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

Abstract

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 μs 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.