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Role of hydrophobic interactions in yeast phosphoglycerate kinase stability
Author(s) -
Receveur Veronique,
Garcia Pascal,
Durand Dominique,
Vachette Patrice,
Desmadril Michel
Publication year - 2000
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(20000201)38:2<226::aid-prot10>3.0.co;2-h
Subject(s) - circular dichroism , phosphoglycerate kinase , denaturation (fissile materials) , fluorescence , yeast , crystallography , native state , chemistry , scattering , small angle x ray scattering , intermediate state , biophysics , analytical chemistry (journal) , biochemistry , enzyme , chromatography , biology , nuclear chemistry , optics , physics , atomic physics
Cold denaturation of yeast phosphoglycerate kinase (yPGK) was investigated by a combination of far UV circular dichroism (CD), steady‐state and time‐resolved fluorescence, and small angle X‐ray scattering. It was shown that cold denaturation of yPGK cannot be accounted for by a simple two‐state process and that an intermediate state can be stabilized under mild denaturing conditions. Comparison between far UV CD and fluorescence shows that in this state the protein displays a fluorescence signal corresponding mainly to exposed tryptophans, whereas its CD signal is only partially modified. Comparison with spectroscopic data obtained from a mutant missing the last 12 amino‐acids (yPGK Δ404) suggests that lowering the temperature mainly results in a destabilization of hydrophobic interactions between the two domains. Small angle X‐ray scattering measurements give further information about this stabilized intermediate. At 4°C and in the presence of 0.45 M Gdn‐HCl, the main species corresponds to a protein as compact as native yPGK, whereas a significant proportion of ellipticity has been lost. Although various techniques have shown the existence of residual structures in denatured proteins, this is one example of a compact denatured state devoid of its main content in alpha helices. Proteins 2000;38:226–238. © 2000 Wiley‐Liss, Inc.