Folding mechanism of ribonuclease T1 in the absence of the disulfide bonds

Abstract

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.

Dimeric Structure of the Bacterial Extracellular Foldase PrsA

“…As a consequence, the refolding of 85% of all molecules is limited in rate by trans 3 cis isomerization at Pro 39 , which shows a time constant of 530 s (at 15°C, pH 8.0). The major part of the fluorescence change during RCM-T1 refolding reflects this isomerization and makes RCM-T1 an excellent substrate for assaying the folding activity PPIases (51)(52)(53)(54). Relatively high PrsA concentrations were necessary to accelerate the refolding of RCM-T1 (Fig.…”

Cns1 Is an Activator of the Ssa1 ATPase Activity

“…Peptidyl-Prolyl cis/trans-Isomerase (PPIase) Assay: Refolding of RCM-T1-To measure the PPIase activity of Cns1, we assayed the refolding of a reduced and S-carboxymethylated S54G/P55N variant of RNase T1 (RCM-T1), as described previously (41). RCM-T1 was preincubated in an unfolded state in 100 mM Tris, pH 8.0, and allowed to refold spontaneously upon transfer into 100 mM Tris, pH 8.0, 2 M NaCl.…”

A Conserved Domain of the Arabidopsis GNOM Protein Mediates Subunit Interaction and Cyclophilin 5 Binding

“…Catalytic activity was strongly inhibited by cyclosporin A with a calculated K I of 8 nM (Figure 7B). We analyzed protein refolding activity of the GSTCyp5 19-201 fusion protein toward the artificial substrate RNase T1 (reduced carboxymethylated RNase T1, RCM-T1, variant S54G/P55N; Mücke and Schmid, 1994). The refolding of 0.7 M RCM-T1 was accelerated approximately sixfold when 77 nM GST-Cyp5 19-201 fusion protein was added ( Figure 7C).…”

“…For protein refolding experiments, ribonuclease T1 variant RCM-T1 (Ser-54-Gly/Pro-55-Asn) was prepared as described by Mücke and Schmid (1994). RCM-T1 was unfolded in 0.1 M Tris-HCl, pH 8.0, at 15ЊC.…”

“…RCM-T1 was unfolded in 0.1 M Tris-HCl, pH 8.0, at 15ЊC. The acceleration of the refolding rate of RCM-T1 was determined (Mücke and Schmid, 1994). Briefly, refolding was initiated at 15ЊC by a 67-fold dilution of unfolded protein to final conditions of 0.1 M Tris-HCl, pH 8.0, 2 M NaCl, 0.7 M RCM-T1, and the desired concentrations of or GST diluted in the same buffer.…”