TEMPERATURE-JUMP INDUCED FAST REFOLDING OF COLD-UNFOLDED PROTEIN

Transient protein structures with only microsecond live times may be s olved at the resolution of single residues by temperature jumping of c old denatured protein [Nolting, B., Golbik, R., and Fersht, A. R. (199 5) Proc. Natl. Acad Sci. USA 92, 10668-10672]. Here it is shown how th is method may be extended to the study of intermediates and transition states which are located on the reaction coordinate between the main folding transition state and the native state. When incubating bovine beta-lactoglobulin A under conditions which favour cold unfolding and rapidly refolding the partially cold unfolded protein by a temperature jump from -4 to 2 degrees C, a fast folding kinetics with a rate cons tant of about 100 s(-1) is observed. The amplitude of the relaxation d ecays following a single exponential function of the time of incubatio n at low temperature before the T-jump. The rate constant of decay mat ches the rate constant of the main unfolding transition, showing that early in the cold denaturation reaction, beta-lactoglobulin is kinetic ally trapped in a partially unfolded intermediate state. Despite the o nly small fluorescence change, refolding within about 10 ms after a te mperature jump involves a considerable degree of solvent exclusion and burial of hydrophobic surface, suggesting a contraction of the molecu le. (C) 1996 Academic Press, Inc.