Isolation and characterization of a cyclic AMP receptor protein from luminous Vibrio harveyi cells.

A cAMP receptor protein (CRP) species was purified from the luminous Vibrio harveyi cells to apparent homogeneity. This protein had a dimeric structure with a molecular weight of 23,000 per subunit. Among all eight nucleotides tested, only cAMP (Kd = 3 to 4 microM at 0 degrees C and 52 microM at 23 degrees C) and cGMP (Kd = 6 to 10 microM at 0 degrees C and 67 microM at 23 degrees C) bound to this protein. Its binding to poly(dI-dC), poly(dA-dT), and DNA fragments isolated from V. harveyi cells were all significantly enhanced by the addition of cAMP. Based on patterns of limited proteolysis by trypsin, this CRP assumes different conformations in the absence and presence of cAMP. Also consistent with this conclusion is the finding that the binding of cAMP to CRP induced about 50% quenching of the CRP fluorescence with a concomitant 3-nm blue shift from the original 336-nm emission peak. The binding of cGMP resulted in similar fluorescence changes but had no apparent effect on the pattern of proteolysis by trypsin. Using an in vitro transcription system known to be dependent on cAMP and Escherichia coli CRP, the synthesis of a run-off transcript product was also significantly enhanced by cAMP and this V. harveyi CRP.