Margaret Nutley

Bactericidal action of high-power Nd:YAG laser light on Escherichia coli in saline suspension

Infra‐red light (1064 nm) from a high‐power Nd:YAG laser caused more than 90% loss of viability of Escherichia coli during exposures that raised the temperature of PBS suspensions of the bacteria to 50 °C in a thermocouple‐equipped cuvette. In contrast, there was minimal loss of viability after heating the same suspensions to 50 °C in a water‐bath, or in a PCR thermal cycler. The mechanism of laser killing at 50 °C was explored by differential scanning calorimetry, by laser treatment of transparent and turbid bacterial suspensions, and by optical absorbancy studies of E. coli suspensions at 1064 nm. Taken together, the data suggested that the bactericidal action of Nd:YAG laser light at 50 °C was due partly to thermal heating and partly to an additional, as yet undefined, mechanism. Scanning electron microscopy revealed localized areas of surface damage on laser‐exposed E. coli cells.

Microcalorimetry of chiral surfactant-cyclodextrin interactions.

The interactions of the chiral surfactants taurodeoxycholate (TDOCA) and deoxycholate (DOCA) with a range of cyclodextrins in aqueous solution have been investigated by isothermal titration microcalorimetry. In the presence of β-cyclodextrin, the apparent critical micelle concentration (cmc) of taurodeoxycholate is increased, and the enthalpy of demicellization decreased, in a manner consistent with 1:1 complexation of TDOCA with β-CD at low concentrations. There is no evidence for direct interaction of cyclodextrins with surfactant micelles. This is confirmed by more direct binding titrations. Below the cmc, TDOCA forms 1:1 host-guest complexes with β-cyclodextrin (ΔH°(bind) = -32 kJ mol(-)(1), K(diss) = 0.38 mM; 25 °C, pH 7), methyl-β-cyclodextrin (ΔH(bind) = -13 kJ mol(-)(1), K(diss) = 0.36 mM), hydroxypropyl-β-cyclodextrin (ΔH°(bind) = -12 kJ mol(-)(1), K(diss) = 0.51 mM), and γ-cyclodextrin (ΔH°(bind) = -7.3 kJ mol(-)(1), K(diss) = 0.08 mM), but not with the smaller α-cyclodextrin. At higher cyclodextrin concentrations, the calorimetric binding data are more ambiguous, suggesting 2:1 cyclodextrin/TDOCA complexation. Similar results are found with DOCA, though experiments here are limited by the tendency of DOCA to form gels in aqueous buffers. Enhanced chromatographic or electrophoretic chiral resolution observed in mixed chiral surfactant/cyclodextrin phases could be the result of increased solubility and/or the multiplicity of chiral complexes in such systems.

The Orf18 Gene Product from Conjugative Transposon Tn916 Is an ArdA Antirestriction Protein that Inhibits Type I DNA Restriction-Modification Systems

Gene orf18, which is situated within the intercellular transposition region of the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA A) protein. Conjugative transposons are generally resistant to DNA restriction upon transfer to a new host. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli, and the recombinant ArdA protein was purified to homogeneity. The protein appears to exist as a dimer at nanomolar concentrations but can form larger assemblies at micromolar concentrations. R/M assays revealed that ArdA can efficiently inhibit R/M by all four major classes of Type I R/M enzymes both in vivo and in vitro. These R/M systems are present in over 50% of sequenced prokaryotic genomes. Our results suggest that ArdA can overcome the restriction barrier following conjugation and so helps increase the spread of antibiotic resistance genes by horizontal gene transfer.

LCST: a powerful tool to control complexation between a dialkoxynaphthalene-functionalised poly(N-isopropylacrylamide) and CBPQT4+ in water

We describe the application of the LCST of a naphthalene-functionalised polyNIPAM derivative as a convenient, tuneable and reversible method to disrupt complex formation with CBPQT(4+) in water.

Energetics of Protein-Cyclodextrin Interactions

The energetics of interaction of a range of cyclodextrins with folded and unfolded proteins has been examined by sensitive microcalorimetry techniques. Weak interaction with exposed amino acid residues promotes unfolding and dissociation of proteins. The possibility that such interactions may facilitate the use of cyclodextrins as “chaperone-mimics” in the refolding of denatured protein has been explored with the enzyme phosphoglycerate kinase. Up to 40% regain of activity can be achieved in some cases.

A mutational analysis of DNA mimicry by ocr, the gene 0.3 antirestriction protein of bacteriophage T7

The ocr protein of bacteriophage T7 is a structural and electrostatic mimic of approximately 24 base pairs of double-stranded B-form DNA. As such, it inhibits all Type I restriction and modification (R/M) enzymes by blocking their DNA binding grooves and inactivates them. This allows the infection of the bacterial cell by T7 to proceed unhindered by the action of the R/M defence system. We have mutated aspartate and glutamate residues on the surface of ocr to investigate their contribution to the tight binding between the EcoKI Type I R/M enzyme and ocr. Contrary to expectations, all of the single and double site mutations of ocr constructed were active as anti-R/M proteins in vivo and in vitro indicating that the mimicry of DNA by ocr is very resistant to change.

Dissection of the DNA Mimicry of the Bacteriophage T7 Ocr Protein using Chemical Modification

The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an ∼ 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove ∼ 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to ∼ 800-fold.

Exploring the DNA mimicry of the Ocr protein of phage T7

DNA mimic proteins have evolved to control DNA-binding proteins by competing with the target DNA for binding to the protein. The Ocr protein of bacteriophage T7 is the most studied DNA mimic and functions to block the DNA-binding groove of Type I DNA restriction/modification enzymes. This binding prevents the enzyme from cleaving invading phage DNA. Each 116 amino acid monomer of the Ocr dimer has an unusual amino acid composition with 34 negatively charged side chains but only 6 positively charged side chains. Extensive mutagenesis of the charges of Ocr revealed a regression of Ocr activity from wild-type activity to partial activity then to variants inactive in antirestriction but deleterious for cell viability and lastly to totally inactive variants with no deleterious effect on cell viability. Throughout the mutagenesis the Ocr mutant proteins retained their folding. Our results show that the extreme bias in charged amino acids is not necessary for antirestriction activity but that less charged variants can affect cell viability by leading to restriction proficient but modification deficient cell phenotypes.

Acetonitrile-induced unfolding of porcine pepsin A: A proposal for a critical role of hydration structures in conformational stability

In order to increase understanding of the basis of the stability of the native conformational state of porcine pepsin A, a strategy based on induction and monitoring of protein denaturation was developed. Structural perturbation was achieved by adding acetonitrile (MeCN) to the protein-solvent system. MeCN was found to induce non-coincident disruption of the secondary and tertiary structural features of pepsin A. It is proposed that gross unfolding is prompted by disruption of the protein hydration pattern induced by the organic co-solvent. It should be noted that the functional properties and thermal stability of the protein were already impaired before the onset of global unfolding. Low and intermediate contents of MeCN in the protein-solvent system affected the sharpness of the thermal transition and the degree of residual structure of the heat-denatured state. The importance of hydration to the conformational stability of pepsin A in its biologically active state is discussed.

Tuneable pseudorotaxane formation between a biotin–avidin bioconjugate and CBPQT4+

A biotinylated 1,5-dialkoxynaphthalene derivative has been shown to have the ability to bind strongly to avidin and thus act as an artificial binding site for cyclobis(paraquat-p-phenylene) thereby facilitating the formation of a tuneable pseudorotaxane-based bioconjugate.