improve agreement with single-molecule FRET data for IDPs, NMR relaxation data for an IDP-folded-domain complex, and chain dynamics and dimensions in biomolecular condensates of charged IDPs. For such condensates, simulations with our osmometry-optimized force field provide the missing link for predicting condensate dynamics across length and time scales. The strategy is broadly extensible to other interaction classes, including those governing protein-nucleic-acid assemblies.

biophysics↗

bioRxiv2026

Time-Resolved Single-Molecule FRET Reveals Length-Dependent Nucleosome Decompaction by Poly(ADP-ribose)

Highly charged chains of poly(ADP-ribose) (PAR) are synthesized in the cell as part of their central role in DNA damage response. However, the effects of PAR on nucleosome structure and dynamics remain incompletely understood. Here we combine droplet-based microfluidic mixing with single-molecule Forster resonance energy transfer spectroscopy to resolve the kinetics of PAR-induced nucleosome decompaction in non-equilibrium measurements with millisecond time resolution. This approach avoids surface-adhesion and enables the tether-free observation of nucleosome remodeling. We find that PAR triggers nucleosome decompaction via a length-dependent kinetic threshold: Chains with less than ten ADP-ribose units act slowly and weakly, whereas longer PAR polymers induce efficient and rapid nucleosome opening. The extent and reversibility of decompaction further depend on PAR concentration and ionic strength, reflecting a mechanism dominated by electrostatic interactions. Enzymatic PAR digestion demonstrates that PAR can promote both reversible linker DNA opening and irreversible nucleosome disassembly. Coarse-grained molecular simulations suggest that these effects arise from a competition between PAR and DNA for histone tail binding. Altogether, our results establish PAR length as a key factor controlling chromatin accessibility during DNA repair and highlight droplet-based microfluidics as a powerful platform for studying such biomolecular interactions.

biophysics↗

bioRxiv2025

Dynamical buffering of reconfiguration dynamics in intrinsically disordered proteins

The dynamics of intrinsically disordered proteins are important for their function, allowing their heterogeneous conformational ensembles to rapidly reconfigure in response to binding partners or changes in solution conditions. However, the relation between sequence composition and chain dynamics has rarely been studied. Here, we characterize the dynamics of a set of 16 naturally occurring disordered regions of identical chain length but with highly diverse sequences. In spite of the strong variation of chain dimensions with sequence in this set inferred from single-molecule FRET, nanosecond fluorescence correlation spectroscopy yields chain reconfiguration times that are almost independent of sequence. This surprising observation contrasts with the slowdown in dynamics, attributed to internal friction, that has been observed in more compact disordered proteins. We investigated this effect with the aid of multi-microsecond, all-atom explicit-solvent simulations of all 16 disordered proteins. The simulations reproduce the experimental FRET efficiencies with near-quantitative accuracy, with explicit inclusion of the FRET dyes improving agreement with experiment while minimally perturbing the protein ensemble. Critically, the simulations also reproduce the lack of correlation between reconfiguration times and chain dimensions across the sequences and allow us to rationalize this observation as arising from two competing factors as the chains get more compact. The narrowing of end-to-end distance distributions and a concomitant reduction of the corresponding intrachain diffusion coefficients have opposite effects that end up resulting in only a small variation of reconfiguration times with chain dimensions. These compensating factors "buffer" the effect of sequence on linker dynamics, which may help to conserve function as sequences evolve.

biophysics↗

bioRxiv2020

The ribosome modulates folding inside the ribosomal exit tunnel

Proteins commonly fold cotranslationally on the ribosome, while the nascent chain emerges from the ribosomal tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is still not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not reproduce the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Exploring the Sequence Fitness Landscape of a Bridge Between Protein Folds

Most foldable protein sequences adopt only a single native fold. Recent protein design studies have, however, created protein sequences which fold into different structures apon changes of environment, or single point mutation, the best characterized example being the switch between the folds of the GA and GB binding domains of streptococcal protein G. To obtain further insight into the design of sequences which can switch folds, we have used a computational model for the fitness landscape of a single fold, built from the observed sequence variation of protein homologues. We have recently shown that such coevolutionary models can be used to design novel foldable sequences. By appropriately combining two of these models to describe the joint fitness landscape of GA and GB, we are able to describe the propensity of a given sequence for each of the two folds. We have successfully tested the combined model against the known series of designed GA/GB hybrids. Using Monte Carlo simulations on this landscape, we are able to identify pathways of mutations connecting the two folds. In the absence of a requirement for domain stability, the most frequent paths go via sequences in which neither domain is stably folded, reminiscent of the propensity for certain intrinsically disordered proteins to fold into different structures according to context. Even if the folded state is required to be stable, we find that there is nonetheless still a wide range of sequences which are close to the transition region and therefore likely fold switches, consistent with recent estimates that fold switching may be more widespread than had been thought. Author SummaryWhile most proteins self-assemble (or "fold") to a unique three-dimensional structure, a few have been identified that can fold into two distinct structures. These so-called "metamorphic" proteins that can switch folds have attracted a lot of recent interest, and it has been suggested that they may be much more widespread than currently appreciated. We have developed a computational model that captures the propensity of a given protein sequence to fold into either one of two specific structures (GA and GB), in order to investigate which sequences are able to fold to both GA and GB ("switch sequences"), versus just one of them. Our model predicts that there is a large number of switch sequences that could fold into both structures, but also that the most likely such sequences are those for which the folded structures have low stability, in agreement with available experimental data. This also suggests that intrinsically disordered proteins which can fold into different structures on binding may provide an evolutionary path in sequence space between protein folds.

biophysics↗

bioRxiv2020

Disordered Proteins Enable Histone Chaperoning on the Nucleosome

Proteins with highly charged disordered regions are abundant in the nucleus, where many of them interact with nucleic acids and control key processes such as transcription. The functional advantages conferred by protein disorder, however, have largely remained unclear. Here we show that disorder can facilitate a remarkable regulatory mechanism involving molecular competition. Single-molecule experiments demonstrate that the human linker histone H1 binds to the nucleosome with ultra-high affinity. However, the large-amplitude dynamics of the positively charged disordered regions of H1 persist on the nucleosome and facilitate the interaction with the highly negatively charged and disordered histone chaperone prothymosin . Consequently, prothymosin can efficiently invade the H1-nucleosome complex and displace H1 via competitive substitution. By integrating experiments and simulations, we establish a molecular model that rationalizes this process structurally and kinetically. Given the abundance of charged disordered regions in the nuclear proteome, this mechanism may be widespread in cellular regulation.

biophysics↗

bioRxiv2019

Cotranslational folding cooperativity of contiguous domains of α-spectrin

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein -spectrin using Force Profile Analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each others folding in distinct ways that may be important for the efficient assembly of -spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in pN. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of [~]15 pN during cotranslational folding. SignificanceIn living cells, proteins are produced in a sequential way by ribosomes. This vectoral process allows the growing protein chain to start to fold before translation has been completed. Thereby, cotranslational protein folding can be significantly different than the folding of a full-length protein in isolation. Here we show how structurally similar repeat domains, normally produced as parts of a single long polypeptide, affect the cotranslational folding of their neighbors. This provides insight into how the cell may efficiently produce multidomain proteins, paving the way for future studies in vivo or with chaperones. We also provide an estimated magnitude of the mechanical force on the nascent chain generated by cotranslational folding, calculated from biochemical measurements and molecular dynamics simulations.

biochemistry↗

bioRxiv2018

IMPROVING THE PERFORMANCE OF THE RNA AMBER FORCE FIELD BY TUNING THE HYDROGEN-BONDING INTERACTIONS

Molecular dynamics (MD) simulations became a leading tool for investigation of structural dynamics of nucleic acids. Despite recent efforts to improve the empirical potentials (force fields, ffs), RNA ffs have persisting deficiencies, which hamper their utilization in quantitatively accurate simulations. Previous studies have shown that at least two salient problems contribute to difficulties in description of free-energy landscapes of small RNA motifs: (i) excessive stabilization of the unfolded single-stranded RNA ensemble by intramolecular base-phosphate and sugar-phosphate interactions, and (ii) destabilization of the native folded state by underestimation of stability of base pairing. Here, we introduce a general ff term (gHBfix) that can selectively fine-tune non-bonding interaction terms in RNA ffs, in particular the H-bonds. gHBfix potential affects the pair-wise interactions between all possible pairs of the specific atom types, while all other interactions remain intact, i.e., it is not a structure-based model. In order to probe the ability of the gHBfix potential to refine the ff non-bonded terms, we performed an extensive set of folding simulations of RNA tetranucleotides and tetraloops. Based on these data we propose particular gHBfix parameters to modify the AMBER RNA ff. The suggested parametrization significantly improves the agreement between experimental data and the simulation conformational ensembles, although our current ff version still remains far from being flawless. While attempts to tune the RNA ffs by conventional reparametrizations of dihedral potentials or non-bonded terms can lead to major undesired side effects as we demonstrate for some recently published ffs, gHBfix has a clear promising potential to improve the ff performance while avoiding introduction of major new imbalances.

biochemistry↗

bioRxiv2018

A small single-domain protein folds through the same pathway on- and off- the ribosome

In vivo, proteins fold and function in a complex environment where they are subject to many stresses that can modulate protein energy landscapes. One aspect of the environment pertinent to protein folding is the ribosome, since proteins have the opportunity to fold while still bound to the ribosome during translation. We use a combination of force and chemical denaturant (chemo-mechanical unfolding), as well as point mutations, to characterize the folding mechanism of the src SH3 domain both as a stalled ribosome nascent chain and free in solution. Our results indicate that src SH3 folds through the same pathway on and off the ribosome. Molecular simulations also indicate that the ribosome does not affect the folding pathway for this small protein. Taken together, we conclude that the ribosome does not alter the folding mechanism of this small protein, which appears to fold at the mouth of the ribosome as the protein emerges from the exit tunnel. These results, if general, suggest the ribosome may exert a bigger influence on the folding of multi-domain proteins or protein domains that can partially fold before the entire domain sequence is outside the ribosome exit tunnel.

biophysics↗

bioRxiv2018

Reversible two-state folding of the ultrafast protein gpW under mechanical force

Ultrafast folding proteins have limited cooperativity and thus are excellent models to resolve, via single-molecule experiments, the fleeting molecular events that proteins undergo during folding. Here we report single-molecule atomic force microscopy (AFM) experiments on gpW, a protein that, in bulk, folds in a few microseconds over a marginal folding barrier (~1 kBT). Applying pulling forces of only 5 pN we maintain gpW in quasi-equilibrium near its mechanical unfolding midpoint, and detect how it interconverts stochastically between the folded and an extended state. This binary pattern indicates that, under an external force, gpW (un)folds over a significant free energy barrier. Using molecular simulations and a theoretical model we rationalize how force induces such barrier in an otherwise downhill free energy surface. Force-induced folding barriers are likely a general occurrence for ultrafast folding biomolecules studied with single molecule force spectroscopy.

biophysics↗

bioRxiv2017

Sequence determinants of protein phase behavior from a coarse-grained model

Membraneless organelles important to intracellular compartmentalization have recently been shown to comprise assemblies of proteins which undergo liquid-liquid phase separation (LLPS). However, many proteins involved in this phase separation are at least partially disordered. The molecular mechanism and the sequence determinants of this process are challenging to determine experimentally owing to the disordered nature of the assemblies, motivating the use of theoretical and simulation methods. This work advances a computational framework for conducting simulations of LLPS with residue-level detail, and allows for the determination of phase diagrams and coexistence densities of proteins in the two phases. The model includes a short-range contact potential as well as a simplified treatment of electrostatic energy. Interaction parameters are optimized against experimentally determined radius of gyration data for multiple unfolded or intrinsically disordered proteins (IDPs). These models are applied to two systems which undergo LLPS: the low complexity domain of the RNA-binding protein FUS and the DEAD-box helicase protein LAF-1. We develop a novel simulation method to determine thermodynamic phase diagrams as a function of the total protein concentration and temperature. We show that the model is capable of capturing qualitative changes in the phase diagram due to phosphomimetic mutations of FUS and to the presence or absence of the large folded domain in LAF-1. We also explore the effects of chain-length, or multivalency, on the phase diagram, and obtain results consistent with Flory-Huggins theory for polymers. Most importantly, the methodology presented here is flexible so that it can be easily extended to other pair potentials, be used with other enhanced sampling methods, and may incorporate additional features for biological systems of interest.\n\nAuthor summaryLiquid liquid phase separation (LLPS) of low-complexity protein sequences has emerged as an important research topic due to its relevance to membraneless organelles and intracellular compartmentalization. However a molecular level understanding of LLPS cannot be easily obtained by experimental methods due to difficulty of determining structural properties of phase separated protein assemblies, and of choosing appropriate mutations. Here we advance a coarse-grained computational framework for accessing the long time scale phase separation process and for obtaining molecular details of LLPS, in conjunction with state of the art enhanced sampling methods. We are able to capture qualitatively the changes of phase diagram due to specific mutations, inclusion of a folded domain, and to variation of chain length. The model is flexible and can be used with different knowledge-based potential energy functions, as we demonstrate. We expect a wide application of the presented framework for advancing our understanding of the formation of liquid-like protein assemblies.

biophysics↗