Nuclear magnetic resonance study on the structure and interaction of cyclic AMP receptor protein and its mutants: a deuterium-labeling and photo-CIDNP study.
The identification and assignment of the proton magnetic resonances of some aliphatic and aromatic amino acid residues of cyclic AMP receptor protein (CRP) are reported. The signals of the leucine and valine residues at around 0 ppm were identified on the basis of intermolecular nuclear Overhauser effects, deuterium labeling, and partial proteolytic digestion. On the addition of cAMP, methyl proton signals due to Val-49 and three leucine residues were detected as upfield-shifted signals at around -0.2 ppm. These signals can be used as indicators of the proper binding of cAMP because they are not observed on the addition of cGMP or 2'-deoxy-cAMP. They are also not observed on cAMP binding to mutant CRP*5 (Ser-62-Phe), which can only be activated by a high concentration of cAMP, but they are observed on cAMP binding to other mutant CRP*s (four species), which can be activated by lower concentrations of cAMP. The resonance of some aromatic protons, i.e., C-2H of two tryptophans, C-2H and C-4H of six histidines, and C-2,6H and C-3,5H of six tyrosine residues in CRP, were assigned by means of deuterium labeling and NOE measurements. The 1H NMR spectrum of labeled CRP [Trp(ring-d5), Phe(ring-d5), and Tyr(3,5-d2)] showed good resolution in the aromatic region. The addition of cAMP to this CRP in D2O caused pronounced line broadening of resonances arising from the residues in the cAMP-binding domain, but the resonances of the DNA-binding domain were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
Lee
B J
BJ
Institute for Protein Research, Osaka University, Japan.
Aiba
H
H
Kyogoku
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D006152:Cyclic GMP / Q000737:chemistry / Q000494:pharmacology; D003903:Deuterium; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D010777:Photochemistry; D011485:Protein Binding; D011487:Protein Conformation; D011953:Receptors, Cyclic AMP / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship
D011953:Receptors, Cyclic AMP; C018892:2'-deoxy cyclic AMP; D003903:Deuterium; D000242:Cyclic AMP; D006152:Cyclic GMP