A Biological Pulsed EPR/ENDOR Facility for the Manchester Interdisciplinary Biocentre

Proteins are dynamic, complex structures that facilitate cellular communication, catalysis, structure, growth and division through their interaction with other biological macromolecules, enzyme substrates and ligands. Biophysical methods are crucial for determining the structure and function of protein molecules. EPR spectroscopy has emerged as a major spectroscopic technique for the structural characterization of protein complexes, membrane protein systems, analysis of protein dynamics and the chemistry catalysed by enzymes. Modern pulsed EPR methods provide important information on protein structure through triangulation of engineered spin labels or natural 'spin active' cofactors present in proteins. EPR spectroscopy can also provide detailed electronic structural information about reactive centres (cofactors and protein based radicals) present in enzyme catalysts, and time-resolved information about the chemistry catalysed by protein systems. The contents of cells are protected and enclosed by an outer sheath or membrane composed of proteins as well as fat molecules (lipids) that form a relatively impermeable barrier. Such membranes are also found inside the cell, and form compartments that have specialised functions. The proteins found in membranes often act as gatekeepers, allowing, or sometimes actively pumping, molecules through the membrane. They also have a range of other functions such as enzymes, sensors (e.g. of hormones) and as scaffolding to provide structural support. Structural information for membrane-bound systems is scarce owing to the difficulties of applying traditional structural approaches (e.g. crystallography) to membrane systems. Spin label EPR spectroscopy provides valuable distance information from which the fold/structure of membrane proteins can be investigated. Unravelling the structure of membrane proteins is one of the major research themes in Manchester, and solid-state NMR, X-ray crystallography and cryo-electron microscopy are all employed to extract structural data. Work in this area would be significantly enhanced by the provision of EPR facilities to measure distance relationships and conformational dynamics. The Manchester group forms one node of the membrane protein structure initiative, a structural proteomics initiative sponsored by the UK research council BBSRC. RNA, in its varied forms, interacts with protein to carry out fundamental roles in the cell. Understanding the contributions of various RNAs to the control of translation in the cell forms an important theme within the structural biology and biophysics group. The understanding of molecular recognition events, including those involved in assembly of macromolecular complexes consisting of both protein and RNA are a challenge for biochemical, biophysical and structural study. The extracellular matrix group forms one of the major research centres at Manchester. The enormous size of extracellular matrix complexes, such as collagen fibrils, necessitates the use of novel structural methods. EPR spectroscopy is ideal in this regard by providing distance relationships in large protein complexes. By combining the lower resolution data from these studies, with higher resolution data for protein components, or fragments of the fibrils, a picture of the architecture of these cellular structures will emerge. Finally, catalysts in biology have properties that chemists would love to emulate. Biological reactions have exquisite specificity, even down to generating a single stereoisomer, and also do not need high temperatures and pressures. To fully understand how these processes are achieved in biology, structural biology must provide atomic structures of the protein catalysts and details (at the quantum level) of reaction mechanism. Manchester has a large grouping in this area and modern EPR facilities will provide much needed electronic structure and time-resolved information to established programmes in this area of biocatalysis.

Paramagnetic species occur at centres of key importance in biological systems. EPR has proved to be a sensitive technique for the study of paramagnetic centres. EPR measures the absorbance of microwave radiation by paramagnetic electrons in a magnetic field. The EPR lineshape gives an indication of the number of unpaired electrons, the symmetry of the environment(s) of the electron(s) and, in systems that contain more than one paramagnetic species, any interactions between unpaired electrons in the sample - including distances between the electrons. The information content of the EPR spectrum is further extended by measurement of the hyperfine interaction. This is the interaction between the unpaired paramagnetic electron and nearby atoms of elements having a nuclear spin, which provides information on the arrangement of nuclei around a paramagnet and the distribution of the unpaired electron over those atoms that form part of the paramagnet. The ENDOR (electron nuclear double resonance) technique allows for the measurement of the hyperfine interaction by monitoring the effect of applying radiofrequency radiation (in the MHz frequency range) on a saturated EPR signal. ENDOR spectroscopy allows a window to be opened into a complex structure so that those atoms around the radical site can be studied. Measurement of the hyperfine interaction using ENDOR spectroscopy can provide biochemically relevant information, such as the distribution of electrons in delocalised systems, the orientation or motion of the radical, the presence of hydrogen bonds and their strengths and lengths, the orientation of substrates in enzyme binding sites and the identities of radical-forming species. The application of these sophisticated biophysical methods to a large portfolio of BBSRC funded research at Manchester is the focus of this application.