Previous studies have produced conflicting interpretations regarding the aggregation state of BPTI in solution. Here, pulsed-field gradient NMR self-association measurements have been performed with BPTI under a variety of temperature, pH, salt, urea conditions, and protein concentrations. Relative to the standard proteins, lysozyme, ribonuclease, and ubiquitin, diffusion constants indicate that BPTI dimerizes at concentrations above about 3 mg/mL and below 280 K. At higher temperatures, a marked self-association is observed above 10 mg/mL. The apparent lack of significant effects from variations in pH and NaCl concentration suggests minimal contribution to the aggregation process from charge-charge interactions. In contrast, in nondenaturing concentrations of urea (2 M), BPTI behaves as a monomer, suggesting that hydrophobic and polar residues modulate BPTI association. The BPTI surface shows that while one side is highly charged, the opposite side, composed mostly of hydrophobic and some hydrophilic residues, is feasible as an interface for BPTI self-association.

Two-dimensional 1H NMR spectra of an analog of reduced BPTI at pH 4.5, 1 degrees C, have been assigned. Spectra indicate considerable conformational averaging, as expected for a flexible, unfolded protein. The presence of extensive nonrandom structure is detected by the presence of NHi-NHi + 1 and aromatic-aliphatic NOEs. Sequential amide-amide NOEs indicate that turn-like conformations are significantly populated at 18 pairs of residues along the chain. Many of these are located in a turn, loop, or helix in native BPTI, but six are observed for contiguous pairs in the segment composed of residues 29-35, which in native BPTI constitute a strand of extended sheet. A novel finding for unfolded proteins is our observation of NOEs implying non-native hydrophobic interactions. Multiple aromatic-aliphatic NOEs are observed for pairs of residues that are within 1-3 residues of each other. Most are non-native and involve residues in both strands of the central antiparallel strand-turn-strand of native BPTI comprised of residues 18-35. All NOEs reported for oligopeptides spanning the BPTI sequence [Kemmink, J., & Creighton, T. (1993) J. Mol. Biol. 234, 861-878] are observed in reduced BPTI, but many others are present as well. Similar spectra are obtained for naturally occurring BPTI reduced by dithiothreitol, BPTI with cysteines replaced by alpha-amino-n-butyric acid, and BPTI mutant F45A reduced by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

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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