Effect of proline mutations on the stability and kinetics of folding of staphylococcal nuclease.
Abstract
The role of proline in the stability and kinetics of folding of wild-type staphylococcal nuclease and its P117G, P117T, and P31A mutants was examined as a function of guanidinium thiocyanate (Gdn-SCN) concentration. Replacement of Pro-117 with Gly or Thr caused small increases in stability, whereas substitution of Pro-31 by Ala led to a small decrease in stability. The slopes of the plots of delta G against denaturant concentration (m) for the mutant proteins are significantly smaller than for the wild-type, suggesting a decrease in the solvent-accessible surface area of the denatured state relative to that of the wild-type. The rates of unfolding and refolding were monitored using tryptophan fluorescence. The kinetic traces for refolding in the presence of Gdn-SCN were triphasic for the wild-type protein and P31A but biphasic for P117G and P117T mutants. The slower phases were typically 10% of the total amplitude except in the transition region. The rates of the fastest and medium phases of the wild-type were essentially unaffected by the mutations. Double-jump experiments in which the protein was unfolded in a high concentration of denaturant for a short time period and then refolded to final Gdn-SCN concentrations near the Cm revealed a fast increase in fluorescence emission corresponding to formation of the native state, followed by a slower decrease with an amplitude that varied with the guanidine concentration and time of unfolding.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 302, 303, 304, 305 Extra Details: staphylococcal nuclease; kinetics of folding; proline mutations;,solvent-accessible surface area; stability
Submission Details
ID: UvCdrBVJ3
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:15 p.m.
Version: 1
Publication Details
Nakano T;Antonino LC;Fox RO;Fink AL,Biochemistry (1993) Effect of proline mutations on the stability and kinetics of folding of staphylococcal nuclease. PMID:8448112Additional Information
Study Summary
| Number of data points | 15 |
| Proteins | Thermonuclease ; Thermonuclease ; Thermonuclease ; Thermonuclease |
| Unique complexes | 4 |
| Assays/Quantities/Protocols | Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O |
| Libraries | Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ |
Structure view and single mutant data analysis
Study data
| Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
|---|
Data Distribution
Studies with similar sequences (approximate matches)
Correlation with other assays (exact sequence matches)
Relevant PDB Entries
| Structure ID | Release Date | Resolution | Structure Title |
|---|---|---|---|
| 4WRD | 2014-10-23T00:00:00+0000 | 1.65 | Crystal structure of Staphylcoccal nulease variant Delta+PHS V66E L125E at cryogenic temperature |
| 2LKV | 2011-10-21T00:00:00+0000 | 0 | Staphylococcal Nuclease PHS variant |
| 2M00 | 2012-10-14T00:00:00+0000 | 0 | Solution structure of staphylococcal nuclease E43S mutant in the presence of ssDNA and Cd2+ |
| 2OXP | 2007-02-20T00:00:00+0000 | 2.0 | Crystal Structure of Staphylococcal Nuclease mutant V66D/P117G/H124L/S128A |
| 3D4W | 2008-05-15T00:00:00+0000 | 1.9 | Crystal structure of Staphylococcal nuclease variant Delta+PHS A109R at cryogenic temperature |
| 3D8G | 2008-05-23T00:00:00+0000 | 1.99 | Crystal structure of Staphylococcal nuclease variant Delta+PHS I72R at cryogenic temperature |
| 3MVV | 2010-05-04T00:00:00+0000 | 1.72 | Crystal structure of Staphylococcal nuclease variant Delta+PHS F34A at cryogenic temperature |
| 3QOJ | 2011-02-10T00:00:00+0000 | 1.6 | Cryogenic structure of Staphylococcal nuclease variant D+PHS/V23K |
| 3QOL | 2011-02-10T00:00:00+0000 | 1.9 | Crystal structure of Staphylococcal nuclease variant D+PHS/V23E at pH 6 determined at 100 K |
| 3R3O | 2011-03-16T00:00:00+0000 | 1.9 | Crystal structure of Staphylococcal nuclease variant Delta+PHS T62A at cryogenic temperature and with high redundancy |
Relevant UniProtKB Entries
| Percent Identity | Matching Chains | Protein | Accession | Entry Name |
|---|---|---|---|---|
| 100.0 | Thermonuclease | P00644 | NUC_STAAU | |
| 99.3 | Thermonuclease | Q5HHM4 | NUC_STAAC | |
| 99.1 | Thermonuclease | Q99VJ0 | NUC_STAAM | |
| 99.1 | Thermonuclease | Q7A6P2 | NUC_STAAN | |
| 99.3 | Thermonuclease | Q6GB41 | NUC_STAAS | |
| 99.3 | Thermonuclease | Q8NXI6 | NUC_STAAW | |
| 99.3 | Thermonuclease | Q6GIK1 | NUC_STAAR |