Role of hydrophobic interactions in yeast phosphoglycerate kinase stability

✍ Scribed by Veronique Receveur; Pascal Garcia; Dominique Durand; Patrice Vachette; Michel Desmadril

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 355 KB
• Volume: 38
• Category: Article
• ISSN: 0887-3585
• DOI: 10.1002/(sici)1097-0134(20000201)38:2<226::aid-prot10>3.0.co;2-h

✦ Synopsis

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 ⌬404) 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.