The rate of isomerisation 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 chymotrypsin inhibitor 2 (CI2), all of which are in the trans conformation in the native structure. The isomerisation of one or more of these peptidyl-proline bonds to the cis conformation in the denatured state gives rise to heterogeneity, leading to both fast and slow-folding species. The refolding of the fast-folding species, which has all trans peptidyl-proline bonds, is much faster than that of the slow-folding species, 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 refolding, 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 conformation 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 mutant 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 denatured state of intact CI2.
Tan
Y J
YJ
Cambridge Centre for Protein Engineering, UK.
Oliveberg
M
M
Otzen
D E
DE
Fersht
A R
AR
D000591:Amino Acid Isomerases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D019696:Peptidylprolyl Isomerase; D010940:Plant Proteins; D011392:Proline / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013696:Temperature
D002352:Carrier Proteins; D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; C093336:chymotrypsin inhibitor 2; D011392:Proline; D002918:Chymotrypsin; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase