Document Detail

NMR assignment of protein side chains using residue-correlated labeling and NOE spectra.
MedLine Citation:
PMID:  14643705     Owner:  NLM     Status:  MEDLINE    
A new approach for the isotopic labeling of proteins is proposed that aims to facilitate side chain resonance assignments. Residue-correlated (RC) labeling is achieved by the expression of a protein on a medium containing a mixture of labeled, e.g., [U-13C,15N]amino acids, and NMR silent, [U-2H]amino acids. De novo synthesis of amino acids was suppressed by feedback inhibition by the amino acids in the growth medium and by the addition of beta-chloro-L-alanine, a transaminase inhibitor. Incorporation of these amino acids into synthesized proteins results in a relative diminution of inter-residue NOE interactions and a relative enhancement of intra-residue NOEs. Comparison of the resulting NOE spectra with those obtained from a uniformly labeled sample allows identification of intra-residue NOE peaks. Thus, this approach provides direct information for sidechain assignments in the NOE spectra, which are subsequently used for structural analysis. We have demonstrated the feasibility of this strategy for the 143 amino acid nuclease inhibitor NuiA, both at 35 degrees C, corresponding to a rotational correlation time of 9.5 ns, and at 5 degrees C, corresponding to a rotational correlation time of 22 ns.
Geoffrey A Mueller; Thomas W Kirby; Eugene F DeRose; Robert E London
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Publication Detail:
Type:  Comparative Study; Evaluation Studies; Journal Article; Validation Studies    
Journal Detail:
Title:  Journal of magnetic resonance (San Diego, Calif. : 1997)     Volume:  165     ISSN:  1090-7807     ISO Abbreviation:  J. Magn. Reson.     Publication Date:  2003 Dec 
Date Detail:
Created Date:  2003-12-03     Completed Date:  2004-03-12     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  9707935     Medline TA:  J Magn Reson     Country:  United States    
Other Details:
Languages:  eng     Pagination:  237-47     Citation Subset:  IM    
National Institute of Environmental Health Sciences, Laboratory of Structural Biology, 111 Alexander Drive, PO Box 12233 MD MR-01, Research Triangle Park, NC 27709, USA.
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MeSH Terms
Amino Acids / chemistry*
Bacterial Proteins / chemistry*
Carbon Isotopes
Feasibility Studies
Glucose / chemistry
Magnetic Resonance Spectroscopy / methods*
Nitrogen Isotopes
Phenylalanine / chemistry
Protein Structure, Tertiary*
Proteins / chemistry
Spin Labels / chemical synthesis*
Statistics as Topic
Valine / chemistry
beta-Alanine / analogs & derivatives*,  chemistry*
Reg. No./Substance:
0/Amino Acids; 0/Bacterial Proteins; 0/Carbon Isotopes; 0/Nitrogen Isotopes; 0/NuiA protein, Anabaena; 0/Proteins; 0/Spin Labels; 107-95-9/beta-Alanine; 3981-36-0/3-chloroalanine; 50-99-7/Glucose; 63-91-2/Phenylalanine; 7004-03-7/Valine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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