Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
39
|
| pubmed:dateCreated |
1996-11-12
|
| pubmed:abstractText |
Tryptophan was substituted for Tyr92 to create a sensitive and unique optical probe in order to study the unfolding and refolding kinetics of disulfide-intact bovine pancreatic ribonuclease A by fluorescence-detected stopped-flow techniques. The stability of the Trp mutant was found to be similar to that of wild-type RNase A when denatured by heat or GdnHCl, and the mutant was found to have 85% of the activity of the wild-type protein. Single-jump unfolding experiments showed that the unfolding pathway of the Trp mutant contains a fast and a slow phase similar to those seen previously for the wild-type protein, indicating that the mutation did not alter the unfolding pathway significantly. The activation energy of the slow-unfolding phase suggested that proline isomerization is involved, with the Trp residue presumably reporting on changes in its local environment. Single-jump refolding experiments revealed the presence of GdnHCl-independent burst phase and a native-like intermediate, most likely IN, on the folding pathway. Single-jump refolding data at various final GdnHCl concentrations were fit to a kinetic folding model involving two pathways to the native state; one pathway involves the intermediate IN, and the other is a direct one to the native state. This model provides site-specific information, since Trp92 monitors the formation of local structure only in the neighborhood of that residue. Double-jump refolding experiments permitted the detection of a previously reported, hydrophobically collapsed intermediate, I phi. The refolding data support the hypothesis that the region around position 92 is a chain-folding initiation site in the folding pathway.
|
| pubmed:grant | |
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
Oct
|
| pubmed:issn |
0006-2960
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
1
|
| pubmed:volume |
35
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
12978-92
|
| pubmed:dateRevised |
2007-11-14
|
| pubmed:meshHeading |
pubmed-meshheading:8841145-Animals,
pubmed-meshheading:8841145-Cattle,
pubmed-meshheading:8841145-Enzyme Stability,
pubmed-meshheading:8841145-Fluorescence,
pubmed-meshheading:8841145-Guanidine,
pubmed-meshheading:8841145-Guanidines,
pubmed-meshheading:8841145-Hydrogen-Ion Concentration,
pubmed-meshheading:8841145-Kinetics,
pubmed-meshheading:8841145-Models, Chemical,
pubmed-meshheading:8841145-Mutagenesis, Site-Directed,
pubmed-meshheading:8841145-Mutation,
pubmed-meshheading:8841145-Protein Denaturation,
pubmed-meshheading:8841145-Protein Folding,
pubmed-meshheading:8841145-Ribonuclease, Pancreatic,
pubmed-meshheading:8841145-Spectrometry, Fluorescence,
pubmed-meshheading:8841145-Temperature,
pubmed-meshheading:8841145-Thermodynamics,
pubmed-meshheading:8841145-Tryptophan
|
| pubmed:year |
1996
|
| pubmed:articleTitle |
Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A.
|
| pubmed:affiliation |
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
|
| pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, U.S. Gov't, Non-P.H.S.
|