Temperature-jump induced fast refolding of cold-unfolded protein.

Transient protein structures with only microsecond live times may be solved at the resolution of single residues by temperature jumping of cold denatured protein [Nölting, B., Golbik, R., and Fersht, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 10668-10672]. Here it is shown how this 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 constant of about 100 s-1 is observed. The amplitude of the relaxation decays following a single exponential function of the time of incubation at low temperature before the T-jump. The rate constant of decay matches the rate constant of the main unfolding transition, showing that early in the cold denaturation reaction, beta-lactoglobulin is kinetically trapped in a partially unfolded intermediate state. Despite the only small fluorescence change, refolding within about 10 ms after a temperature jump involves a considerable degree of solvent exclusion and burial of hydrophobic surface, suggesting a contraction of the molecule.