James G. Hoggett

Mode of drug binding to DNA determined by optical tweezers force spectroscopy

Abstract Optical tweezers were employed to investigate the effects of small DNA-binding molecules on the low-force (≤ 15 pN) stretching behaviour of single DNA molecules. As the canonical B-DNA helix is not perturbed in this force regime, the effects on DNA elasticity observed upon drug binding provide useful insight into how DNA-binding drugs may alter in vivo processes. In this study, the effects of agents with different DNA binding modes were analysed. DNA force—extension curves were recorded in the presence of netropsin, a purely minor groove-binding antibiotic drug, ethidium bromide, an intercalating fluorescent dye, and berenil, an antiprotozoal drug proposed to exhibit both intercalative and minor groove-binding modes. Applying an approximation of the worm-like chain model, which describes the low-force stretching behaviour, the results were analysed in terms of the DNA contour length and persistence length. From these single molecule studies it was observed that minor groove-binding and intercalating modes of DNA-binding could be distinguished based on changes to DNA elasticity.

Interaction of σ70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance

The interaction between the core form of bacterial RNA polymerases and σ factors is essential for specific promoter recognition, and for coordinating the expression of different sets of genes in response to varying cellular needs. The interaction between Escherichia coli core RNA polymerase and σ70 has been investigated by surface plasmon resonance. The His‐tagged form of σ70 factor was immobilised on a Ni2+–NTA chip for monitoring its interaction with core polymerase. The binding constant for the interaction was found to be 1.9×10−7 M, and the dissociation rate constant for release of σ from core, in the absence of DNA or transcription, was 4×10−3 s−1, corresponding to a half‐life of about 200 s.

The binding of glucose and nucleotides to hexokinase from Saccharomyces cerevisiae.

The binding of glucose, ADP and AdoPP[NH]P, to the native PII dimer and PII monomer and the proteolytically-modified SII monomer of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from Saccharomyces cerevisiae was monitored at pH 6.7 by the concomitant quenching of protein fluorescence. The data were analysed in terms of Qmax, the maximal quenching of fluorescence at saturating concentrations of ligand, and [L]0.5, the concentration of ligand at half-maximal quenching. No changes in fluorescence were observed with free enzyme and nucleotide alone. In the presence of saturating levels of glucose, Qmax induced by nucleotide was between 2 and 7%, and [L]0.5 was between 0.12 and 0.56 mM, depending on the nucleotide and enzyme species. Qmax induced by glucose alone was between 22 and 25%, while [L]0.5 was approx. 0.4 mM for either of the monomeric hexokinase forms and 3.4 for PII dimer. In the presence of 6 mM ADP or 2 mM AdoPP[NH]P, Qmax for glucose was increased by up to 4% and [L]0.5 was diminished 3-fold for hexokinase PII monomer, 6-fold for SII monomer, and 15-fold for PII dimer. The results are interpreted in terms of nucleotide-induced conformational change of hexokinase in the presence of glucose and synergistic binding interactions between glucose and nucleotide.

The cooperative binding of glucose to yeast hexokinase PI dimer

Summary The binding of glucose to yeast hexokinase PI isozyme has been measured by micro scale equilibrium dialysis using a double labeling techniques. It was found that under conditions where the enzyme exists exclusively as dimer glucose bound to one site per monomer with strong cooperatively (Hill coefficient = 1.6). Under the same conditions, the PII isozyme dimer binds glucose non-cooperatively (1). The possibiely exists therefore that the yeast haxokinase PI and OII isozymes may differ markedly in their regulatory properties.

Use of the tryptophan analogue 7-azatryptophan to study the interaction of σN with Escherichia coli RNA polymerase core enzyme

The minor sigma factor σ N of Escherichia coli RNA polymerase (RNAP) has been labelled with the tryptophan analogue 7-azatryptophan (7AW) by biosynthetic incorporation. This has the effect of shifting the absorbance spectra of the protein so there is appreciable absorbance at 315 nm. Consequently this makes 7AWσ N an ideal tool to study the protein/protein interaction of σ N and RNAP using the absorbance optics in the XL-A analytical ultracentrifuge.

Synergistic binding of glucose and aluminium · ATP to hexokinase from Saccharomyces cerevisiae

The binding of glucose, AlATP and AlADP to the monomeric and dimeric forms of the native yeast hexokinase PII isoenzyme and to the proteolytically modified SII monomeric form was monitored at pH 6.7 by the concomitant quenching of intrinsic protein fluorescence. No fluorescence changes were observed when free enzyme was mixed with AlATP at concentrations up to 7500 microM. In the presence of saturating concentrations of glucose, the maximal quenching of fluorescence induced by AlATP was between 1.5 and 3.5% depending on species, and the average value of [L]0.5, the concentration of ligand at half-saturation, over all monomeric species was 0.9 +/- 0.4 microM. The presence of saturating concentrations of AlATP diminished [L]0.5 for glucose binding by between 260- and 670-fold for hexokinase PII and SII monomers, respectively (dependent on the ionic strength), and by almost 4000-fold for PII dimer. The data demonstrate extremely strong synergistic interactions in the binding of glucose and AlATP to yeast hexokinase, arising as a consequence of conformational changes in the free enzyme induced by glucose and in enzyme-glucose complex induced by AlATP. The synergistic interactions of glucose and AlATP are related to their kinetic synergism and to the ability of AlATP to act as a powerful inhibitor of the hexokinase reaction.

Towards deconvoluting the interaction of the Bacillus subtilis sporulation proteins SinR and SinI using tryptophan analogue incorporated proteins

Sporulation in Bacillus subtilis is used as the strategy of last resort for survival of the organism and it is a very tightly controlled developmental process. One of the control checkpoints that must be overcome for sporulation to occur is the repression of sporulation genes by the protein SinR (13.5 kDa). This is done by binding of the anti-repressor SinI (6.5 kDa) to form a tightly bound heterodimer. To investigate the interaction of SinI with SinR in solution, an analytical ultracentrifuge study was undertaken. SinR was found to be a tetramer, whereas SinI was in a monomer/dimer equilibrium. Derivatives of both SinI and SinR, where the native tryptophan was replaced by the analogue 7-aza-tryptophan (7AW), were expressed and found to be as active as the wild-type proteins. The 7AW proteins have the property of having significant absorbance at 315 nm, thus allowing them to be monitored even in the presence of tryptophan containing proteins, making them ideal for studying protein/protein interactions.