Role of hydrophobic interactions in yeast phosphoglycerate kinase stability.
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 delta404) 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 degrees 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.
Receveur
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V
Laboratoire de Modélisation et Ingénierie des Protéines, Université de Paris-Sud Orsay, France.
Garcia
P
P
Durand
D
D
Vachette
P
P
Desmadril
M
M
D002942:Circular Dichroism; D003080:Cold Temperature; D004795:Enzyme Stability; D008958:Models, Molecular; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D014867:Water; D014961:X-Ray Diffraction; D015003:Yeasts / Q000201:enzymology*
D014867:Water; D010735:Phosphoglycerate Kinase