RAMAN-SPECTROSCOPY OF THE FF GENE V-PROTEIN AND COMPLEXES WITH POLY(DA) - NONSPECIFIC DNA RECOGNITION AND BINDING

Raman spectra of crystals and solutions of the single-stranded DNA bin ding protein of bacteriophage Ff (gene V protein, gVp) and of solution complexes of gVp with single-stranded poly(deoxyadenylic acid) [poly( dA)] reveal the following: (i) The gVp secondary and tertiary structur es are similar in solution and in the crystal and are dominated by bet a-sheet domains, in agreement with NMR and X-ray findings, (ii) Subuni t conformation and side chain environments of gVp are virtually unchan ged over a wide range of salt concentration (0 < [NaCl] < 100 mM); how ever, the solution conformation of poly(dA) exhibits sensitivity to ad ded salt. The perturbed Raman markers indicate subtle changes in helix backbone geometry with accompanying small differences in base stackin g as the concentration of NaCl is changed. (iii) In complexes with pol y(dA), neither the conformation of gVp nor its side chain environments are altered significantly in comparison to the free protein, This is the case at both high salt (nucleotide-to-subunit binding stoichiometr y n = 4) and low salt (n = 3). (iv) The Raman signature of poly(dA) un dergoes small perturbations upon gVp binding, indicative of small chan ges in base stacking and phosphodiester backbone conformation. The pre sent results show that the different stoichiometric binding modes of g Vp to poly(dA) are accomplished without significant changes in gVp sub unit structure and with only modest changes in the single-stranded pol y(dA) ligand. This contrasts sharply with sequence-specific double-str anded DNA binding proteins, such as the phage lambda and D108 represso rs, which undergo substantial structural changes upon DNA binding, and which also alter more dramatically the Raman fingerprints of their DN A target sites. Thus, nonspecific and specific nucleic acid recognitio n modes are distinguishable by Raman spectroscopy. The Raman signature of gVp also allows examination of hydrogen bonding interactions of un ique side chains within the hydrophobic core (cysteine 33) and at the binding interface (tyrosine 41). These are discussed in relation to th e recently published gVp crystal structure.