Jose R. Casas-Finet

Tryptophan 54 and phenylalanine 60 are involved synergistically in the binding of E. coli SSB protein to single-stranded polynucleotides

The binding of both wild‐type and point‐mutated E. coli single‐stranded DNA‐binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy. Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies. Investigation of a point mutation in the E. coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe‐60 is replaced by alanine strongly suggests the participation of Phe‐60 in the binding process, possibly by the formation of an extended stacking structure by Trp‐54, thymine and Phe‐60. This hypothesis is supported by results on the point mutations in which His‐55 is replaced by either leucine or tyrosine.

Role of tryptophan 54 in the binding of E. coli single-stranded DNA-binding protein to single-stranded polynucleotides

Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single‐stranded polynucleotides with both E. coli single‐stranded DNA‐binding protein and an E. coli ssb gene product in which Trp‐54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp‐54 in stabilizing the protein‐nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.

Triplet state properties of tryptophan residues in complexes of mutated Escherichia coli single-stranded DNA binding proteins with single-stranded polynucleotides

Complexes of point-mutated E. coli single-stranded DNA-binding protein (Eco SSB) with homopolynucleotides have been investigated by optical detection of magnetic resonance (ODMR) of the triplet state of tryptophan (Trp) residues. Investigation of the individual sublevel kinetics of the lowest triplet state of Trp residues 40 and 54 in the poly (dT) complex of Eco SSB-W88F,W135F (a mutant protein whose Trp residues at positions 88 and 135 have been substituted by Phe) shows that Trp 54 is the most affected residue upon stacking with thymine bases, confirming previous results based on SSB mutants having single Trp----Phe substitutions. (Zang, L. H., A. H. Maki, J. B. Murphy, and J. W. Chase. 1987. Biophys. J. 52:867-872). The Tx sublevel of Trp 54 shows a fourfold increase in the decay rate constant, as well as an increase in its populating rate constant by selective spin-orbit coupling. The two nonradiative sublevels show no change in lifetime, relative to unstacked Trp. For Trp 40, a weaker perturbation of Tx by thymine results in a sublevel lifetime about one-half that of normal Trp. Trp54 displays a 2[E]transition of negative polarity in the double mutant SSB complex with Poly (dT), but gives a vanishingly weak [D] - [E] signal, thus implying that the steady-state sublevel populations of Tx and Tz are nearly equal in this residue. Poly (5-BrU) induces the largest red-shift of the Eco SSB-W88F,W135F Trp phosphorescence 0,0-band of all polynucleotides investigated. Its phosphorescence decay fits well to two exponential components of 1.02 and 0.12 s, with no contribution from long-lived Trp residues. This behavior provides convincing evidence that both Trp 40 and 54 are perturbed by stacking with brominated uridine. The observed decrease in the Trp [D] values further confirms the stacking of the Trp residues with 5-BrU. Wave-length-selected ODMR experiments conducted on the [D[ + [E] transition of Eco SSB-W88F,W135F complexed with poly(5HgU) indicate the presence of multiple heavy atom-perturbed sites. Measurements made on poly (5-HgU) which each of its 4 Trp residues has been replaced in turn by Phe demonstrate that Trp 40 and 54 are the only Trp residues undergoing stacking with nucleotide bases, as previously proposed.

Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy

The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found.

Evidence for stacking interactions between 5-mercurated polyuridylic acid and HIV-1 p7 nucleocapsid protein obtained by phosphorescence and optically detected magnetic resonance (ODMR)

The photoexcited triplet state of Trp‐37 in the C‐terminal zinc finger of the HIV‐1 p7 nucleocapsid protein was used as a probe of p7 interactions with the heavy atom‐derivatized RNA homopolymer, poly‐5‐mercuriuridylic acid (5‐HgU). Binding of p7 to 5‐HgU (Hg blocked with 2‐mercaptoethanol) produces an external heavy atom effect (HAE) on Trp‐37 characterized by fluorescence quenching, reduction of the phosphorescence lifetime by three orders of magnitude, and the appearance of the D + E phosphorescence‐detected ODMR signal, absent in unperturbed Trp, but induced by a HAE. The details of the HAE are consistent with out‐of‐plane van der Waals contact of Hg with the indole chromophore of Trp‐37. Steric requirements suggest further that the Trp‐RNA contact occurs via an aromatic stacking interaction.

Binding of recA protein to single- and double-stranded polynucleotides occurs without involvement of its aromatic residues in stacking interactions with nucleotide bases

Phosphorescence and optically detected triplet state magnetic resonance (ODMR) spectroscopy studies of recA protein and its complexes with poly(5-HgU) and poly(dA-5BrdU) show that the two tryptophan residues are not involved in stacking interactions with the nucleotide bases of either single- or double-stranded polynucleotides. Solvent conditions which induce preferential binding to single-stranded ligands result in a shortening of the tyrosine phosphorescence lifetime, which is further reduced upon binding to poly(5-HgU). This suggests a change in the global conformation or self-aggregation state of the protein. Binding to poly(dA-5BrdU) induces small changes in the tryptophan zero field splittings of recA, but significant changes on those of 5BrdU, which are consistent with recA binding to the minor groove of the polynucleotide.

Characterization of a 32-Residue Peptide From Rat DNA Polymerase β With Single-Stranded DNA-Binding Affinity

Publisher Summary This chapter describes the characterization of a 32-residue peptide from rat DNA polymerase β with single-stranded DNA-binding affinity. This small peptide appears to possess most of the nucleic-acid-binding activity of the larger subdomain. In a study described in the chapter, it was purified by affinity chromatography on ssDNA-cellulose and, like its precursor and its circular dichroism spectrum, indicated a significant degree of α-helical structure. The nucleic-acid interactive properties of the 3-kDa peptide are similar to those of the 8-kDa species; their salt dependence of oligonucleotide binding was found to be comparable. In 10 mM sodium phosphate, pH 7.5, about 80% of the binding free energy of the larger subdomain is accounted for by the smaller fragment.