Isotope-edited UV Raman spectroscopy was applied to the study of the binding mode of cyclic AMP (cAMP) receptor protein (CRP) to a 22-mer oligonucleotide (LacDNA) representing the primary CRP binding site of the E. coli lac promoter. LacDNA contains four guanine residues in the consensus pentamer regions (G5 and G7 on the sense strand and G5 and G7 on the anti-sense strand) and they were individually labeled with deuterium at C(8) on the guanine ring. In the UV (251 nm) Raman difference spectrum between unlabeled and C(8)-D-labeled LacDNA, a sharp positive/negative peak pair appeared at ∼1490/1465 cm −1 , which was assigned to a guanine ring vibration (n 6 /n 6 ) sensitive to the hydrogen bonding state at C(6) O. CRP is a dimeric protein and forms two complexes with the cofactor cAMP, i.e. half-filled CRP-(cAMP) 1 and fully liganded CRP-(cAMP) 2 . The n 6 /n 6 wavenumbers measured in the presence of CRP showed that CRP-(cAMP) 1 binds to G5 and G5 in a symmetric manner, whereas CRP-(cAMP) 2 exhibits an additional binding to G7. Since the base sequence of LacDNA and the structure of CRP-(cAMP) 1 are both asymmetric, the symmetric LacDNA-CRP-(cAMP) 1 interaction suggests that CRP-(cAMP) 1 , which is considered to be dominant under physiological conditions, has a conformational flexibility to conform to structural asymmetry of natural DNA sequences. In contrast, the symmetric complex CRP-(cAMP) 2 binds to LacDNA asymmetrically, suggesting a decreased flexibility of CRP in the fully liganded form. Isotope-edited UV Raman spectroscopy provides unique information on the DNA recognition by CRP.
Isotope-edited Raman spectroscopy, a combination of site-selective isotopic labeling and Raman difference spectroscopy, is a useful method for studying the structure and interaction of individual nucleic acid residues in oligonucleotides. To obtain basic data for applying isotope-edited Raman spectroscopy to guanine residues, we studied the vibrational modes of UV resonance Raman bands of the C8-deuterated guanine ring by examining the wavenumber shifts upon seven isotopic substitutions (2-13 C, 2-15 N, 6-18 O, 7-15 N, 8-13 C, 9-15 N and 1 -13 C). The hydrogen bond sensitivities of the Raman bands were also investigated by comparing the Raman spectra recorded in several solvents of different hydrogen bonding properties. Some of the Raman bands were found to be markers of hydrogen bonding at specific donor or acceptor sites on the guanine ring. The Raman bands, which shift on C8-deuteration, remain in the difference spectrum between the unlabeled and C8-deuterated guanine rings. Among them, a negative peak around 1525 cm −1 and a strong positive/negative peak pair around 1485/1465 cm −1 serve as markers of hydrogen bonding at N7 and C6 O, respectively. Another weak positive/negative peak pair around 1025/1040 cm −1 is sensitive to hydrogen bonding at the proton donor sites (N1 -H and N2 -H 2 ). The applicability of the hydrogen bond markers has been tested by using a 22-mer oligonucleotide duplex containing eight guanine residues and its analog in which a single guanine residue is C8-deuterated. The difference spectrum shows that the hydrogen bonding state of the guanine residue at the labeled position is consistent with the Watson-Crick base pair structure of DNA. Isotope-edited Raman spectroscopy is a useful tool for studying the hydrogen bonding state of selected guanine residues in oligonucleotides.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.