Folding kinetics of BPTI variants and development of a model for the relationship of hydrogen exchange to protein folding

Two native-like aromatic-aliphatic NOEs are observed in reduced and unfolded BPTI. One is between side chains of Tyr23 and Ala25; the other is between Gly37 CalphaH and Tyr35 aromatic ring. To investigate the roles of these two interactions in folding, equilibrium and kinetic aspects of the GdmCl denaturation of six BPTI mutants were examined with circular dichroism spectroscopy. Of the mutants studied, RM(14--38) has the 30--51 and 5--55 disulfide bonds, while the other mutants have all three native disulfide bonds. All mutants, which have native-like structures, are destabilized by 3--6 kcal/mol, compared to WT. At pH 2 and 20°C, the relaxation rate constants of the major kinetic phase vary among different mutants, ranging from ∼10 ms to 0.71 s in the absence of denaturant. All mutants, except for G37A, fold by a two-state mechanism. The results support the hypothesis that the beta-turn region, especially the local interaction between Tyr23 and Ala25, is crucial to BPTI folding initiation. G37A deviates from a standard chevron plot at strongly folding conditions. This is the first reported case where a single-site mutation causes "rollover" in an otherwise two-state folding process. Combined with other structural and dynamic information on Gly37, our results suggest that the NH-aromatic polar interaction involving Gly37 and Tyr35 are important in keeping the flexible loops from non-native conformations. In the absence of this interaction, BPTI folding encounters a kinetic trap.;A database of hydrogen-deuterium exchange results has been compiled for proteins for which there are reports of out-exchange in the native and/or partially folded states, and protection against exchange during folding. The question whether the slow exchange core is the folding core has been examined in a detailed comparison of the specific NHs and the elements of secondary structure on which they are located. There is a strong tendency for the same elements of secondary structure to carry NHs most protected in the native and partially folded states, and NHs first protected during folding. There is not a one-to-one correspondence of individual NHs that is most protected in native and partially folded states, and first protected during folding.