Role of tryptophyl residues in the binding of gene 32 protein from phage T4 to single-stranded DNA. Photochemical modification of tryptophan by trichloroethanol

Abstract

From previous studies on model peptides and on single-strand binding proteins, aromatic amino acids are expected to play a role in the specific binding of the gene 32 protein from bacteriophage T4 to single-stranded nucleic acids. In this paper, we report the results obtained with gene 32 protein whose tryptophyl residues have been photochemically modified by trichloroethanol. Trichloroethanol is an efficient quencher of the fluorescence emission of indole derivatives and is able to form covalent adducts with tryptophan in its first excited singlet state upon UV irradiation. From W absorption and fluorescence measurements, it is concluded that irradiation of the gene 32 protein from bacteriophage T4 in the presence of trichloroethanol leads to the formation of only one type of photoproduct. This photochemical reaction results in a complete quenching of the protein fluorescence. However, determination of the tryptophan content of the irradiated protein

Fluorescence Quenching Reactions

“…TCE appears to promote a photochemical modification of Trp residues in proteins. This reaction was successfully employed by Toulme et al (1984) to selectively modify certain Trp residues in the gene 32 protein from phage T4. Acrylamide will slowly react with primary amino groups and sulfhydryl groups at high pH (Geisthardt and Kruppa, 1987;Hashimoto and Albridge, 1970;Danileviciute et al, 1981).…”

Fluorescence Quenching: Theory and Applications

“…(204) Of course, a quencher may also potentiate photolysis if the quenching mechanism involves, for example, irreversible electron transfer. (205) Solute quenchers can compete with triplet state decay (see Section 2.7), and some quenchers (i.e., heavy atom quenchers) can promote intersystem crossing and thus can lead to an increase in the population of the triplet state.…”

Involvement of tryptophyl residues in the binding of model peptides and gene 32 protein from phage T4 to single-stranded polynucleotides. A spectroscopic method for detection of tryptophan in the vicinity of nucleic acid bases

“…Cysteinyl residues of the protein have been protected by reaction with There is a class of proteins termed single-strand binding (SSB)1 proteins that bind in a specific manner to singlestranded DNA or polynucleotides as compared to doublestranded ones. These proteins are known to be involved in basic processes of viral DNA metabolism [Héléne et al, 1982; see Toulmé et al (1984) and references cited therein]. The origin of the specificity for this class of proteins is so far not elucidated.…”

“…The origin of the specificity for this class of proteins is so far not elucidated. In previous works (see below) and in the accompanying papers (Toulmé et al, 1984;Casas-Finet et al, 1984), we have tried to shed some light on the possible role of aromatic residues in the binding specificity of SSB proteins. Aromatic residues (tyrosine and tryptophan) were shown to be involved in the binding of oligopeptides to single-stranded nucleic acids through stacking interactions with nucleic acid bases [for a review, see Héléne & Maurizot (1981)].…”

“…Aromatic residues (tyrosine and tryptophan) were shown to be involved in the binding of oligopeptides to single-stranded nucleic acids through stacking interactions with nucleic acid bases [for a review, see Héléne & Maurizot (1981)]. Tryptophyl residues were also found to be involved in the binding of the gene 32 protein (gp 32), a SSB protein from phage T4, onto single-stranded polynucleotides (Héléne et al, 1976;Toulmé et al, 1984). The methods used in these works provided only indirect evidence for the presence of tryptophyl residues in the binding site of the SSB protein-DNA complexes.…”