Optical spectroscopy for biomolecular interactions

The applicants wish to purchase spectroscopic equipment totalling £139,710 (including service contracts) to enable fluorescence detected linear dichroism measurements (FDLD) and the interpretation of the FDLD data using fluorescence and absorption data. The spectrometers will also be used for linear dichroism (LD), circular dichroism (CD), fluorescence, fluorescence polarisation anisotropy and absorbance measurements as stand-alone experiments as well as in conjunction with the new technique of FDLD. Jasco UK Ltd. are contributing £49,219 to the project in the form of £6,000 of staff time for training and supporting workshops to be run at Warwick and the remainder in discounts on the purchase of the instruments. Jasco wish to establish Warwick as the Jasco Centre of excellence in Polarised Spectroscopy which will further enhance the collaboration between the applicants and Jasco UK Ltd. Crystal Precision Optics will contribute £4,000 of staff time to support design and development of new equipment emanating from this project. A small amount of PI time will be required to oversee the procurement, installation, and establishing the equipment as a multi-user facility. Rationale underlying proposal: All biological processes are fundamentally interactions between molecules, mainly between macromolecules or between macromolecules and small molecules. Despite significant advances in our ability to characterise such interactions and the 'single-molecule' revolution that is taking place, we still struggle to measure key properties of molecular interactions. This is particularly true for the significant classes of biological molecules that have proved difficult to study by established structural techniques such as crystallography and NMR. These include long polymeric structures: DNA; DNA-ligand complexes; fibres including fibrous proteins; and also membrane proteins. Wishing to probe the time dependence of interactions including enzyme kinetics, fibre assembly or protein insertion into membranes introduces an added dimension of complexity. The aim of this proposal is to establish the newly invented technique of FDLD so the UK community can benefit from its advantages. The core FDLD instrument will be supported by standard fluorescence and absorption instruments to enable data interpretation. All three instruments will also be used for independent LD, CD, fluorescence, polarised fluorescence and measurements of interacting biomolecular systems. Over the last few years Warwick has become the national and indeed international hub for innovation based on the technique of flow linear dichroism (LD), which is the difference in absorbance of light polarised parallel and perpendicular to an orientation axis. LD can be used to deduce kinetic and structural information about a wide range of systems, the only requirements being that they have absorbance spectroscopy and they can be oriented. Flow LD requires samples to be long enough to be oriented by shear forces in solution and is ideally suited to DNA and fibrous proteins. We have also had significant success in orienting membrane systems as the flow-distortion of liposomes creates an orientation axis. In the context of ligand binding, the key attraction of LD is that it is selective only for those molecules bound to the long system. Thus, e.g., only PCR products are detected, not the background population of free nucleotides. One of the new techniques we have developed is fluorescence detected linear dichroism (FDLD). This is the focus of the current application. FDLD captures the advantages for intermolecular interactions of (i) LD (namely detecting only oriented samples) and (ii) fluorescence (namely only detecting fluorophores and with higher sensitivity than absorption-based techniques). In the proof of concept paper for FDLD we showed how FDLD can be used selectively to study the orientation on DNA of ligands whose spectroscopy lies under the DNA absorbance bands.

The purchase of a state of the art spectropolarimeters adapted for linear dichroism and the newly developed technique of fluorescence detected linear dichroism (FDLD) will be used to support a variety of research areas some involving the unique attractions of FDLD and others using more standard circular dichroism, linear dichroism, fluorescence and absorbance. The fluorescence and absorbance instruments are required for analysis of the FDLD data and will also be used as stand-alone instrument in some projects. The proposed instrumentation will be used in a number of research projects studying interactions of biomolecules both at Warwick and elsewhere. Areas of research include the following: 1. Fibrous protein structure, kinetics and dynamics with particular application to 1.1. Amyloid fibrils; 1.2. FtsZ (the bacterial homologue of tubulin); 1.3. Self assembled fibres 1.4. The mechanism of actin action; 2. Membrane protein structure, kinetics and dynamics 2.1. Antibiotic peptides 2.2. Transmembrane helix-helix interactions 2.3. Antiviral and stem-cell-mobilizing metallomacrocycles 2.4 . Ion channels 3. DNA-protein and DNA-ligand systems 3.1. DNA-enzyme complexes 3.2. Metallodrugs: targeting DNA and proteins 3.3. Small molecule/DNA complexes 4. Carbon-nanotube and CNT-ligand systems 5. Study of bacterial cell wall synthesis 6. Protein structure for the biopharmaceutical industry 7. Molecular switches based upon pyramidal inversion