Comparison of cAMP receptor protein (CRP) and a cAMP-independent form of CRP by Raman spectroscopy and DNA binding.
The secondary structures of the cAMP receptor protein (CRP), a complex of CRP and cAMP, and a cAMP-independent receptor protein mutant (CRP*141 gln) were examined by using Raman spectroscopy. Spectra were obtained from CRP and CRP*141 gln dissolved in 0.3 M NaCl and 30 mM sodium phosphate at protein concentrations of 30-40 mg/mL. CRP and CRP.cAMP1 were compared at lower protein concentrations (10-12 mg/mL) in a solvent of 0.35 M NaCl and 20 mM sodium phosphate. Raman analysis indicates that CRP structural changes induced by one bound cAMP or by the Gly to Gln mutation at residue 141 are small. Spectra of the three CRP samples are essentially identical from 400 to 1900 cm-1. This result differs from the Raman spectroscopy study of CRP and CRP.cAMP2 cocrystals [DeGrazia et al. (1990) Biochemistry 29, 3557]. The latter work showed spectral differences between CRP and CRP.cAMP2 consistent with alterations in the protein conformation. These studies indicate that CRP and CRP.cAMP1 in solution are similar in structure and differ from CRP.cAMP2 cocrystals. Protease digestion and a DNA binding assay were also employed to characterize the wild-type and mutant proteins. CRP*141 gln exhibited the same conformational characteristics of previously reported cAMP-independent mutant proteins. It was sensitive to proteolytic attack in the absence of cAMP, or upon addition of cGMP. In the absence of cAMP, both wild-type and mutant CRPs bound noncooperatively to a 62 bp lac promoter DNA. The equilibrium constants were approximately 10(6) M-1 in 0.1 M Na+. CRP*141 gln had a 2-4-fold higher affinity for the 62 bp DNA than CRP.(ABSTRACT TRUNCATED AT 250 WORDS)
Tan
G S
GS
School of Biology, Georgia Institute of Technology, Atlanta 30332.
Kelly
P
P
Kim
J
J
Wartell
R M
RM
D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D000242:Cyclic AMP