Cis proline mutants of ribonuclease A. II. Elimination of the slow‐folding forms by mutation

Ribonuclease A is known to form an equilibrium mixture of fast‐folding (UF) and slow‐folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis → trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site‐directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis → trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A.

[1]  K. Wüthrich,et al.  Nmr studies of the molecular conformations in the linear oligopeptides H‐(L‐Ala)n‐L‐Pro‐OH , 1976, Biopolymers.

[2]  F. Schmid,et al.  Fast- and slow-refolding forms of unfolded ribonuclease A differ in tyrosine fluorescence. , 1982, Biochemistry.

[3]  U. Hahn,et al.  Folding of ribonuclease T1. 1. Existence of multiple unfolded states created by proline isomerization. , 1990, Biochemistry.

[4]  L. Ramdas,et al.  Folding/unfolding kinetics of mutant forms of iso-1-cytochrome c with replacement of proline-71. , 1986, Biochemistry.

[5]  A. Wrba,et al.  Role of proline peptide bond isomerization in unfolding and refolding of ribonuclease. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[6]  R. L. Baldwin,et al.  Role of proline isomerization in folding of ribonuclease A at low temperatures. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Brandts,et al.  Isomerization of proline-93 during the unfolding and refolding of ribonuclease A. , 1983, Biochemistry.

[8]  F M Richards,et al.  Replacement of proline-76 with alanine eliminates the slowest kinetic phase in thioredoxin folding. , 1987, Biochemistry.

[9]  K. Kuwajima,et al.  The Pro117 to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics , 1991, FEBS letters.

[10]  F. Asturias,et al.  Location of high-affinity metal binding sites in the profile structure of the Ca+2-ATPase in the sarcoplasmic reticulum by resonance x-ray diffraction. , 1991, Biophysical journal.

[11]  R. L. Baldwin,et al.  Cis proline mutants of ribonuclease A. I. thermal stability , 1992, Protein science : a publication of the Protein Society.

[12]  R. L. Baldwin,et al.  Test of the extended two-state model for the kinetic intermediates observed in the folding transition of ribonuclease A. , 1978, Journal of molecular biology.

[13]  Evidence for the existence of three or more slow phases in the refolding of ribonuclease A and some characteristics of the phases. , 1987, Biochemistry.

[14]  U. Hahn,et al.  Replacement of a cis proline simplifies the mechanism of ribonuclease T1 folding. , 1990, Biochemistry.

[15]  F. Schmid.  Mechanism of folding of ribonuclease A. Slow refolding is a sequential reaction via structural intermediates. , 1983, Biochemistry.

[16]  R. L. Baldwin,et al.  Both the fast and slow refolding reactions of ribonuclease A yield native enzyme. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Brandts,et al.  Involvement of prolines-114 and -117 in the slow refolding phase of ribonuclease A as determined by isomer-specific proteolysis. , 1984, Biochemistry.

[18]  R. L. Baldwin,et al.  Effects of solvent viscosity and different guanidine salts on the kinetics of ribonuclease A chain folding , 1978 .

[19]  T. Kiefhaber,et al.  Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1. , 1992, Journal of molecular biology.

[20]  K. Wüthrich,et al.  The X‐Pro peptide bond as an nmr probe for conformational studies of flexible linear peptides , 1976, Biopolymers.

[21]  F. Schmid.  Proline isomerization in unfolded ribonuclease A. The equilibrium between fast-folding and slow-folding species is independent of temperature. , 2005, European journal of biochemistry.

[22]  H. Halvorson,et al.  Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. , 1975, Biochemistry.

[23]  H. Blaschek,et al.  A native-like intermediate on the ribonuclease A folding pathway. 2. Comparison of its properties to native ribonuclease A. , 2005, European journal of biochemistry.

[24]  H. Nicholson,et al.  Folding kinetics of T4 lysozyme and nine mutants at 12 degrees C. , 1992, Biochemistry.

[25]  R. Jaenicke,et al.  Folding of homologous proteins. The refolding of different ribonucleases is independent of sequence variations, proline content and glycosylation. , 1983, Journal of molecular biology.

[26]  T. Herning,et al.  Effects of proline mutations on the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not result from proline cis-trans isomerization. , 1991, Biochemistry.

[27]  R. L. Baldwin,et al.  A quantitative treatment of the kinetics of the folding transition of ribonuclease A. , 1976, Biochemistry.

[28]  C. Dobson,et al.  Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis , 1987, Nature.

[29]  R. L. Baldwin,et al.  Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[30]  K. Wüthrich,et al.  Nmr studies of the rates of proline cis–trans isomerization in oligopeptides , 1981 .

[31]  H. Scheraga,et al.  Nonnative isomers of proline-93 and -114 predominate in heat-unfolded ribonuclease A. , 1990, Biochemistry.