| Study of protein topography with flash photolytically generated nonspecific surface-labeling reagents: surface labeling of ribonuclease A. | |
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MedLine Citation:
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PMID: 556946 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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A method for nonspecifically labeling essentially all exposed residues of a protein is described. A reactive aryl nitrene is generated from N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-Taurine), within 500 mus by flash photolysis in the presence of protein. The reactive nitrene is inserted in about 2 ms into those carbon-hydrogen bonds of the protein that are exposed to the solvent. The method is applied here to ribonuclease A to demonstrate the different degree of labeling of the native and denatured protein. On the basis of amino acid analysis, it appears that residues of the native protein that are buried in the interior of the molecule (as judged from the x-ray structure) do not react with the nitrene. However, when these residues (even nonreactive ones such as valine and proline) are exposed by denaturation of the protein, they do react with the nitrene. It is shown that native ribonuclease A retains 90% of its enzymatic activity when flashed in the absence of NAP-Taurine. This small loss in activity arises from the disruption of a limited portion of the native enzyme structure, as judged by circular dichroism, ultraviolet, and Raman spectra. The site of this limited disruption may be a portion of the enzyme surface near the Cys-26-Cys-84 disulfide bond. The utility of this surface labeling technique for studying the pathways of protein folding or unfolding is discussed. |
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Authors:
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R R Matheson; H E Van Wart; A W Burgess; L I Weinstein; H A Scheraga |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Biochemistry Volume: 16 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 1977 Feb |
Date Detail:
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Created Date: 1977-04-15 Completed Date: 1977-04-15 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 396-403 Citation Subset: IM |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acids
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analysis Animals Cattle Circular Dichroism Pancreas / enzymology Photolysis Protein Binding Protein Conformation Ribonucleases* Spectrophotometry Spectrophotometry, Ultraviolet Spectrum Analysis, Raman Taurine |
| Chemical | |
Reg. No./Substance:
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0/Amino Acids; 107-35-7/Taurine; EC 3.1.-/Ribonucleases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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