Investigation of complexes formed between gene 32 protein from bacteriophage T4 and heavy-atom-modified single-stranded polynucleotides using optical detection of magnetic resonance

• Published in: Biochemistry (Easton) 1986-10, Vol.25 (20), p.5865-5872
• Main Authors: Khamis, Mustafa I, Maki, August H
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; DNA-binding protein; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; gene products; Interactions. Associations; Intermolecular phenomena; Luminescent Measurements; Magnetic Resonance Spectroscopy - methods; Molecular biophysics; optically detected magnetic resonance; phage T4; Poly U - analogs & derivatives; Poly U - metabolism; polynucleotides; Protein Binding; T-Phages - metabolism; Viral Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00368a005

Optical detection of triplet-state magnetic resonance (ODMR) is employed to study the complexes formed between gene 32 protein (GP32), a single-stranded DNA-binding protein from bacteriophage T4, and the heavy-atom-derivatized polynucleotides poly(5-HgU) and poly(5-BrU). The triplet-state properties of some of the tryptophan (Trp) residues in the complexes are dramatically different from those in the free protein, in that they are subject to an external heavy-atom effect. Direct evidence for the presence of a heavy-atom effect, and hence a close-range interaction between mercurated or brominated nucleotide bases and Trp residues in the complex, is provided by the observation of the zero-field (D) + (E) ODMR transition of Trp, which is not normally observed in the absence of a heavy-atom perturbation. The amplitude-modulated phosphorescence-microwave double-resonance (AM-PMDR) technique is employed to selectively capture the phosphorescence spectrum originating from the heavy-atom-perturbed Trp residue(s) in the GP32-poly(5-HgU) complex. Arguments based on our experimental results lead to the conclusion that the heavy-atom perturbation arises from aromatic stacking interactions between Trp and mercurated bases. Wavelength-selected ODMR measurements reveal the existence of two environmentally distinct and spectrally different types of Trp in GP32. One of these types is perturbed selectively by the heavy atom and hence undergoes stacking interactions with the heavy-atom-derivatized bases of the polynucleotide while the second type of Trp residue is unaffected.