REDUCED BPTI IS COLLAPSED - A PULSED-FIELD GRADIENT NMR-STUDY OF UNFOLDED AND PARTIALLY FOLDED BOVINE PANCREATIC TRYPSIN-INHIBITOR

Pulsed field gradient NMR was used to measure the hydrodynamic behavio r of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R](Abu), at pH 4.5 and pH 2.5, and unfolded [14-38](Abu) at pH 2.5. These were prepared by chemical synthesis. [R](Abu) is a model for reduced BPTI; all cysteine residues are replaced by alpha-amino-n-butyric acid (Abu). [14-38](Abu) retain s cysteines 14 and 38, which form a disulfide bond, while the other cy steine residues are replaced by Abu. In the PFG experiments, the diffu sion coefficient is measured as a function of protein concentration, a nd the Value of D degrees-the diffusion coefficient extrapolated to in finite dilution-is determined. From D degrees, a value of the hydrodyn amic radius, R-k, is computed from the Stokes-Einstein relationship. A t pH 4.5, [R](Abu) has an R-h value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental R-h, Va lue is the same as for a random coil. In view of the changes in NMR-de tected structure of [R](Abu) at pH 4.5 versus pH 2.5 (Pen H, Barbar E, Barany G, Woodward C. 1995. Extensive non-random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:139 74-13981), the collapse of reduced BPTI at pH 4.5 may be associated wi th the formation of non-native hydrophobic clusters of pairs of side c hains one to three amino acids apart in sequence. The diffusion consta nt of [14-38](Abu), was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially fold ed [14-38](Abu), relative to native BPTI, is similar to the increase i n radius of gyration measured for other proteins under ''molten globul e'' conditions.