Chemical modifications of functional residues of fd gene 5 DNA-binding protein

Abstract

The binding of gene 5 protein from bacteriophage fd to poly[d(A-T)], fdDNA, and poly(A) is accompanied by a dramatic reversal in the signs of the large ellipticity bands of the nucleic acid chromophores from 250 to 290 nm. The change in the circular dichroism of the DNA induced by the protein, which reaches a maximum at a protein to nucleotide molar ratio of 1:4, has been used as an assay of the alterations in binding of gene 5 protein to DNA accompanying changes in the ionic environment and subsequent to chemical modification of the protein. Divalent cations completely dissociate the gene 5 protein-fd DNA complex at 0.1 M, while 0.5 M monovalent cations are required. All cations are more effective in dissociating the complex with poly[d(A-T)] commensurate with the accompanying stabilization of the double helix to which gene 5 protein does not bind. Acetylation of all six lysyl residues and three of the five tyrosyl residues of the protein with N-acetylimidazole prevents complex formation. Removal of the three tyrosyl O-acetyl groups with hydroxylamine does not restore the binding of gene 5 protein to DNA. Tetranitromethane nitrates the same three tyrosyl residues (Tyr-26, Tyr-41, and Tyr-56 as determined by peptide mapping) and reduces the binding affinity of the protein for fd DNA by similar to 100-fold. The 19F NMR spectrum of gene 5 protein labeled with m-fluorotyrosine shows three surface and two buried fluorotyrosyl residues. All tyrosyl residues are protected from nitration in the complex with fd DNA, but acetylimidazole acetylates surface lysyl residues in the complex and dissociates it. The intrinsic circular dichroism of the acetylated and nitrated gene 5 proteins is not significantly altered. In contrast maleic anhydride reacts with the seven amino groups of the protein and changes the secondary structure to one similar to that present in 6 M guanidine-HCl. The single SH group of the native protein does not react with Ellman's reagent, but it reacts rapidly with one Hg2+ ion which unfolds the protein; fd DNA prevents reaction with Hg2+. Electrostatic forces may be as important as hydrogen bonding in maintaining the native structure of this protein. The lysyl groups of the protein, exposed in both the free protein and the DNA complex, appear to be of prime importance in DNA binding, probably through electrostatic interactions with the DNA binding, probably through electrostatic interactions with the DNA phosphate groups. Three tyrosyl residues also contribute to binding affinity through hydrogen bonding or intercalation. A model of gene 5 protein structure in relation to interactions with a tetranucleotide is presented.

Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein.

“…The interactions of single-stranded-DNA-binding proteins from several bacteriophages, particularly the gene 5 products of bacteriophages Fd, Ike, Pfl, and Pf3 and the gene 32 product of bacteriophage T4, with their substrates have been the subject of considerable study (1,2,7,15,38,41,55). For the Fd and Pfl gene 5 proteins, chemical modification experiments have established that several tyrosine residues and basic residues are essential for binding to DNA (2,41). In addition, a phenylalanine residue can become cross-linked to one of the essential tyrosine residues of the Pfl gene product, suggesting it is also located within the DNA-binding cleft of the protein (41).…”

500 MHz 1H NMR study of the role of lysines and arginines in the binding of gene‐5 protein to oligoadenylic acids

“…From X-ray diffraction data [20] it was concluded that 2 lysyl residues (24 and 26) and 3 arginyl residues (21,80 and 82) may be situated at the inner surface of the DNA binding groove. In [21 ] DNA binding was inhibited upon acetylation of the lysyl residues. The minor shift of the smaller Lys-e triplet upon complexation with d(A)8 is consistent with an 100 MHz IH NMR study [22] where broadening but no detectable shift for the Lys-e proton resonances was found upon binding of lysine containing oligopeptides to poly(A).…”

RNA Structure Inhibits the TRAP ( rp RNA-binding AttenuationProtein)-RNA Interaction

“…For example, stabilization of the hairpin loop of U1A snRNA (39) and the helical region of loop E in 5 S rRNA (34) by Mg 2ϩ greatly enhances binding of the U1A and the L25 proteins, respectively. In contrast, results with TRAP are akin to that observed with single-stranded DNA-binding proteins such as the gene 5 protein from the phage fd, where Mg 2ϩ inhibits binding to DNA (40).…”