Proline isomerization in bovine pancreatic ribonuclease A. 2. Folding conditions.
The kinetics of cis-trans isomerization of individual X-Pro peptide groups is used to study the backbone dynamics of bovine pancreatic ribonuclease A (RNase A). We previously developed and validated a fluorescence method for monitoring the cis-trans isomerization of the Tyr92-Pro93 and Asn113-Pro114 peptide groups of RNase A under unfolding conditions [Juminaga, D., Wedemeyer, W. J., and Scheraga, H. A. (1998) Biochemistry 37, 11614-11620]. The essence of this method is to introduce a fluorescent residue (Tyr or Trp) in a position adjacent to the isomerizing proline (if one is not already present) and to eliminate the fluorescence of other such residues adjacent to prolines by mutating them to phenylalanine. Here, we extend this method to observe the cis-trans isomerization of these peptide groups under folding conditions using two site-directed mutants (Y92F and Y115F) of RNase A. Both isomerizations decelerate with increasing concentrations of GdnHCl, with nearly identical m values (1.11 and 1.19 M(-1), respectively) and extrapolated zero-GdnHCl time constants (42 and 32 s, respectively); by contrast, under unfolding conditions, the cis-trans isomerizations of both Pro93 and Pro114 are independent of GdnHCl concentration. Remarkably, the isomerization rates under folding conditions at GdnHCl concentrations above 1 M are significantly slower than those measured under unfolding conditions. The temperature dependence of the Pro114 isomerization under folding conditions is also unusual; whereas Pro93 exhibits an activation energy typical of proline isomerization (19.4 kcal/mol), Pro114 exhibits a sharply reduced activation energy of 5.7 kcal/mol. A structurally plausible model accounts for these results and, in particular, shows that folding conditions strongly accelerate the cis-trans isomerization of both peptide groups to their native cis conformation, suggesting the presence of flickering local structure in their beta-hairpins.
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Rajiv
R
Bhat
William J
WJ
Wedemeyer
Harold A
HA
Scheraga
D000818:Animals; D002417:Cattle; D019791:Guanidine; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D013237:Stereoisomerism; D013696:Temperature
D011994:Recombinant Proteins; D011392:Proline; D012259:Ribonuclease, Pancreatic; D019791:Guanidine