Document Detail


Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment.
MedLine Citation:
PMID:  22252484     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with (15)N-(1)H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three different species of amino acids in each sample, (15)N, 1-(13)C, and fully (13)C/(15)N labeled, permits identification of more amino acid types and sequential pairs than would be possible with previously published combinatorial methods. The new protocol involves recording of up to five 2D triple-resonance experiments to distinguish the various isotopomeric dipeptide species. The pattern of backbone NH cross peaks in this series of spectra adds a new dimension to the combinatorial grid, which otherwise mostly relies on comparison of [(15)N, (1)H]-HSQC and possibly 2D HN(CO) spectra of samples with different labeled amino acid compositions. Application to two α-helical membrane proteins shows that using no more than three samples information can be accumulated such that backbone assignments can be completed solely based on 3D HNCA/HN(CO)CA experiments. Alternatively, in the case of severe signal overlap in certain regions of the standard suite of triple-resonance spectra acquired on uniformly labeled protein, or missing signals due to a lack of efficiency of 3D experiments, the remaining gaps can be filled.
Authors:
Frank Löhr; Sina Reckel; Mikhail Karbyshev; Peter J Connolly; Norzehan Abdul-Manan; Frank Bernhard; Jonathan M Moore; Volker Dötsch
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-01-18
Journal Detail:
Title:  Journal of biomolecular NMR     Volume:  52     ISSN:  1573-5001     ISO Abbreviation:  J. Biomol. NMR     Publication Date:  2012 Mar 
Date Detail:
Created Date:  2012-03-12     Completed Date:  2012-08-09     Revised Date:  2013-07-31    
Medline Journal Info:
Nlm Unique ID:  9110829     Medline TA:  J Biomol NMR     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  197-210     Citation Subset:  IM    
Affiliation:
Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt, Germany.
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MeSH Terms
Descriptor/Qualifier:
Carbon Isotopes / chemistry
Membrane Proteins / chemistry*
Nitrogen Isotopes / chemistry
Nuclear Magnetic Resonance, Biomolecular / methods*
Grant Support
ID/Acronym/Agency:
U54 GM094608/GM/NIGMS NIH HHS; U54 GM094608/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Carbon Isotopes; 0/Membrane Proteins; 0/Nitrogen Isotopes
Comments/Corrections

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