COLD DENATURATION-INDUCED CONFORMATIONAL-CHANGES IN PHOSPHOGLYCERATE KINASE FROM YEAST

The temperature-dependent conformational equilibrium of 3-phosphoglyce rate kinase has been studied in the temperature range from 1 to 30-deg rees-C by means of dynamic light scattering, small-angle X-ray scatter ing, differential scanning calorimetry, circular dichroism spectroscop y, and fluorescence spectroscopy. At 28-degrees-C and in the presence of 0.7 M guanidine hydrochloride (GuHCl), the radius of gyration (R(G) ) and the Stokes radius (R(S)) are 2.44 and 3.09 nm, respectively. Dec reasing the temperature effects unfolding of the molecule, a process t hat involves two stages. The two stages correspond to the successive u nfolding of the N-terminal and C-terminal domains. The peak maxima of the excess heat capacity, determined from differential calorimetric sc ans, extrapolated to 0 scan rate, are positioned at 16.5-degrees-C for the N-terminal domain and at 6.3-degrees-C for the C-terminal domain. At 4.5-degrees-C, the radius of gyration and the Stokes radius increa se to 7.8 and 4.8 nm, respectively. The persistence length and the len gth of the statistical chain segment of the unfolded polypeptide chain are 1.74 and 3.48 nm, corresponding to five and ten amino acids, resp ectively. At 1-degrees-C, the dimensions of the unfolded chain nearly agree with the predicted dimensions under theta conditions. Thus, the conformational changes upon cold denaturation can be described by a tr ansition from a compactly folded molecule to a random coil. The confor mation-dependent ratio rho = R(G)R(S)-1 increases from rho = 0.79 to r ho = 1.63. The volume of the unfolded chain is 30 times larger than th at of the folded chain in the native state. The unfolding/refolding pr ocesses of PGK in the GuHCl-containing solvent are governed by slow ki netics. Thermodynamic equilibrium is reached only some hours after the temperature is changed.