Near ultraviolet circular dichroism study of the cyclic AMP receptor protein, its NH2-terminal domain and their interaction with cyclic AMP.
Circular dichroism in the near ultraviolet wavelength range was employed to examine conformational features of CRP (a dimer with a chain of 209 amino acids) and of its subtilisin core -alpha CRP- which retains the cAMP binding site (a dimer spanning the sequence 1-117). Binding of the ligand cAMP (allosteric activator), as well as cGMP was also investigated. The well resolved transitions could be assigned to the various classes of aromatic amino acid residues in the two proteins. In addition to signals which are attributable to the missing aromatic residues (Phe-136 and Tyr-206) the difference spectrum (CRP minus alpha CRP) shows a significant perturbation of a tryptophanyl contribution centred at 296 nm. From the available X-ray structure of the cAMP-CRP complex we are led to conclude that a conformational reorganisation takes place in the alpha CRP. A very large negative maximum is observed at 255 nm when cAMP binds to CRP and to alpha CRP. The maximum effect is observed in both cases at a ratio of one ligand bound per protomer. In the 280-300 nm wavelength range a smaller but significant perturbation affects specifically the spectra and reveals different cAMP-induced conformational changes in the two proteins. We propose that the major (255 nm) contribution to the perturbation spectrum of bound cAMP, and the qualitatively similar signal for cGMP, reflects an immobilisation of the sugar and adenine moieties of the bound ligand in an almost anti-conformation for both CRP and alpha CRP.
Blazy
B
B
Centre de Biophysique Moléculaire, Orléans, France.
Baudras
A
A
Maurizot
J C
JC
D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000737:chemistry; D006152:Cyclic GMP / Q000737:chemistry; D004926:Escherichia coli; D011487:Protein Conformation; D011953:Receptors, Cyclic AMP / Q000737:chemistry / Q000648:ultrastructure*; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D006152:Cyclic GMP