IDENTIFICATION OF UNFOLDING DOMAINS IN LARGE PROTEINS BY THEIR UNFOLDING RATES

Three unfolding domains in rabbit muscle aldolase destabilized in 3 M urea have been identified from their unfolding rate constants (0.10, 0 .036, and 0.0064 min(-1)). The populations of folded and various, part ially unfolded forms were determined by amide hydrogen exchange and ma ss spectrometry. Results of this study show that unfolding domains may include multiple, noncontiguous segments of the backbone and that dif ferent regions of helices may belong to different unfolding domains. I n addition, these results show that the domain unfolding most rapidly is located distant from the subunit binding surfaces and has the great est access to the denaturant. The bimodal intermolecular distributions of deuterium found in this study show that unfolding of these domains is cooperative. It is proposed that these unfolding domains are corre lated with local energy minima in the free-energy folding surface of a ldolase. In addition to the three unfolding domains, there are three s hort segments that do not unfold in 3 M urea. These segments, which ar e located in the subunit binding surface, identify the most stable reg ions of aldolase. This study also demonstrates that it is now possible to identify and characterize unfolding domains in relatively large (M -r 158 000) proteins.