The three-dimensional structure determination of RNAs by NMR spectroscopy requires sequential resonance assignment, often hampered by assignment ambiguities and limited dispersion of 1H and 13C chemical shifts, especially of C4â?²/H4â?². Here we present a novel through-bond 4D HPCH NMR experiment involving phosphate backbone where C4â?²â??H4â?² correlations are resolved along the 1H3â?²â??31P spectral planes. The experiment provides high peak resolution and effectively removes ambiguities encountered during assignments. Enhanced peak dispersion is provided by the inclusion of additional 31P and 1H3â?² dimensions and constant-time evolution of chemical shifts. High spectral resolution is obtained by using non-uniform sampling in three indirect dimensions. The experiment fully utilizes the isotopic 13C-labeling with evolution of C4â?² carbons. Band selective 13C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the C4â?²â??C3â?² and C4â?²â??C5â?² homonuclear couplings. Multiple quantum line narrowing is employed to minimize sensitivity loses. The 4D HPCH experiment is verified and successfully applied to a non-coding 34-nt RNA consisting typical structure elements and a 14-nt RNA hairpin capped by cUUCGg tetraloop.
C4â?²/H4â?² selective, non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of 13C-labeled RNAs
C4â?²/H4â?² selective, non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of 13C-labeled RNAs
Abstract
A through bond, C4â?²/H4â?² selective, â??out and stayâ?? type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4â?²(i)â??C4â?²(i)â??C4â?²(iâ??1)â??H4â?²(iâ??1) correlations. The 31P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4â?² dimension. The experiment fully utilizes 13C-labeling of RNA by inclusion of two C4â?² evolution periods. An...
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09-11-2014 12:06 AM
Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments
Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments
Abstract
A fast, robust and reliable strategy for automated sequential resonance assignment for uniformly -labeled RNA via its phosphodiester backbone is presented. It is based on a series of high-dimensional through-bond APSY experiments: a 5D HCP-CCH COSY, a 4D H1â?²C1â?²CH TOCSY for ribose resonances, a 5D HCNCH for ribose-to-base connection, a 4D H6C6C5H5 TOCSY for pyrimidine resonances, and a 4D...
High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins
High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins
Abstract Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using δ subunit (20 kDa) of Bacillus subtilis RNA polymerase...
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02-21-2012 03:40 AM
An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in 15N, 13C labeled proteins
An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in 15N, 13C labeled proteins
Abstract An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely 13Câ?²(i), 15N(i), 1HN(i) connectivities in uniformly 15N/13C-labeled proteins. In comparison to the â??out-and-backâ?? style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the 13Câ?² dimension and on average 1.6 times higher sensitivity especially for residues in α-helices. Performance of the new experiment...
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01-09-2011 12:46 PM
Sparsely-sampled High-resolution 4-D Experiments for Efficient Backbone Resonance Assignment of Disordered Proteins
Sparsely-sampled High-resolution 4-D Experiments for Efficient Backbone Resonance Assignment of Disordered Proteins
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 4 January 2011</br>
Jie, Wen , Jihui, Wu , Pei, Zhou</br>
Intrinsically disordered proteins (IDPs) play important roles in many critical cellular processes. Due to their limited chemical shift dispersion, IDPs often require four pairs of resonance connectivities (H?, C?, C? and CO) for establishing sequential backbone assignment. Because most conventional 4-D...
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01-05-2011 11:03 AM
[NMR paper] A sequential HNCA NMR pulse sequence for protein backbone assignment.
A sequential HNCA NMR pulse sequence for protein backbone assignment.
Related Articles A sequential HNCA NMR pulse sequence for protein backbone assignment.
J Magn Reson. 2001 May;150(1):100-4
Authors: Meissner A, Sørensen OW
The conventional HNCA pulse sequence suffers from the ambiguity that it cannot distinguish inter- and intraresidue correlations because the one-bond and two-bond J(NC(alpha)) coupling constants are of similar magnitude. This paper presents a novel pulse sequence, sequential HNCA, that leads to a spectrum exhibiting...
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11-19-2010 08:32 PM
[NMR paper] Sequential assignment of the backbone nuclei (1H, 15N and 13C) of c-H-ras p21 (1-166)
Sequential assignment of the backbone nuclei (1H, 15N and 13C) of c-H-ras p21 (1-166).GDP using a novel 4D NMR strategy.
Related Articles Sequential assignment of the backbone nuclei (1H, 15N and 13C) of c-H-ras p21 (1-166).GDP using a novel 4D NMR strategy.
J Biomol NMR. 1992 Nov;2(6):639-46
Authors: Campbell-Burk SL, Domaille PJ, Starovasnik MA, Boucher W, Laue ED
The c-H-ras p21 protein is the product of the human ras proto-oncogene, a member of a ubiquitous eukaryotic gene family which is highly conserved in evolution. These proteins play...
High resolution 4D HPCH experiment for sequential assignment of 13 C-labeled RNAs via phosphodiester backbone
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