Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins |
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Authors: | Frank Löhr Vicky Katsemi Marco Betz Judith Hartleib Heinz Rüterjans |
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Affiliation: | (1) Institut für Biophysikalische Chemie, Johann Wolfgang Goethe-Universität Frankfurt am Main, Biozentrum N230, 1. OG, Marie Curie-Strasse 9, D-60439 Frankfurt, Germany |
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Abstract: | Methods are described to correlate aromatic 1H 2/13C 2 or 1H 1/15N 1 with aliphatic 13C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [15N, 1H]- and/or [13C, 1H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [15N, 1H]-TROSY-H(N)Car experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups. |
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Keywords: | aromatic resonance assignment DFPase perdeuteration triple-resonance NMR TROSY xylanase |
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