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Regulation of Ras Paralog Thermostability by Networks of Buried Ionizable Groups.

ABSTRACT: Protein folding is governed by a variety of molecular forces including hydrophobic and ionic interactions. Less is known about the molecular determinants of protein stability. Here we used a recently developed computer algorithm (pHinder) to investigate the relationship between buried charge and thermostability. Our analysis revealed that charge networks in the protein core are generally smaller in thermophilic organisms as compared to mesophilic organisms. To experimentally test whether core network size influences protein thermostability, we purified 18 paralogous Ras superfamily GTPases from yeast and determined their melting temperatures (Tm, or temperature at which 50% of the protein is unfolded). This analysis revealed a wide range of Tm values (35-63 °C) that correlated significantly (R = 0.87) with core network size. These results suggest that thermostability depends in part on the arrangement of ionizable side chains within a protein core. An improved capacity to predict protein thermostability may be useful for selecting the best candidates for protein crystallography, the development of protein-based therapeutics, as well as for industrial enzyme applications.

SUBMITTER: Isom DG

PROVIDER: S-EPMC5574170 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Regulation of Ras Paralog Thermostability by Networks of Buried Ionizable Groups.

Isom Daniel G DG Sridharan Vishwajith V Dohlman Henrik G HG

Biochemistry 20160106 3

Protein folding is governed by a variety of molecular forces including hydrophobic and ionic interactions. Less is known about the molecular determinants of protein stability. Here we used a recently developed computer algorithm (pHinder) to investigate the relationship between buried charge and thermostability. Our analysis revealed that charge networks in the protein core are generally smaller in thermophilic organisms as compared to mesophilic organisms. To experimentally test whether core ne ...[more]

PMID: 26701741

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Project description:The role of pH in regulating biological activity is ubiquitous, and understanding pH-mediated activity has traditionally relied on analyzing static biomolecular structures of highly populated ground states solved near physiological pH. However, recent advances have shown the increasing importance of transiently populated states, the characterization of which is extremely challenging but made plausible with the development of techniques such as relaxation dispersion NMR spectroscopy. To unlock the pH dependence of these transient states with atomistic-level details, we applied the recently developed explicit solvent constant pH molecular dynamics (CPHMD(MSλD)) framework to a series of staphylococcal nuclease (SNase) mutants with buried ionizable residues and probed their dynamics in different pH environments. Among our key findings is the existence of open states in all SNase mutants containing "buried" residues with highly shifted pKa's, where local solvation around the protonation site was observed. The calculated pKa demonstrated good agreement with experimental pKa's, with a low average unsigned error of 1.3 pKa units and correlation coefficient R(2) = 0.78. Sampling both open and closed states in their respective pH range, where they are expected to be dominant, was necessary to reproduce experimental pKa's, and in the most extreme examples of pKa shifts measured, it can be interpreted that the open-state structures are transient at physiological pH, contributing a small population of 1-2%. This suggests that buried ionizable residues can trigger conformational fluctuations that may be observed as transient-state structures at physiological pH. Furthermore, the coupled relationship of both open and closed states and their role in recapitulating macroscopic experimental observables suggest that structural analysis of buried residues may benefit from looking at structural pairs, as opposed to the conventional approach of looking at a single static ground-state conformation.

Dataset Information

Regulation of Ras Paralog Thermostability by Networks of Buried Ionizable Groups.

Publications

Regulation of Ras Paralog Thermostability by Networks of Buried Ionizable Groups.

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