Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor

Abstract

Pulsed field gradient NMR was used to measure the hydrodynamic behavior 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 [14-38lAh,, at pH 2.5. These were prepared by chemical synthesis.[RIAbu is a model for reduced BPTI; all cysteine residues are replaced by a-amino-n-butyric acid (Abu). [14-38lAb, retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of Do-the diffusion coefficient extrapolated to infinite dilution-is determined. From Do, a value of the hydrodynamic radius, R,,, is computed from the Stokes-Einstein relationship. At pH 4.5, [RIAbu has an R,, value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental R,, value is the same as for a random coil. In view of the changes in NMR-detected structure of [RIA,,,, at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non-random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 3413974-13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non-native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14-38IAbu was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14-38lAh,, relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under "molten globule" conditions. Keywords: bovine pancreatic trypsin inhibitor; protein folding; protein hydrodynamics; pulsed field gradient NMR; unfolded proteinsThe structure of polypeptides at the onset of folding is a fundamental issue in protein folding. Two questions concerning unfolded proteins are of special interest: The tendency of the unfolded chain to form non-random conformations, and the extent of collapse of the polypeptide prior to formation of secondary structure.

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