Circular dichroism spectroscopy of bovine pancreatic trypsin inhibitor and five altered conformational states. Relationship of conformation and the refolding pathway of the trypsin inhibitor.
As part of a conformational study of the pathway of unfolding and refolding of bovine pancreatic trypsin inhibitor that accompanies breakage and formation of its three disulfide bonds, circular dichroism spectra have been measured for several limiting conformational states: native and refolded, with the three correct disulfide bonds; the (30--51, 5--55) two-disulfide species trapped during unfolding and refolding, which have a stable nativelike conformation; the fully reduced protein, with no disulfide bonds. Refolded protein with the three correct disulfide bonds has been found to be slightly different from the native protein; this conformational difference could be removed by gently heating the refolded protein. The same difference appears to be present between the two-disulfide intermediates, lacking the 14--38 disulfide bond, produced during unfolding and refolding. The conformational difference appear to be introduced at an early stage of refolding. The fully reduced protein, with no disulfides, exists as a flexible polypeptide chain with no detectable fixed conformation. The near-ultraviolet portions of the spectra are resolved into probable contributions by tyrosine, disulfide, and phenylalanine side-chain electronic transitions. The probable contributions to the native protein spectrum by tyrosines were also elucidated by observing the spectral shifts caused by their ionization at pH 12.5, where the folded conformation is maintained. The rotational strengths of the isolated transitions provide a measure of conformational flexibilities for the chromophores. Resolution of the far-ultraviolet spectrum of the native protein into contributions of its known secondary structures was not successful.
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D008958:Models, Molecular; D010179:Pancreas / Q000032:analysis; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014360:Trypsin Inhibitor, Kunitz Soybean; D014361:Trypsin Inhibitors
D014361:Trypsin Inhibitors; D014360:Trypsin Inhibitor, Kunitz Soybean