Formation of a hydrophobic cluster in denatured bovine pancreatic trypsin inhibitor.

Bovine pancreatic trypsin inhibitor (BPTI) unfolds upon reduction of its three disulfide bonds. A recombinant model of the reduced state of BPTI, called [R]Ala, in which all six Cys residues are replaced with Ala, has been expressed in Escherichia coli. 1H nuclear magnetic resonance spectroscopy shows that [R]Ala does not contain stable secondary structure. Some chemical shift dispersion exists, however, suggesting the existence of non-random interactions in [R]Ala. In particular, the side-chain protons of Ile19 resonate upfield of those of Ile18. This observation was investigated using an eight residue peptide model, P17-24, corresponding to residues 17 to 24 of BPTI. The non-random chemical shift dispersion of the Ile residues observed in [R]Ala also occurs in P17-24, indicating that P17-24 contains interactions that are similar to those found in the corresponding region of [R]Ala. The only interresidue nuclear Overhauser effects observed in P17-24 are between the ring protons of Tyr21 and the gamma CH3 group of Ile19, indicating that these protons are in close proximity. Substitution of Tyr21 by Ala in P17-24 results in the loss of the chemical shift dispersion of the Ile resonances, suggesting that the upfield shifts of the Ile19 resonances are due to ring current shifts arising from the proximity of Tyr21. Collectively, these results suggest that the side-chain of Ile19 is positioned at least some of the time above the plane of the aromatic ring of Tyr21. We conclude that these two residues participate in a hydrophobic cluster in P17-24 and in the denatured state of BPTI.