Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation

Abstract

The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution.

Principles and Overview of Sampling Methods for Modeling Macromolecular Structure and Dynamics

“…A central issue is the large uncertainty inherent in such calculations. One way in which computational methods address this issue is by integrating different types of experimental data [ 315 , 316 ].…”

“…A variation of this idea is to measure not a time-average but an ensemble-average observable. The latter is referred to as the replica-averaged approach, and a variety of restraining algorithms, including those that conduct both time and ensemble averaging, have been developed and applied to sample and characterize native, transition, intermediate, and unfolded states of proteins [ 17 , 32 , 34 , 312 , 316 , 507 – 512 ].…”

A Two-Fold Structural Classification Method for Determining the Accurate Ensemble of Protein Structures

“…Recently, there have been significant efforts to develop integrated approaches by combining small-angle scattering experiments with molecular simulations to derive the atomic-level structures of bio-macromolecules [3,4,6,11,16,20,21,24,28,32,33]. These approaches roughly fall into two categories: one is to search for the protein conformations from existing structural candidates, pre-generated from molecular simulation using standard force fields, which best fits to the experimental SAS data [11,21,22,24,32,33]; while the other one is to apply biased potentials to drive the simulation towards the protein conformations in better agreement with experiment [4,6,16]. The present work focuses on the discussion of the first type of approaches.…”

ir-HSP: Improved Recognition of Heat Shock Proteins, Their Families and Sub-types Based On g-Spaced Di-peptide Features and Support Vector Machine

“…The advancement of relatively cheaper sequencing technologies has witnessed a huge volume of protein sequences that are added to the public databases (Feng et al, 2013 ; Ahmad et al, 2015 ; Kumar et al, 2016 ). Due to lack of experimentally validated structures in the databases, resource intensive traditional method like nuclear magnetic resonance (NMR) has become inappropriate for identifying HSP families in large protein datasets (Redfield, 2004 ; Lange et al, 2012 ; Nasedkin et al, 2015 ). Thus, the development of computational method for identifying HSPs and their families is essential due to their inexpensive and high throughput nature.…”