Probing Protein Ensemble Rigidity and Hydrogen-Deuterium exchange

TL;DR

This paper introduces a novel FIRST-based method for analyzing protein ensemble rigidity and predicting hydrogen-deuterium exchange by integrating conformational dynamics and solvent accessibility data, validated through experiments on Sso AcP.

Contribution

It develops a new ensemble-based rigidity analysis approach that accounts for protein dynamics and refines hydrogen bond modeling, improving HDX prediction accuracy.

Findings

Ensemble rigidity predictions match experimental HDX data.

Refined hydrogen bond model enhances protein flexibility analysis.

Method effectively predicts protected and exchange-prone regions.

Abstract

Protein rigidity and flexibility can be analyzed accurately and efficiently using the program FIRST. Previous studies using FIRST were designed to analyze the rigidity and flexibility of proteins using a single static (snapshot) structure. It is however well known that proteins can undergo spontaneous sub-molecular unfolding and refolding, or conformational dynamics, even under conditions that strongly favour a well-defined native structure. These (local) unfolding events result in a large number of conformers that differ from each other very slightly. In this context, proteins are better represented as a thermodynamic ensemble of `native-like' structures, and not just as a single static low-energy structure. Working with this notion, we introduce a novel FIRST-based approach for predicting rigidity/flexibility of the protein ensemble by (i) averaging the hydrogen bonding strengths from the entire ensemble and (ii) by refining the mathematical model of hydrogen bonds. Furthermore, we combine our FIRST-ensemble rigidity predictions with the ensemble solvent accessibility data of the backbone amides and propose a novel computational method which uses both rigidity and solvent accessibility for predicting hydrogen-deuterium exchange (HDX). To validate our predictions, we report a novel site specific HDX experiment which characterizes the native structural ensemble of Acylphosphatase from hyperthermophile Sulfolobus solfataricus (Sso AcP). The sub-structural conformational dynamics that is observed by HDX data, is closely matched with the FIRST-ensemble rigidity predictions, which could not be attained using the traditional single `snapshot' rigidity analysis. Moreover, the computational predictions of regions that are protected from HDX and those that undergo exchange are in very good agreement with the experimental HDX profile of Sso AcP.