ENHANCED PROTEIN FLEXIBILITY CAUSED BY A DESTABILIZING AMINO-ACID REPLACEMENT IN BPTI

A genetically engineered variant of bovine pancreatic trypsin inhibito r (Y35G BPTI) has been shown previously by X-ray crystallography to ha ve a three-dimensional structure dramatically different from that of t he wild-type protein, particularly in the protease-binding region of t he molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Desc ribed here are N-15 NMR relaxation studies to compare the backbone dyn amics of Y35G BPTI to those of the wild-type protein. The Tyr35 --> Gl y substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the l ongitudinal relaxation rates, indicating that the substitution facilit ates relatively slow backbone motions, estimated to be on the microsec ond time-scale. The results indicate that the residues making up the t rypsin-binding site undergo large and relatively slow conformational c hanges in solution, estimated to be on the 5 to 20 mu s time-scale. It is thus likely that the crystal structure represents only one of mult iple interconverting conformations in solution, only a fraction of whi ch may be competent for binding trypsin. The large thermodynamic desta bilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions th at are normally favored by the highly ordered structure of the wild-ty pe protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic proper ties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins. (C) 1997 Academic Pre ss Limited.