Substitution of trans-proline at three positions in ubiquitin (residues 19, 37 and 38) produces significant context-dependent effects on protein stability (both stabilizing and destabilizing) that reflect changes to a combination of parameters including backbone flexibility, hydrophobic interactions, solvent accessibility to polar groups and intrinsic backbone conformational preferences. Kinetic analysis of the wild-type yeast protein reveals a predominant fast-folding phase which conforms to an apparent two-state folding model. Temperature-dependent studies of the refolding rate reveal thermodynamic details of the nature of the transition state for folding consistent with hydrophobic collapse providing the overall driving force. Brønsted analysis of the refolding and unfolding rates of a family of mutants with a variety of side chain substitutions for P37 and P38 reveals that the two prolines, which are located in a surface loop adjacent to the C terminus of the main alpha-helix (residues 24-33), are not significantly structured in the transition state for folding and appear to be consolidated into the native structure only late in the folding process. We draw a similar conclusion regarding position 19 in the loop connecting the N-terminal beta-hairpin to the main alpha-helix. The proline residues of ubiquitin are passive spectators in the folding process, but influence protein stability in a variety of ways.

• PMID: 15560788
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Crespo MD, Platt GW, Bofill R, Searle MS

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