Determinants of backbone dynamics in native BPTI: cooperative influence of the 14-38 disulfide and the Tyr35 side-chain.

15Nitrogen relaxation experiments were used to characterize the backbone dynamics of two modified forms of bovine pancreatic trypsin inhibitor (BPTI). In one form, the disulfide between Cys14 and Cys38 in the wild-type protein was selectively reduced and methylated to generate an analog of the final intermediate in the disulfide-coupled folding pathway. The second form was generated by similarly modifying a mutant protein in which Tyr35 was replaced with Gly (Y35G). For both selectively reduced proteins, the overall conformation of native BPTI was retained, and the relaxation data for these proteins were compared with those obtained previously with the native wild-type and Y35G proteins. Removing the disulfide from either protein had only small effects on the observed longitudinal relaxation rates (R1) or heteronuclear cross relaxation rates (nuclear Overhauser effect), suggesting that the 14-38 disulfide has little influence on the fast (ps to ns) backbone dynamics of either protein. In the wild-type protein, the pattern of residues undergoing slower (micros to ms) internal motions, reflected in unusually large transverse relaxation rates (R2), was also largely unaffected by the removal of this disulfide. It thus appears that the large R2 rates previously observed in native wild-type protein are not a direct consequence of isomerization of the 14-38 disulfide. In contrast with the wild-type protein, reducing the disulfide in Y35G BPTI significantly decreased the number of backbone amides displaying large R2 rates. In addition, the frequencies of the backbone motions in the modified protein, estimated from R2 values measured at multiple refocusing delays, appear to span a wider range than those seen in native Y35G BPTI. Together, these observations suggest that the slow internal motions in Y35G BPTI are more independent in the absence of the 14-38 disulfide and that formation of this bond may lead to a substantial loss of conformational entropy. These effects may account for the previous observation that the Y35G substitution greatly destabilizes the disulfide. The results also demonstrate that the disulfide and the buried side-chain influence the dynamics of the folded protein in a highly cooperative fashion, with the effects of removing either being much greater in the absence of the other.

Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI.

A genetically engineered variant of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography to have a three-dimensional structure dramatically different from that of the wild-type protein, particularly in the protease-binding region of the molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described here are 15N NMR relaxation studies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein. The Tyr35 --> Gly substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the longitudinal relaxation rates, indicating that the substitution facilitates relatively slow backbone motions, estimated to be on the microsecond time-scale. The results indicate that the residues making up the trypsin-binding site undergo large and relatively slow conformational changes in solution, estimated to be on the 5 to 20 micros time-scale. It is thus likely that the crystal structure represents only one of multiple interconverting conformations in solution, only a fraction of which may be competent for binding trypsin. The large thermodynamic destabilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions that are normally favored by the highly ordered structure of the wild-type protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic properties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins.