Conformational changes and association of chemically modified chymotrypsins

The low pH conformational transitions of a series of modified chymotrypsins have been examined and compared to the association properties of these same proteins in order to determine if the effect of the modification was directly upon the association or indirectly through conformational changes. The modifications of α-chymotrypsin that have been studied are serine-195 modified with acetyl or with phenylmethane sulfonyl groups, His-57 modified by tosylamidophenylethyl chloromethyl ketone or with a methyl group, and Met-192 modified with a nitrophenacyl group. Chymotrypsinogen and native and modified delta-chymotrypsin have also been studied. Ultraviolet difference spectra and optical rotatory dispersion measurements show that the various proteins may be divided into three groups depending upon their response in the low pH transition. These groupings based upon conformational transitions are not divided in the same manner as were those based upon the effect of the modifications on the association process itself. The latter process had earlier been found to correlate with the size of the modification in the active site. From these findings it has been concluded that the effect of the modifications on the association was directly upon the interacting subunit interface and that the observed conformational change is only incidental to this effect. Therefore, the amino acids involved in the association in solution are the same as those involved in the dimerization in the crystal, namely, the active site region of Ser-195, His-57, and Met-192. This is the most direct demonstration that the mode of association of a protein in solution is the same as the mode of association in the crystal.