Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

Jade Shi,R Paul Nobrega,T J Lane,Vijay S Pande,C Robert Matthews,Sagar V Kathuria,Christian Schwantes,Osman Bilsel

doi:10.1038/srep44116

Abstract

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. Here, we report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. This prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. Using these results, we then predict incisive single molecule FRET experiments as a means of model validation. This study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.

Highlights

Overall shape and size and be consistent with an experimental SAXS profile, making it impossible to decide, based on experiment alone, which should correspond to the true excited state
We present an approach for atomistic excited state structural inference in the data-poor regime that uses all-atom molecular dynamics (MD) simulation data as a prior for the excited state ensemble, which is subsequently refined against experimental data
We simulate its SAXS profile using the predictor CRYSOL31. This choice was made as a result of a benchmarking study we performed that compared the performance of many different SAXS predictors in simulating the native state CheY SAXS profile, with CRYSOL giving the best agreement

Summary

Introduction

Overall shape and size and be consistent with an experimental SAXS profile, making it impossible to decide, based on experiment alone, which should correspond to the true excited state. In contrast to these methods, which used coarse-grained and/or biased MD methods to improve sampling, the massive computing power afforded by Folding@home allows us to simulate the system using an all-atom, unbiased representation to timescales orders of magnitude beyond which the excited state is experimentally observed This allows us to take advantage of the strong kinetic prior information in our simulation model to generate a candidate set of structures, and make a robust statistical prediction about the relative importance (population) of each structure in the excited state ensemble, which we can refine against the experimental data. Our approach to inferring structural features of the excited state of CheY at atomic detail involves first extensively simulating the protein using MD, and using the data to build a Markov State Model (MSM) of the dynamics, which partitions the sampled protein conformations into discrete “microstates” based on kinetic criterion and computes transition rates between them. An ensemble model gives a more realistic description of the excited state, which may be a highly heterogeneous ensemble of protein conformations

Methods

Results

Conclusion