Document Detail

Conformational flexibility in a staphylococcal nuclease mutant K45C from time-resolved resonance energy transfer measurements.
MedLine Citation:
PMID:  8068683     Owner:  NLM     Status:  MEDLINE    
Thermal fluctuations exist in native proteins and other macromolecules in solution. Some may play a role in ligand or receptor binding, control rates of enzymatic catalysis, or define a range of conformations a segment can adopt in solution. We apply the method of time-resolved resonance energy transfer to study the conformational flexibility of a staphylococcal nuclease mutant, K45C, where lysine 45 located at a flexible loop is replaced by a cysteine. We labeled the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) and used the TNB group covalently attached to the protein as an energy acceptor from a single tryptophan at residue 140 as the donor. Conformational flexibility occurring on the time scale of nanoseconds or longer is dispersed as an apparent distance distribution in time-resolved resonance energy transfer measurements. Below room temperature the apparent distance distribution was fitted with a symmetric Lorentzian model with a full width at half maximum height of about 6 A, indicating substantial degrees of heterogeneity between residues 45 and 140. At room or higher temperature where the protein is in its native state, the apparent distance distribution is asymmetric, indicating the presence of static disorders. Segments in the protein that contribute to the static disorder can be converted to mobile ones with the addition of denaturing guanidinium chloride.
P Wu; L Brand
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  33     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1994 Aug 
Date Detail:
Created Date:  1994-09-27     Completed Date:  1994-09-27     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  10457-62     Citation Subset:  IM    
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
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MeSH Terms
Energy Transfer
Enzyme Stability
Micrococcal Nuclease / chemistry*,  genetics
Molecular Structure
Mutagenesis, Site-Directed
Protein Conformation
Protein Denaturation
Spectrometry, Fluorescence
Grant Support
Reg. No./Substance:
0/Guanidines; 0/Solutions; 113-00-8/Guanidine; EC Nuclease

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