Nature of the unfolded state of ribonuclease A: effect of cis-trans X-Pro peptide bond isomerization.
The equilibrium unfolded state of disulfide-intact bovine pancreatic ribonuclease A is a heterogeneous mixture of unfolded species. Previously, four unfolded species have been detected experimentally. They are Uvf, Uf, UsII, and UsI which have refolding time constants on the millisecond, millisecond to second, second to tens of seconds, and hundreds of seconds time scales, respectively. In the current study, the refolding pathway of the protein was investigated under favorable folding conditions of 0.58 M GdnHCl, pH 5.0, and 15 degrees C. In addition to the above four unfolded species, the presence of a fifth unfolded species was detected. It has a refolding time constant on the order of 2 s under the conditions employed. This new unfolded species is labeled Um, for medium-refolding species. Single-jump refolding experiments monitored by tyrosine burial and by cytidine 2'-monophosphate inhibitor binding indicate that the different unfolded species refold to the native state along independent refolding pathways. The buildup of the different unfolded species upon unfolding of the protein from the native state was monitored by absorbance using double-jump experiments. These experiments were carried out at 15 degrees C and consisted of an unfolding step at 4.2 M GdnHCl and pH 2.0, followed, after a variable delay time, by a refolding step at 0.58 M GdnHCl and pH 5.0. The results of these experiments support the conclusion that the different unfolded species arise from cis-trans isomerizations at the X-Pro peptide bonds of Pro 93, 114, and 117 in the unfolded state of the protein. The rates of these isomerizations were obtained for each of these three X-Pro peptide bonds at 15 degrees C.
Houry
W A
WA
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Scheraga
H A
HA
D000818:Animals; D002417:Cattle; D003568:Cytidine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry
D004791:Enzyme Inhibitors; D006146:Guanidines; D010455:Peptides; D011392:Proline; D012259:Ribonuclease, Pancreatic; D003568:Cytidine Monophosphate; D019791:Guanidine