THE RATE OF ISOMERIZATION OF PEPTIDYL-PROLINE BONDS AS A PROBE FOR INTERACTIONS IN THE PHYSIOLOGICAL DENATURED STATE OF CHYMOTRYPSIN INHIBITOR-2

There are four peptidyl-proline bonds in the 64-residue protein chymot rypsin inhibitor 2 (CI2), all of which are in the trans conformation i n the native structure. The isomerisation of one or more of these pept idyl-proline bonds to the cis conformation in the denatured state give s rise to heterogeneity, leading to both fast and slow-folding species . The refolding of the fast-folding species, which has all trans pepti dyl-proline bonds, is much faster than that of the slow-folding specie s, which have one or more cis peptidyl-proline bonds. In CI2, the slow -folding species can be classified into two groups by their rates of r efolding, temperature-dependence, pH-dependence and [GdmCl]-dependence of the rate constants and the effect of peptidyl-prolyl isomerase on the rate constants. The replacement of Pro6 by Ala removes one of the slow refolding phases, suggesting that the cis peptidyl-Pro6 conformat ion is solely responsible for one of the slow-folding species. Pro6 is located in a region of the protein where non-random interactions have been found in a series of N-terminal fragments of CI2 (residues 1 to 13, 1 to 25, 1 to 28 and 1 to 40). In addition, NMR studies on a mutan t fragment, (1-40)T3A, have confirmed that this non-native interaction is associated with the bulky side-chain of Trp5. The atypical rate of cis to trans isomerisation of the peptidyl-Pro bond is indicative of the presence of a similar hydrophobic cluster in the physiological den atured state of intact CI2. (C) 1997 Academic Press Limited.