Isotope‐edited UV Raman spectroscopy of protein–DNA interactions: binding modes of cyclic AMP receptor protein to a natural DNA recognition site

Abstract

AbstractIsotope‐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 (ν6/ν6′) 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 ν6/ν6′ 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. Copyright © 2005 John Wiley & Sons, Ltd.