Folding and unfolding kinetics of the proline-to-alanine mutants of bovine pancreatic ribonuclease A.
Four single mutants (P42A, P93A, P114A, and P117A) of bovine pancreatic ribonuclease A (RNase A) in which each mutant has one of the four prolines of RNase A changed to alanine were prepared. The physical properties of these four mutants indicate that their native structure is essentially identical to that of wild-type RNase A. The disulfide-intact forms of these proteins were denatured in guanidine hydrochloride (Gdn.HCl) and then refolded by dilution of the Gdn.HCl. Single-jump folding, single-jump unfolding, and double-jump unfolding/folding stopped-flow experiments were carried out on wild-type and the four proline mutants of RNase A using absorption detection to follow the folding kinetics. The single-jump folding experiments carried out at six different final Gdn.HCl concentrations indicate that the folding rate constants of individual steps for the mutants are similar to those of wild-type RNase A. The Tyr92-Pro93 peptide bond has a cis conformation in native wild-type RNase A, and the results from our double-jump stopped-flow experiments indicate that the Tyr92-Ala93 peptide bond in the P93A mutant of RNase A is also cis in the native state. The existence of two cis peptide bonds (preceding Pro93 and Pro114) in wild-type RNase A is probably due to (as-yet-unidentified) long-range interactions, and such interactions are presumably the origin of a cis peptide bond even when alanine is substituted for Pro93. The data from the double-jump stopped-flow experiments are interpreted in terms of a folding/unfolding model. This model specifies the cis/trans isomerization state of the unfolded species (Uvf, Uf, Um, and Us) at each X-Pro peptide bond. Also, this model confirms the existence of several previously postulated chain-folding initiation sites.
Dodge
R W
RW
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Scheraga
H A
HA
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000818:Animals; D002417:Cattle; D005813:Genes, Synthetic; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007536:Isomerism; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D010179:Pancreas / Q000201:enzymology; D011392:Proline / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics
D006146:Guanidines; D011994:Recombinant Proteins; D011392:Proline; D012259:Ribonuclease, Pancreatic; D019791:Guanidine; D000409:Alanine