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High tolerance for ionizable residues in the hydrophobic interior of proteins.

ABSTRACT: Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pK(a) values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.

SUBMITTER: Isom DG

PROVIDER: S-EPMC2584708 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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High tolerance for ionizable residues in the hydrophobic interior of proteins.

Isom Daniel G DG Cannon Brian R BR Castañeda Carlos A CA Robinson Aaron A García-Moreno Bertrand B

Proceedings of the National Academy of Sciences of the United States of America 20081112 46

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protei ...[more]

PMID: 19004768

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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

High tolerance for ionizable residues in the hydrophobic interior of proteins.

Publications

High tolerance for ionizable residues in the hydrophobic interior of proteins.

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