Abstract The known biological functions of RNA have expanded in recent years and now include gene regulation, maintenance of sub-cellular structure, and catalysis, in addition to propagation of genetic information. As for proteins, RNA function is tightly correlated with structure. Unlike proteins, structural information for larger, biologically functional RNAs is relatively limited. NMR signal degeneracy, relaxation problems, and a paucity of long-range 1Hâ??1H dipolar contacts have limited the utility of traditional NMR approaches. Selective isotope labeling, including nucleotide-specific and segmental labeling strategies, may provide the best opportunities for obtaining structural information by NMR. Here we review methods that have been developed for preparing and purifying isotopically labeled RNAs, as well as NMR strategies that have been employed for signal assignment and structure determination.
Content Type Journal Article
Pages 113-125
DOI 10.1007/s10858-009-9375-2
Authors
Kun Lu, University of Maryland Baltimore County Howard Hughes Medical Institute and Department of Chemistry and Biochemistry 1000 Hilltop Circle Baltimore MD 21250 USA
Yasuyuki Miyazaki, University of Maryland Baltimore County Howard Hughes Medical Institute and Department of Chemistry and Biochemistry 1000 Hilltop Circle Baltimore MD 21250 USA
Michael F. Summers, University of Maryland Baltimore County Howard Hughes Medical Institute and Department of Chemistry and Biochemistry 1000 Hilltop Circle Baltimore MD 21250 USA
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Abstract Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives,...
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01-22-2011 03:46 AM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
J Biomol NMR. 2011 Jan 19;
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Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties...
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01-21-2011 01:22 AM
Cell-free expression and stable isotope labelling strategies for membrane proteins
Cell-free expression and stable isotope labelling strategies for membrane proteins
Abstract Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems...
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01-09-2011 12:46 PM
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NMR based screening has become an important tool in the pharmaceutical industry. Methods that provide information on the location of small molecule binding sites on the surface of a drug target (e. g. SAR-by-NMR and related techniques) are of particular interest. In order to extend the applicability of...
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Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed...
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The development of novel isotope labeling strategies for proteins has facilitated the study of the structure and dynamics of these molecules. In addition, the recent emergence of alternative methods of bacterial expression for obtaining isotopically labeled proteins permits the study of...
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Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of glucose. We...
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Cell-free expression and labeling strategies for a new decade in solid-state NMR.
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N Biotechnol. 2010 Aug 3;
Authors: Abdine A, Verhoeven MA, Warschawski DE
Although solid-state NMR and cell-free expression have recently become standard methods in biology, the combination of the two is still at a very early stage of development. In this...
Isotope labeling strategies for NMR studies of RNA
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