HYDROGEN-EXCHANGE IDENTIFIES NATIVE-STATE MOTIONAL DOMAINS IMPORTANT IN PROTEIN-FOLDING

Effects of mutations on hydrogen exchange kinetics, structure, and stability suggest that the slow exchange core is a key element in protein folding. Single amino acid variants of bovine pancreatic trypsin inhibitor (BPTI) have been made with glycine or alanine replacement of residues Tyr 35, Gly 37, Asn 43, and Asn 44. The crystal structures of Y35G and N43G are reported [Housset, D., Kim, K.-S., Fuchs, J., & Woodward, C. (1991) J. Mol. Biol. 220,757-770; Danishefsky, A. T., Housset, D., Kim, K.-S., Tao, F., Fuchs, J., Woodward, C., & Wlodawer, A. (I 993) Protein Sci. 2, 577-587; Kim, K.-S., Tao, F., Fuchs, J. A., Danishefsky, A. T., Housset, D., Wlodawer, A., & Woodward, C. (1993a) Protein Sci. 2, 588-596]. NMR chemical shifts indicate few changes from the wild type (WT) in G37A and N44G. Stabilities of the four mutants were measured by calorimetry and by hydrogen exchange. Values of DELTADELTAG(WT-->mut), the difference in DELTAG of folding/unfolding between the wild type and mutant, estimated by both methods are in good agreement and are in the range 4.7-6.0 kcal/mol. There is no general correlation between stability and hydrogen exchange rates at pH 3.5 and 30-degrees-C. Exchange occurs by two parallel pathways, one involving small noncooperative fluctuations of the native state, and the other involving cooperative, global unfolding. In the mutant proteins, the rates for exchange by the unfolding mechanism are accelerated by a factor corresponding to the increase in the unfolding/folding equilibrium constant. Rates for exchange by the native-state mechanism either are not affected or are accelerated to varying degrees. NH protons with accelerated exchange rates are primarily in the vicinity of the replacement, which in these mutants corresponds to the flexible loops. The overall effects of destabilizing mutations on hydrogen exchange are similar to those resulting from the addition of 8 M urea (Kim & Woodward, 1993). Motional domains defined by exchange rates are the slow exchange core, flexible loops, and secondary structure not in the core. In BPTI, the slow exchange core is the folding core, peptides corresponding to the slow exchange core have native-like structure, and the presence or absence of local structural relaxation around mutation sites reflects the intrinsic local flexibility measured by hydrogen exchange. We propose that this is general for proteins, that the protein segments in the slow exchange core determine the basic fold, and that in compact nonnative states the collapsed region corresponds to the slow exchange core.