Folding mechanism of ribonuclease T1 in the absence of the disulfide bonds.
In the absence of its two disulfide bonds, ribonuclease T1 can exist in a native-like folded conformation when > or = 2 M NaCl is present. We measured the kinetics of unfolding and refolding of two reduced and carboxymethylated variants of ribonuclease T1 with one cis proline (the Ser54Gly/Pro55Asn variant) and with two cis prolines (the wild-type protein) as a function of the NaCl concentration. Single and double mixing techniques were used. Analysis of the kinetic results demonstrates that the two cis prolyl bonds at Pro39 and Pro55 remain cis in the folded state after the reduction and carboxymethylation of the disulfide bonds. Folded molecules with trans isomers could not be found. The substitution of cis-Pro55 influences the proline-limited folding reaction, and the analysis of the changes in the folding kinetics shows that the trans-->cis isomerizations of both prolines are slow and are rate-determining steps for the refolding of ribonuclease T1 in the presence as well as in the absence of the disulfide bonds. The direct folding reaction of protein chains with correct prolyl isomers is also affected by the Ser54Gly/Pro55Asn mutation. The rate of refolding is decreased, whereas the rate of unfolding is almost unaffected. The kinetic analysis points to two main consequences of the Ser54Gly/Pro55Asn mutation for the stability and the folding mechanism of RNase T1. It is moderately destabilizing, because the deletion of a conformationally restricted residue (Pro55-->Asn) and the insertion of a flexible residue (Ser54-->Gly) both tend to increase the entropy of the unfolded state. The cis<-->trans isomerization of Pro55 is abolished, however, leading to a decrease in the entropy of the unfolded protein. These two entropic contributions seem to partially compensate each other, and the net change in free energy as a consequence of the Ser54Gly/Pro55Asn double mutation is very small.
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Laboratorium für Biochemie, Universität Bayreuth, Germany.
Schmid
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D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D006163:Ribonuclease T1 / Q000737:chemistry*; D012965:Sodium Chloride; D013329:Structure-Activity Relationship; D014508:Urea
D004220:Disulfides; D012965:Sodium Chloride; D014508:Urea; D011392:Proline; D006163:Ribonuclease T1