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. Study holds ProTherm entries: 3215, 3216, 3217, 3218, 3219 Extra Details: ribonuclease A; proline; alanine; rate constant;,long-range interactions; kinetics
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:21 p.m.
|Number of data points||14|
|Proteins||Ribonuclease pancreatic ; Ribonuclease pancreatic|
|Assays/Quantities/Protocols||Experimental Assay: activity ; Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV|