[(13)C,(13)C]- and [(13)C,(1)H]-TROSY in a triple resonance experiment for ribose-base and intrabase correlations in nucleic acids.
A novel TROSY (transverse relaxation-optimized spectroscopy) element is introduced that exploits cross-correlation effects between (13)C-(13)C dipole-dipole (DD) coupling and (13)C chemical shift anisotropy (CSA) of aromatic ring carbons. Although these (13)C-(13)C effects are smaller than the previously described [(13)C,(1)H]-TROSY effects for aromatic (13)C-(1)H moieties, their constructive use resulted in further transverse relaxation-optimization by up to 15% for the resonances in a 17 kDa protein-DNA complex. As a practical application, two- and three-dimensional versions of the HCN triple resonance experiment for obtaining ribose-base and intrabase correlations in the nucleotides of DNA and RNA (Sklenar, V.; Peterson, R. D.; Rejante, M. R.; Feigon, J. J. Biomol. NMR 1993, 3, 721-727) have been implemented with [(13)C,(1)H]- and [(13)C,(13)C]-TROSY elements to reduce the rate of transverse relaxation during the polarization transfers between ribose (13)C1' and base (15)N1/9 spins, and between (13)C6/8 and N1/9 within the bases. The resulting TROSY-HCN experiment is user-friendly, with a straightforward, robust experimental setup. Compared to the best previous implementations of the HCN experiment, 2-fold and 5-fold sensitivity enhancements have been achieved for ribose-base and intrabase connectivities, respectively, for (13)C,(15)N-labeled nucleotides in structures with molecular weights of 10 and 17 kDa. TROSY-HCN experiments should be applicable also with significantly larger molecular weights. By using modified TROSY-HCN schemes, the origins of the sensitivity gains have been analyzed.
Riek
R
R
Institute für Molekularbiologie and Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland.
Pervushin
K
K
Fernández
C
C
Kainosho
M
M
Wüthrich
K
K
D000818:Animals; D051943:Antennapedia Homeodomain Protein; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism; D008280:Magnetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins; D009696:Nucleic Acids / Q000737:chemistry*; D014157:Transcription Factors
D051943:Antennapedia Homeodomain Protein; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D009696:Nucleic Acids; D014157:Transcription Factors; D004247:DNA