Understanding protein folding, evolution and function via molecular simulation

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

The project has addressed the following areas in the past year: 1. Analyzing cooperativity in protein dynamics and folding. We have developed methods for analyzing all-atom folding simulations to detect cooperativity amongst the formation of native contacts. We are now applying this method to detect allostery in folded proteins and also identify evidence of alternative folds analyzing the unfolded state of fold-switching proteins. Lastly, we are investigating the use of cooperativity to define better reaction coordinates for protein folding (David Wang). 2. Sequence dependence of co-phase separation of proteins. In cells, membraneless organelles do not simply consist of a single macromolecular component (or two, in the case of complex coacervation), but likely involve many different molecules. As a starting point for understanding the relative affinity one protein for a coacervate formed by another, we are collaborating via a shared Oxcam Ph.D. student with Tuomas Knowles at the University of Cambridge to develop a high-throughput sequence-scanning methodology using a microdrop platform [Saar et al, 2023]. In parallel, we are developing a coarse-grained simulation approach to study this problem (Lydia Good). 3. Structure and dynamics of intrinsically disordered proteins from all-atom simulations. We have used a set of single-molecule FRET data for 16 intrinsically disordered domains of equal length to benchmark all-atom simulations. We find that simulations are able to reproduce the FRET data remarkably well and inclusion of chromophores in simulation models improves accuracy. We can show that a residual deficiency of the force fields appears to be that salt bridges are too strong, especially those involving Arginine. We also were able to resolve an apparent paradox whereby the reconfiguration times of the domains were approximately the same although internal friction is expected to be higher for more compact chains. (Milos Ivanovic) 4. Bridging experiment and all atom simulation with coarse-grained models. We can use the same all-atom simulations from (3) to optimize better coarse-grained models. Briefly, all-atom simulations give a vast amount of training data that can be used to parameterize a detailed coarse-grained model, but they may themselves have deficiencies, while experimental data are a gold standard, but are much more limited in quantity and are ensemble-averaged. We have successfully devised a method that can use both experimental data and all-atom simulations simultaneously to optimize a coarse-grained model (Robert Best). 5. Improving treatment of salt bridges in all-atom force fields. Motivated by the results of (3) above, we have recorded and used osmotic pressure data for solutions of charged amino acids to reparameterize salt bridges in all-atom force-fields, yielding more accurate simulations of complex coacervates of intrinsically disordered proteins (Milos Ivanovic). 6. Complex coacervation of highly charged intrinsically disordered proteins. Recent work in collaboration with Ben Schuler's single molecule FRET group in Zurich has shown that oppositely charged nuclear proteins undergo complex coacervation. Building on our methodological developments for performing all-atom simulations of these systems, we have investigated the effects of sequence variation on the dynamics within these condensates, finding in agreement with experiment that Arginine-rich proteins greatly slow the dynamics [Galvanetto et al, 2025] (Milos Ivanovic). 7. Charge regulation in intrinsically disordered proteins. The effective charge state of intrinsically disordered proteins can be modulated by counterion condensation as well as protonation of ionizable groups. We are using constant pH molecular dynamics simulations to investigate the coupling between protonation state and conformation, and how that can increase the cooperativity of disordered linkers as molecular switches (Carolyn Fitch). 8. Modelling properties of the extracellular matrix using coarse-grained models. We have recently started developing bottom up coarse-grained simulation models to describe the extracellular matrix and how they are related to its underlying molecular structure. We have initially focused on developing accurate atomistic models for collagen, and validating them against the available data from NMR and small angle X-ray scattering; we are extending this to coarse-grained models that will allow the investigation of packing of tropocollagen in collagen fibers. (George Pantelopulos). 9. Dynamics of water adjacent to protein surfaces. In collaboration with Juan Lopez and Ad Bax we are investigating how rapidly water exchanges near proteins. NMR relaxation data on the protein that probe the interactions between water molecules and the protein backbone can be well reproduced from direct calculations of these parameters from all-atom molecular simulations with a high quality force field. The simulations are being analyzed to provide a molecular interpretation for the observations (Robert Best). Group members or jointly supervised external collaborators involved in each project are listed in brackets () at the end of each section. References are given in [] brackets.