A nuclear magnetic resonance study of the cyclic AMP receptor protein (CRP): assignments of the NH protons of histidine and tryptophan residues and the effect of binding of cAMP to CRP.
Seven well-resolved signals could be observed in the field lower than 9.5 ppm in the 1H-NMR spectrum of the H2O solution of cyclic AMP receptor protein (CRP). The signals of the tryptophan and histidine residues were identified on the basis of the CPMG spin echo spectra, the intra-residue NOE, 15N labeling, deuterium labeling, and the results of pH titration. The assignments of peaks to specific tryptophan and histidine residues are discussed in relation to the amino acid sequence and X-ray crystallographic data, and were confirmed by experiments involving partial subtilisin digestion. The four signals E (11.25 ppm), F (11.15 ppm), G (10.75 ppm), and H (10.65 ppm) were tentatively assigned to the resonances of the histidine residue at position 159, the arginine residue at position 82, and the tryptophan residues at positions 85 and 13, respectively. On the addition of cAMP and cGMP, signals F and G shifted up- and downfield respectively and conformational changes in the structure of CRP could be detected. The conformational transition mostly occurs when one cAMP molecule binds to one of the dimer subunits, but is completed only when both cAMP binding sites are saturated.
Lee
B J
BJ
Institute for Protein Research, Osaka University.
Lee
S J
SJ
Hayashi
F
F
Aiba
H
H
Kyogoku
Y
Y
D002352:Carrier Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein; D006152:Cyclic GMP / Q000378:metabolism; D003903:Deuterium; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011487:Protein Conformation; D011522:Protons; D011953:Receptors, Cyclic AMP; D013057:Spectrum Analysis; D014364:Tryptophan
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D011522:Protons; D011953:Receptors, Cyclic AMP; D006639:Histidine; D014364:Tryptophan; D003903:Deuterium; D000242:Cyclic AMP; D006152:Cyclic GMP