Modeling Conformationally Flexible Proteins With X-ray Scattering and Molecular Simulations

Abstract

Proteins and protein complexes with high conformational flexibility participate in a wide range of biological processes. These processes include genome maintenance, gene expression, signal transduction, cell cycle regulation, and many others. Gaining a structural understanding of conformationally flexible proteins and protein complexes is arguably the greatest problem facing structural biologists today. Over the last decade, some progress has been made toward understanding the conformational flexibility of such systems using hybrid approaches. One particularly fruitful strategy has been the combination of small-angle X-ray scattering (SAXS) and molecular simulations. In this article, we provide a brief overview of SAXS and molecular simulations and then discuss two general approaches for combining SAXS data and molecular simulations: minimal ensemble approaches and full ensemble approaches. In minimal ensemble approaches, one selects a minimal ensemble of structures from the simulations that best fit the SAXS data. In full ensemble approaches, one validates a full ensemble of structures from the simulations using SAXS data. We argue that full ensemble models are more realistic than minimal ensemble searches models and that full ensemble approaches should be used wherever possible.

Control of DNA Damage Bypass by Ubiquitylation of PCNA

“…Fortunately, new approaches, both experimental and computational, are emerging that should allow us to gain valuable new structural and biochemical information about such conformationally flexible, multi-protein complexes. For example, hybrid approaches, such as full ensemble methods that combine small-angle X-ray scattering data and molecular dynamics simulations, should be extremely useful in studying such systems [81]. Similarly, single-molecule total internal reflection fluorescence microscopy studies of the assembly and composition of multi-protein complexes should also prove useful [82].…”

Conformational flexibility of fork-remodeling helicase Rad5 shown by full-ensemble hybrid methods

“…It functions as an E3 ubiquitin ligase to catalyze the poly-ubiquitylation of PCNA, and it functions as a fork-remodeling helicase to catalyze the conversion of stalled replication forks to chicken foot structures. To begin to understand the structure and conformational flexibility of Rad5, we have employed a full ensemble hybrid method that combines molecular simulations with SAXS [26].…”

“…Full ensemble hybrid methods have substantial advantages over the more widely used minimal ensemble hybrid methods [26]. Traditionally, molecular simulations are used to generate a large ensemble of structures.…”

“…By contrast, full ensemble methods are far more realistic in that they represent the conformational flexibility of a protein by thousands of individual structures, each related to one other by a series of time steps in a molecular simulation. In full ensemble methods, the simulations are generally not used to fit the experimental SAXS data [26]. Instead the experimental SAXS data is used simply to validate the simulations.…”

“…Obtaining high-resolution structural models of proteins with high conformational flexibility is one of the most difficult challenges in contemporary structural biology. Recently, our group has successfully employed a full-ensemble hybrid method that combines Langevin dynamics (LD) simulations and small-angle X-ray scattering (SAXS) to generate experimentally validated, high-resolution structural models of several proteins with high conformational flexibility [24–26]. These included one protein with a disordered C-terminal tail region, non-classical DNA polymerase eta (pol η), as well as two proteins with short tether regions: ubiquitin-modified PCNA and SUMO (small ubiquitin-like modifier)-modified PCNA.…”