Open AccessContext‐dependent effects of proline residues on the stability and folding pathway of ubiquitin
Author(s) -
Crespo Maria D.,
Platt Geoffrey W.,
Bofill Roger,
Searle Mark S.
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04392.x
Subject(s) - folding (dsp implementation) , phi value analysis , chemistry , protein folding , context (archaeology) , native state , crystallography , ubiquitin , biophysics , amino acid , side chain , stereochemistry , biochemistry , biology , organic chemistry , paleontology , gene , electrical engineering , engineering , polymer
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 α‐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 β‐hairpin to the main α‐helix. The proline residues of ubiquitin are passive spectators in the folding process, but influence protein stability in a variety of ways.