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PubMed · 10677826

Efficient side-chain and backbone assignment in large proteins: application to tGCN5.

Y Lin·G Wagner
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Abstract

In determining the structure of large proteins by NMR, it would be desirable to obtain complete backbone, side-chain, and NOE assignments efficiently, with a minimum number of experiments and samples. Although new strategies have made backbone assignment highly efficient, side-chain assignment has remained more difficult. Faced with the task of assigning side-chains in a protein with poor relaxation properties, the Tetrahymena histone acetyltransferase tGCN5, we have developed an assignment strategy that would provide complete side-chain assignments in cases where fast 13C transverse relaxation causes HCCH-TOCSY experiments to fail. Using the strategy presented here, the majority of aliphatic side-chain proton and carbon resonances can be efficiently obtained using optimized H(CC-CO)NH-TOCSY and (H)C(C-CO)NH-TOCSY experiments on a partially deuterated protein sample. Assignments can be completed readily using additional information from a 13C-dispersed NOESY-HSQC spectrum. Combination of these experiments with H(CC)NH-TOCSY and (H)C(C)NH-TOSCY may provide complete backbone and side-chain assignments for large proteins using only one or two samples.

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AcetyltransferasesAnimalsDeuteriumHistone AcetyltransferasesNuclear Magnetic Resonance, BiomolecularProtein Structure, SecondarySaccharomyces cerevisiae ProteinsSensitivity and SpecificityTetrahymenaJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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Y Lin, G Wagner. 1999. Efficient side-chain and backbone assignment in large proteins: application to tGCN5.. https://doi.org/10.1023/a%3A1008343915382

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