Christopher W. Wharton

Nanoscale spatial induction of ultraviolet photoproducts in cellular DNA by three-photon near-infrared absorption.

The high‐resolution spatial induction of ultraviolet (UV) photoproducts in mammalian cellular DNA is a goal of many scientists who study UV damage and repair. Here we describe how UV photoproducts can be induced in cellular DNA within nanometre dimensions by near‐diffraction‐limited 750 nm infrared laser radiation. The use of multiphoton excitation to induce highly localized DNA damage in an individual cell nucleus or mitochondrion will provide much greater resolution for studies of DNA repair dynamics and intracellular localization as well as intracellular signalling processes and cell–cell communication. The technique offers an advantage over the masking method for localized irradiation of cells, as the laser radiation can specifically target a single cell and subnuclear structures such as nucleoli, nuclear membranes or any structure that can be labelled and visualized by a fluorescent tag. It also increases the time resolution with which migration of DNA repair proteins to damage sites can be monitored. We define the characteristics of localized DNA damage induction by near‐infrared radiation and suggest how it may be used for new biological investigations.

INFRARED SPECTROSCOPY OF ENZYME REACTION INTERMEDIATES

Abbreviations: ATP’ase, adenosine triphosphatase; IR, infrared; MRSA, methicillin-resistant Staphylococcus aureus; PBP, penicillin binding protein; pM, pH meter reading TIM, triosephosphate isomerase.

Gap junction communication dynamics and bystander effects from ultrasoft X-rays.

Gap junctions provide a route for small molecules to pass directly between cells. Toxic species may spread through junctions into ‘bystander’ cells, which may be exploited in chemotherapy and radiotherapy. However, this may be prevented by junction closure, and therefore an understanding of the dose-dependency of inhibition of communication and bystander effects is important. Low-energy ionising radiation (ultrasoft X-rays) provides a tool for the study of bystander effects because the area of exposure may be carefully controlled, and thus target cells may be clearly defined. Loss of gap junction-mediated intercellular communication between irradiated cells was dose-dependent, indicating that closure of junctions is proportional to dose. Closure was associated with hyperphosphorylation of connexin43. Inhibition of communication occurred in bystander cells but was not proportional to dose. Inhibition of communication at higher radiation doses may restrict the spread of inhibitory factors, thus protecting bystander cells. The reduction in communication that takes place in bystander cells was dependent on cells being in physical contact, and not on the release of signalling factors into the medium.

Hydrogen bonding and protein perturbation in beta-lactam acyl-enzymes of Streptococcus pneumoniae penicillin-binding protein PBP2x.

A soluble form of Streptococcus pneumoniae PBP2x, a molecular target of penicillin and cephalosporin antibiotics, has been expressed and purified. IR difference spectra of PBP2x acylated with benzylpenicillin, cloxacillin, cephalothin and ceftriaxone have been measured. The difference spectra show two main features. The ester carbonyl vibration of the acyl-enzyme is ascribed to a small band between 1710 and 1720 cm-1, whereas a much larger band at approx. 1640 cm-1 is ascribed to a perturbation in the structure of the enzyme, which occurs on acylation. The protein perturbation has been interpreted as occurring in beta-sheet. The acyl-enzyme formed with benzylpenicillin shows the lowest ester carbonyl vibration frequency, which is interpreted to mean that the carbonyl oxygen is the most strongly hydrogen-bonded in the oxyanion hole of the antibiotics studied. The semi-synthetic penicillin cloxacillin is apparently less well organized in the active site and shows two partially overlapping ester carbonyl bands. The penicillin acyl-enzyme has been shown to deacylate more slowly than that formed with cloxacillin. This demonstrates that the natural benzylpenicillin forms a more optimized and better-bonded acyl-enzyme and that this in turn leads to the stabilization of the acyl-enzyme required for effective action in the inhibition of PBP2x. The energetics of hydrogen bonding in the several acyl-enzymes is discussed and comparison is made with carbonyl absorption frequencies of model ethyl esters in a range of organic solvents. A comparison of hydrolytic deacylation with hydroxaminolysis for both chymotryspin and PBP2x leads to the conclusion that deacylation is uncatalysed.

Novel ligands for the purification of catechol-O-methyl transferase by affinity chromatography

Abstract Affinity chromatography materials were synthesised by reaction of catechol, guaiacol and pyrogallol with agarose gels having pendant diazotised aromatic amines. The use of these materials in the purification of catechol- O -methyl transferase (COMT) after ammonium sulphate precipitation and gel chromatography on Sephadex G75 resulted in a 1400-fold increase in specific activity compared to that of the rat liver homogenate. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The nature of the binding reaction between COMT and these immobilised ligands was examined by measuring their affinities under various conditions of ionic strength and bulk hydrogen ion concentration. Borate ions were shown to specifically elute the enzyme. The results in this paper are consistent with previous proposals regarding the mechanism of COMT.

Order and Disorder in the Domain Organization of the Plasmid Partition Protein KorB

The plasmid partition protein KorB has a dual role: it is essential for the correct segregation of the low copy number broad host range RK2 plasmid while also being an important regulator of transcription. KorB belongs to the ParB family of proteins, and partitioning in RK2 has been studied as a simplified model of bacterial chromosome segregation. Structural information on full-length ParB proteins is limited, mainly due to the inability to grow crystals suitable for diffraction studies. We show, using CD and NMR, that KorB has regions of significant intrinsic disorder and hence it adopts a multiplicity of conformations in solution. The biophysical data are consistent with bioinformatic predictions based on the amino acid sequence that the N-terminal region and also the region between the central DNA-binding domain and the C-terminal dimerization domain are intrinsically disordered. We have used small angle x-ray scattering data to determine the ensemble of solution conformations for KorB and selected deletion mutants, based on models of the known domain structures. This conformational range of KorB is likely to be biologically required for DNA partitioning and for binding to a diverse set of partner proteins.

α-Carboxy-6-nitroveratryl: A Photolabile Protecting Group for Carboxylic Acids

The synthesis of a new photolabile protecting group for carboxylic acids, alpha-carboxy-6-nitroveratryl (alphaCNV), is described. Bromide 3, prepared in four steps from 3,4-dimethoxyphenylacetic acid, was used to alkylate carboxylic acids under mild conditions in good yield. Palladium-catalyzed deallylation afforded the acids 4a-h, which underwent rapid and quantitative photolysis at wavelengths longer than 300 nm to liberate the carboxylic acid in good to quantitative yield. The rate of photolysis and quantum yield were determined to be 325 s(-1) and 0.17.

Photorelease of tyrosine from α-carboxy-6-nitroveratryl (αCNV) derivatives

The synthesis of photolabile tyrosine derivatives protected on the phenolic oxygen by the α-carboxy-6-nitroveratryl (αCNV) protecting group is described. The compounds undergo rapid photolysis at wavelengths longer than 300 nm to liberate the corresponding phenol in excellent yield (quantum yield for the deprotection of tyrosine = 0.19). Further protection of caged tyrosine is possible, yielding N-Fmoc protected derivatives suitable for direct incorporation of caged tyrosine in solid-phase peptide synthesis.

Anatomy of a Simple Acyl Intermediate in Enzyme Catalysis: Combined Biophysical and Modeling Studies on Ornithine Acetyl Transferase.

Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the alpha-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; (13)C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-alpha-acetyl-L-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm(-1). Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm(-1) IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm(-1) IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated residue, Thr-181. Overall, the results reveal that acyl-enzyme complex structures may be more dynamic than previously thought and support the use of a comprehensive biophysical and modeling approach in studying such intermediates.

A critical analysis of the use of radiation inactivation to measure the mass of protein.

Measurements are presented of the radiation inactivation of four enzymes exposed to a 6 MeV proton beam. It has long been thought that the measurement of the susceptibility of an enzyme to ionizing radiation can be used to determine its molecular mass. Results are frequently interpreted using the empirical analysis of Kempner and Macey (Biochim. Biophys. Acta 163, 188-203, 1963). We examine this analysis and discuss the validity and limitations of the assumptions on which it is based. Our results indicate that the specific biochemical properties of each enzyme make a significant contribution to its radiation sensitivity.