A PULSED-FIELD GRADIENT NMR-STUDY OF BOVINE PANCREATIC TRYPSIN-INHIBITOR SELF-ASSOCIATION

Previous studies have produced conflicting interpretations regarding t he 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 conce ntrations. Relative to the standard proteins, lysozyme, ribonuclease, and ubiquitin; diffusion constants indicate that BPTI dimerizes at con centrations above about 3 mg/mL and below 280 K. At higher temperature s, a marked self-association is observed above 10 mg/mL. The apparent lack of significant effects from variations in pH and NaCl concentrati on suggests minimal contribution to the aggregation process from charg e-charge interactions. In contrast, in nondenaturing concentrations of urea (2 M), BPTI behaves as a monomer, suggesting that hydrophobic an d polar residues modulate BPTI association. The BPTI surface shows tha t while one side is highly charged, the opposite side, composed mostly of hydrophobic and some hydrophilic residues, is feasible as an inter face for BPTI self-association.