Circular dichroism spectroscopy of bovine pancreatic trypsin inhibitor and five altered conformational states. Relationship of conformation and the refolding pathway of the trypsin inhibitor

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Abstract
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 3 disulfide bonds, CD spectra were measured for several limiting conformational states: native and refolded, with the 3 correct disulfide bonds; the (30-51, 5-55) 2-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 3 correct disulfide bonds differed from the native protein; this conformational difference could be removed by gently heating the refolded protein. The same difference exists between the 2-disulfide intermediates, lacking the 14-38 disulfide bond, produced during unfolding and refolding. The conformational difference appears 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 UV 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 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 UV spectrum of the native protein into contributions of its known secondary structures was not successful.