Chen, B. and King, J. (1991) In: Protein Refolding (G. Georgiou & E. de Bernardez Clark, eds.) ACS Symposium Series 470, American Chemical Society, Washington, D.C., pp. 119-132
Pathway for the thermal unfolding of wild type and mutant forms of the thermostable P22 tailspike endorhamnosidase
Increasing the stabilities of proteins under realistic conditions requires understanding the mechanisms of protein denaturation far from equilibirum. A protein displaying very high thermal stability, resistance to proteases and resistance to detergents is the tailspike endorhamnosidase of bacteriophage P22. The folding pathway for the tailspike is well defined both in vivo and in vitro. Kinetic analysis of the thermal and detergent unfolding pathway reveals a relatively slow process which passes through a long-lived partially folded trimeric intermediate. This species has its N-termini unfolded with the remaining regions of the polypeptide chains in a compact native-like form. The intermediate can refold back to the native on cooling. Further unfolding of the intermediate at high temperature generates an aggregating species which renders the process kinetically irreversible. In the presence of SDS the unfolding intermediate is quantitatively converted to an unfolded monomer/SDS complex. Thus denaturation initiates via the melting of sites at the N-terminus, but the rate limiting step for the overall process is the melting of the intermediate species. Examining the thermal unfolding kinetics of more than ten temperature sensitive for folding (tsf) mutant proteins shows they have small effects on the transition from the native to the intermediate, but large effects on the transition from the intermediate to the fully ufolded form. The genetic modification of unfolding intermediates may provide an avenue for increasing the stability of proteins to irreversible denaturation.