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.

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Abstract:The 5192-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfideintact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyt92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C [40,95]A variant, which is an analog of the major ratedetermining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.Keywords: cis-trans isomerization; crystal structures of RNase A variants; disorder; X-Pro and X-Ala peptide groups Kinetic and NMR studies of the folding of bovine pancreatic ribonuclease A (RNase A) are being carried out to identify the interatomic interactions that lead to the folded protein structure. 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).Mutants of RNase A have been examined to identify the roles of key residues in the folding mechanism. In guanidine hydrochlorideinduced unfolding of disulfide-intact RNase A, the peptide groups preceding proline isomerize to an ensemble of species containing mixtures of cis and trans groups at each of these sites (Garel & Baldwin, 1973). The first unfolded species to form is Uuf (Houry et al., 1994), a very fast refolding species in which these four X-Pro groups retain their native cisltrans conformation. 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 (P...

The refolding kinetics of guanidine-denatured disulfide-intact bovine pancreatic ribonuclease A (RNase A) and its proline-42-to-alanine mutant (Pro42Ala) have been studied by monitoring tyrosine burial and 2'-cytidine monophosphate (2'CMP) inhibitor binding. The folding rate for wild-type RNase A is faster in the presence of the inhibitor 2'CMP than in its absence, indicating that the transition-state structure in the rate-determining step is stabilized by 2'CMP. The folding rate monitored by 2'CMP binding to the major slow-folding species of Pro42Ala RNase A is faster than the folding rate monitored by tyrosine burial; however, the folding rate monitored by inhibitor binding to the minor slow-folding species is decreased significantly over the folding rate monitored by tyrosine burial, indicating that the major and minor slow-folding species of Pro42Ala fold to the native state with different transition-state conformations in the rate-determining step.

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