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Journal of Structural and Functional Genomics

, Volume 14, Issue 3, pp 109–118 | Cite as

Reduced dimensionality (4,3)D-hnCOCANH experiment: an efficient backbone assignment tool for NMR studies of proteins

  • Dinesh KumarEmail author
Article

Abstract

Sequence specific resonance assignment of proteins forms the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone (1H, 15N, 13Cα and 13C′) resonances of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality experiment—(4,3)D-hnCOCANH and exploits the linear combinations of backbone (13Cα and 13C′) chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text). The resulted increased dispersion of peaks—which is different in sum (CA + CO) and difference (CA − CO) frequency regions—greatly facilitates the analysis of the spectrum by resolving the problems (associated with routine assignment strategies) arising because of degenerate amide 15N and backbone 13C chemical shifts. Further, the spectrum provides direct distinction between intra- and inter-residue correlations because of their opposite peak signs. The other beneficial feature of the spectrum is that it provides: (a) multiple unidirectional sequential (ii + 1) 15N and 13C correlations and (b) facile identification of certain specific triplet sequences which serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. On top of all this, the F 2F 3 planes of the spectrum corresponding to sum (CA + CO) and difference (CA − CO) chemical shifts enable rapid and unambiguous identification of sequential HSQC peaks through matching their coordinates in these two planes (see the text). Overall, the experiment presented here will serve as an important backbone assignment tool for variety of structural and functional proteomics and drug discovery research programs by NMR involving well behaved small folded proteins (MW < 15 kDa) or a range of intrinsically disordered proteins.

Keywords

Multidimensional NMR Backbone assignment hncoCANH hnCOcaNH Sequential correlations 

Abbreviations

NMR

Nuclear magnetic resonance

HSQC

Heteronuclear single quantum correlation

CARA

Computer aided resonance assignment

RD

Reduced dimensionality

ADF

Actin depolymerizing factor

Notes

Acknowledgement

This work is being financially supported by the Department of Science and Technology under SERC Fast Track Scheme (Registration Number: SR/FT/LS-114/2011) for carrying out the research work. I would also like to acknowledge the High Field NMR Facility at Centre of Biomedical Magnetic Resonance, Lucknow, India.

Supplementary material

10969_2013_9161_MOESM1_ESM.pdf (1.7 mb)
Supplementary material 1 (PDF 1763 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Centre of Biomedical Magnetic Resonance (CBMR)LucknowIndia

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