Double-resonance experiments at 500 MHz on gene-5 protein and its complex with octadeoxyriboadenylic acid

Abstract

In this paper, a detailed description is presented of the aromatic part of the 500-MHZ 1H nuclear magnetic resonance (NMR) spectrum of the helix-destabilizing gene-5 protein (GVP) encoded by the coliphage M13. As a result of the resolution obtained at 500 MHZ, it was possible to perform selective decoupling and time-resolved selective Overhauser experiments. The magnitudes of the observed Overhauser effects compare favorably with magnitudes expected on the basis of theoretical calculations. These experiments in conjunction with selective decoupling experiments allowed a detailed interpretation of the aromatic part of the protein spectrum. The spectrum of the aromatic part of the GVP-d(A)8 complex could be interpreted in a similar fashion. The ring protons of one phenylalanyl residue and of two tyrosyl residues show rather large shifts upon complex formation. This indicates that these residues are involved in the interaction with the DNA molecule in accordance with earlier observations. Direct evidence for the proximity of these aromatic rings and the DNA fragment in the complex was obtained by additional Overhauser experiments. It turns out that the H3',H4', and/or the H5' sugar protons of the oligonucleotide are situated near the ring protons of (most likely) two or all three of the aromatic residues of which the resonances undergo large shifts upon complex formation.

Influence of N6‐methylation of residue A(5) on the conformational behaviour of d(C‐C‐G‐A‐A‐T‐T‐C‐G‐G) in solution studied by 1H‐NMR spectroscopy

“…Exact temperatures were determined from the chemical shift value of the water peak relative to Me4NC1 as described earlier [l I] '. One-dimensional selective-saturation transfer experiments were carried out at 300 MHz: a l-s selective saturation pulse was employed in the one-dimensional NOEdifference pulse sequence proposed by Alma et al [12].…”

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

“…The isolation and purification of the gene-5 protein were essentially as in [12] in the aromatic part of the spectrum (8.1-6.4 ppm downfield from DSS) to the protons of the single histidyl the 5 tyrosyl and the 3 phenylalanyl residues in the protein [ 10]. The resonances of 2 tyrosyl and 1 phenylalanyl residues also undergo large shifts upon binding of oligonucleotides to the protein [10,11 ] and ~>2 of these residues are located close to the DNA, in the protein-DNA complex.…”

“…There is a wealth of spectroscopic evidence that aromatic residues are involved in the interaction with DNA [4,8,9]. The molecular surroundings of 2 tyrosines and 1 phenylalanine are influenced by binding of DNA [10,11]. This phenylalanine and i>1 of these tyrosines are located in close proximity to the DNA [10].…”

“…The molecular surroundings of 2 tyrosines and 1 phenylalanine are influenced by binding of DNA [10,11]. This phenylalanine and i>1 of these tyrosines are located in close proximity to the DNA [10]. However, the interaction of gene-5 protein with DNA is strongly dependent on ionic strength [1].…”

Conformational analysis of the dinucleotides 5′‐methylphospho‐N6‐dimethyladenylyl‐uridine (mpm62A‐U) and 5′methylphospho‐uridylyl‐N6‐dimethyladenosine (mpU‐m62A) and of the trinucleotide U‐m62A‐U

“…The timedomain signals were apodized with Gaussian windows, zerofilled to 32 K and Fourier transformed. 500-MHz NOE difference spectra were recorded at 0°C following Alma et al [15]. A selective preirradiation pulse of 0.5 s was used.…”