MQ-hCN-based pulse sequences for the measurement of 13C1'-1H1', 13C1'-15N, 1H1'-15N, 13C1'-13C2', 1H1'-13C2',13C6/8-1H6/8, 13C6/8-15N, 1H6/8-15N, 13C6-13C5, 1H6-13C5 dipolar couplings in 13C, 15N-labeled DNA (and RNA).
A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.
Yan
Jiangli
J
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908-0736, USA.
Corpora
Takeshi
T
Pradhan
Padmanava
P
Bushweller
John H
JH
D000818:Animals; D016880:Anisotropy; D002247:Carbon Isotopes; D050657:Core Binding Factor alpha Subunits; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D008280:Magnetics; D009363:Neoplasm Proteins; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011522:Protons; D012313:RNA / Q000737:chemistry; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D002247:Carbon Isotopes; D050657:Core Binding Factor alpha Subunits; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009587:Nitrogen Isotopes; D011522:Protons; D014157:Transcription Factors; C406688:core binding factor alpha; D012313:RNA; D004247:DNA