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-r esolved fluorescence, and small angle X-ray scattering. It was shown that c old denaturation of yPGK cannot be accounted for by a simple two-state proc ess and that an intermediate state can be stabilized under mild denaturing conditions. Comparison between far UV CD and fluorescence shows that in thi s state the protein displays a fluorescence signal corresponding mainly to exposed tryptophans, whereas its CD signal is only partially modified. Comp arison with spectroscopic data obtained from a mutant missing the last 12 a mino-acids (yPGK Delta 404) suggests that lowering the temperature mainly r esults in a destabilization of hydrophobic interactions between the two dom ains. Small angle X-ray scattering measurements give further information ab out this stabilized intermediate. At 4 degrees C and in the presence of 0.4 5 M Gdn-HCl, the main species corresponds to a protein as compact as native yPGK, whereas a significant proportion of ellipticity has been lost. Altho ugh various techniques have shown the existence of residual structures in d enatured proteins, this is one example of a compact denatured state devoid of its main content in alpha helices. Proteins 2000;38:226-238. (C) 2000 Wi ley-Liss, Inc.