Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins.
Methods are described to correlate aromatic 1H(delta)2/13C(delta)2 or 1H(epsilon)1/15N(epsilon)1 with aliphatic 13C(beta) 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 beta-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 diisopropyl-fluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine delta-CH and tryptophan epsilon-NH groups.
Löhr
Frank
F
Institut für Biophysikalische Chemie, Johann Wolfgang Goethe-Universität Frankfurt am Main, Germany. murph@bpc.uni-frankfurt.de
Katsemi
Vicky
V
Betz
Marco
M
Hartleib
Judith
J
Rüterjans
Heinz
H
D000595:Amino Acid Sequence; D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry; D002247:Carbon Isotopes; D003903:Deuterium; D004950:Esterases / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D008280:Magnetics; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D044345:Phosphoric Triester Hydrolases; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D014364:Tryptophan / Q000737:chemistry*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D011522:Protons; D006639:Histidine; D014364:Tryptophan; D003903:Deuterium; D004950:Esterases; D044345:Phosphoric Triester Hydrolases; C020061:diisopropyl-fluorophosphatase; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase