Investigation of the cAMP receptor protein secondary structure by Raman spectroscopy.
Raman spectroscopy was employed to examine the secondary structure of the cAMP receptor protein (CRP). Spectra were obtained over the range 400-1900 cm-1 from solutions of CRP and from CRP-cAMP cocrystals. The spectra of CRP dissolved in 30 mM sodium phosphate and 0.15 M NaCl buffered at either pH 6 or pH 8 or dissolved in 0.15-0.2 M NaCl at protein concentrations of 5, 15, and 30 mg/mL were examined. Estimates of the secondary structure distribution were made by analyzing the amide I region of the spectra (1630-1700 cm-1). CRP secondary structure distributions were essentially the same in either pH and at all protein concentrations examined. The amide I analyses indicated a structural distribution of 44% alpha-helix, 28% beta-strand, 18% turn, and 10% undefined for CRP in solution. Raman spectra of CRP-cAMP cocrystals differed from the spectra of CRP in solution. Some differences were assigned to interfering background bands, whereas other spectral differences were attributed to changes in CRP structure. Differences in the amide III region and in the intensity at 935 cm-1 were consistent with alterations in secondary structure. Analysis of the amide I region of the CRP-cAMP cocrystal spectrum indicated a secondary structure distribution of 37% alpha-helix, 33% beta-strand, 17% turn, and 12% undefined. This result is in agreement with a published secondary structure distribution derived from X-ray analysis of CRP-cAMP cocrystals (37% alpha-helix and 36% beta-strand).(ABSTRACT TRUNCATED AT 250 WORDS)
DeGrazia
H
H
School of Physics, Georgia Institute of Technology, Atlanta 30332.
Harman
J G
JG
Tan
G S
GS
Wartell
R M
RM
D000577:Amides; D000242:Cyclic AMP / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D011953:Receptors, Cyclic AMP / Q000235:genetics / Q000302:isolation & purification; D013059:Spectrum Analysis, Raman / Q000379:methods
D000577:Amides; D008024:Ligands; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP