Multiplet-filtered and gradient-selected zero-quantum TROSY experiments for 13C1H3 methyl groups in proteins
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Multiplet-filtered and gradient-selected heteronuclear zero-quantum coherence (gsHZQC) TROSY experiments are described for measuring 1H–13C correlations for 13CH3 methyl groups in proteins. These experiments provide improved suppression of undesirable, broad outer components of the heteronuclear zero-quantum multiplet in medium-sized proteins, or in flexible sites of larger proteins, compared to previously described HZQC sequences (Tugarinov et al. in J Am Chem Soc 126:4921–4925, 2004; Ollerenshaw et al. in J Biomol NMR 33:25–41, 2005). Hahn-echo versions of the gsHZQC experiment also are described for measuring zero- and double-quantum transverse relaxation rate constants for identification of chemical exchange broadening. Application of the proposed pulse sequences to Escherichia coli ribonuclease HI, with a molecular mass of 18 kD, indicates that improved multiplet suppression is obtained without substantial loss of sensitivity.
KeywordsMethyl TROSY Zero-quantum HZQC HMQC RNase H
A.G.P. and M.L.G. acknowledge support from National Institute of Health grants GM50291 and GM089047, respectively. A.G.P. is a member of the New York Structural Biology Center (NYSBC). The NYSBC is a STAR center supported by the New York State Office of Science, Technology and Academic Research. Data acquired at 18.8 and 21.1 T were collected at the NYSBC. The 900 MHz (21.1 T) spectrometers were purchased with funds from the National Institute of Health (USA), the Keck Foundation (New York State), and the NYC Economic Development Corporation. A.G.P. and M.L.G. thank Mark Rance (University of Cincinnati) and Daròn Freedberg (CBER/FDA) for helpful scientific discussions.
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