Ultraviolet difference spectroscopy of intermediates trapped in unfolding and refolding of bovine pancreatic trypsin inhibitor

Abstract

The solvent surface accessibilities of the aromatic amino acids of bovine pancreatic trypsin inhibitor were examined after the protein was trapped at various stages of unfolding and refolding. Two types of near-UV difference spectroscopy were used in making these measurements. One type compares the near UV spectrum of each protein with the spectrum of the native inhibitor; the other is solvent perturbation spectroscopy. The 2 types of difference spectroscopy utilized are compared and found to be equivalent measures of tyrosine solvent exposure if the perturbation spectra are corrected for a probable contribution by buried residues. The experimental values for tyrosine solvent exposure in the native inhibitor are in agreement with those calculated from its crystal structure. The results of these studies identify an order in which the 4 tyrosines and the 4 phenylalanines of bovine pancreatic trypsin inhibitor are removed from solvent as refolding proceeds. The relative solvent accessibilities of the aromatic residues suggest an ordering in which the protein chain obtains compact globular structures.