Triple-resonance methods for complete resonance assignment of aromatic protons and directly bound heteronuclei in histidine and tryptophan residues.
A set of three experiments is described which correlate aromatic resonances of histidine and tryptophan residues with amide resonances in 13C/15N-labelled proteins. Provided that backbone 1H and 15N positions of the sequentially following residues are known, this results in sequence-specific assignment of histidine 1H(delta2)/13C(delta2) and 1H(epsilon1)/13C(epsilon1) as well as tryptophan 1H(delta1)/13C(delta1), 1H(zeta2)/13C(zeta2), 1H(eta2)/13C(eta2), 1H(epsilon3)/13C(epsilon3), 1H(zeta3)/13C(zeta3) and 1H(epsilon1)/15N(epsilon1) chemical shifts. In the reverse situation, these residues can be located in the 1H-(15)N correlation map to facilitate backbone assignments. It may be chosen between selective versions for either of the two amino acid types or simultaneous detection of both with complete discrimination against phenylalanine or tyrosine residues in each case. The linkages between delta-proton/carbon and the remaining aromatic as well as backbone resonances do not rely on through-space interactions, which may be ambiguous, but exclusively employ one-bond scalar couplings for magnetization transfer instead. Knowledge of these aromatic chemical shifts is the prerequisite for the analysis of NOESY spectra, the study of protein-ligand interactions involving histidine and tryptophan residues and the monitoring of imidazole protonation states during pH titrations. The new methods are demonstrated with five different proteins with molecular weights ranging from 11 to 28 kDa.
Löhr
Frank
F
Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Frankfurt am Main, Biozentrum N230, 1. OG, Marie Curie-Strasse 9, D-60439, Frankfurt, Germany.
Rogov
Vladimir V
VV
Shi
Meichen
M
Bernhard
Frank
F
Dötsch
Volker
V
D000577:Amides; D024322:Amino Acids, Aromatic / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology; D002247:Carbon Isotopes; D018797:Cell Cycle Proteins / Q000737:chemistry; D016969:Desulfovibrio vulgaris; D003903:Deuterium; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D005418:Flavodoxin / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry; D011494:Protein Kinases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry; D006163:Ribonuclease T1 / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
D000577:Amides; D024322:Amino Acids, Aromatic; D002247:Carbon Isotopes; D018797:Cell Cycle Proteins; D029968:Escherichia coli Proteins; D005418:Flavodoxin; D009097:Multienzyme Complexes; D009587:Nitrogen Isotopes; D011506:Proteins; D011522:Protons; D011994:Recombinant Proteins; C489728:rcsC protein, E coli; D006639:Histidine; D014364:Tryptophan; D003903:Deuterium; D011494:Protein Kinases; D006163:Ribonuclease T1; D010749:Phosphoprotein Phosphatases; D043364:Endo-1,4-beta Xylanases