Folding and Unfolding Kinetics of the Proline-to-Alanine Mutants of Bovine Pancreatic Ribonuclease A

Abstract

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.

Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A

“…RNase A contains four disulfide bonds and four proline residues (two cis and two trans) (Wlodawer et al, 1988), and kinetic folding studies have been carried out both with the disulfide bonds intact (Garel & Baldwin, 1973;Houry et al, 1994;Dodge & Scheraga, 1996) and with the disulfide bonds first reduced and then oxidized (Rothwarf et al, 1998).…”

“…The concentration of U,, decreases rapidly as the X-Pro groups isomerize to the equilibrium isomeric-state populations. In a stopped-flow refolding kinetic experiment, U, folds very rapidly to the native structure without requiring peptide-group isomerization (Houry et al, 1994) which, as shown by Brandts et al (1975), would otherwise slow the refolding process.The distribution of cis and trans proline peptide groups in the unfolded state has been deduced from stopped-flow unfolding/ refolding kinetic studies of the wild-type and of proline-to-alanine and tyrosine-to-phenylalanine mutants of RNase A (Dodge & Scheraga, 1996;Houry & Scheraga, 1996;Juminaga et al, 1997).In these studies, the U,, species of the proline-93-to-alanine (P93A) mutant of RNase A exhibited very different kinetic properties than the U , species of wild-type RNase A. The UUf species of P93A converts to other unfolded species (Urn and U,) at a rate that is 20 times greater than that at which the UUf species converts to other unfolded species in wild-type RNase A (Table 7 of Dodge & Scheraga, 1996).…”

“…The distribution of cis and trans proline peptide groups in the unfolded state has been deduced from stopped-flow unfolding/ refolding kinetic studies of the wild-type and of proline-to-alanine and tyrosine-to-phenylalanine mutants of RNase A (Dodge & Scheraga, 1996;Houry & Scheraga, 1996;Juminaga et al, 1997).…”

“…The UUf species of P93A converts to other unfolded species (Urn and U,) at a rate that is 20 times greater than that at which the UUf species converts to other unfolded species in wild-type RNase A (Table 7 of Dodge & Scheraga, 1996). U,, is the unfolded species that has peptide groups in their native isomeric states, and therefore, the rapid rate of disappearance of U , in P93A suggested that the isomeric states of peptide groups occumng in the native state were energetically unfavorable in the unfolded state.…”

Protein prosthesis: β‐peptides as reverse‐turn surrogates

“…This finding is not rote, as replacing Asn113 and Pro114 with R-Nip-RNip (a diastereomer of R-Nip-S-Nip) eliminates enzymatic activity. Similarly, the transition temperatures for the P114A and P114G variants are decreased by $10 C. [23][24][25] Thus, b 2 -hAla-b 3 -hAla and R-Nip-S-Nip are more than passive linkers 26 -both support native protein structure. This conclusion is supported by the indistinguishable CD spectra of both RNase A and b 2 b 3 hAla RNase A (Supporting Information Fig.…”