Award details

Studies of biomolecules and their interactions by using NMR spectroscopy with cutting edge sensitivity.

Reference	BB/D524775/1
Principal Investigator / Supervisor	Professor Dusan Uhrin
Co-Investigators / Co-Supervisors	Professor Paul Barlow, Professor Peter Sadler
Institution	University of Edinburgh
Department	Sch of Chemistry
Funding type	Research
Value (£)	154,155
Status	Completed
Type	Research Grant
Start date	01/01/2006
End date	31/12/2007
Duration	24 months

Abstract

We seek funds to purchase a cryoprobe for our 800 MHz NMR spectrometer. Such an accessory will provide us with the maximum sensitivity currently available to the biomolecular NMR community. The enhanced NMR facility will be available to all researchers in the joint Universities of Edinburgh and St Andrews School of Chemistry; and, via collaboration, with numerous other scientists in Edinburgh and in Dundee, Glasgow and Strathclyde Universities. A collaboration with Bruker Spectrospin will be established, resulting in a large contribution to the cost of the instrument from this company. The School of Chemistry will also contribute, thus achieving excellent value for research council money. Only the research of the applicants, and main users (DU, PNB and PJS) of the 800 MHz cryoprobe is summarised below: The research of DU focuses on studies of glycosaminoglycan:protein interactions. The ability of glycosaminoglycans (GAGs) to bind and modulate protein-protein interactions or enzymatic activity means they are regarded as important determinants of cellular responsiveness in development, homeostasis, and disease. GAGs are a heterogeneous set of proteoglycans, differing in sulphation patterns and the nature of constituting monosaccharides. Chemically homogeneous species can be purified from natural materials, but their preparation is challenging and the yields are low. A further limitation is that the solubility of protein:GAG complexes is often relatively low and they can typically be prepared only in submillimolar concentrations. Their characterisation by NMR is complicated because of the difficulties in detecting the intermolecular NOEs. DU is therefore developing methods that will use the effects of paramagnetic centres as structural restraints in structure calculations of GAG:protein complexes. The sensitivity of the 800 MHz cryoprobe will ensure that we will be able to perform many key experiments that would not be possible otherwise at achievable concentrations. At present two systems are studied: hepatocyte growth factor/scatter factor (HGF/SF) and, in collaboration with PNB, factor H. Both systems represent potential therapeutic targets. HGF/SF is implicated in wound healing, organ regeneration and cancer. Factor H, modules 7 and 20, which are the known heparin binding sites are linked to age-related macular degeneration (AMD), a condition that accounts for half of the blind registrations in the UK, and atypical hemolityc uremic syndrome (HUS), an illness leading to acute renal failure, respectively. The PNB lab works on proteins of the compliment cascade, an essential molecular component of mammalian immune defences. The mostly large and flexible, multiple-domain, complement proteins are dissected into their component modules or pairs of adjacent modules for NMR studies. Most inter-protein recognition events can be emulated in the NMR tube by the formation of complexes involving individual modules or contiguous pairs of modules. The new cryoprobe will be utilised by the PNB group to study such complexes. The sensitivity-gain will be a requirement for the success of this approach. It will allow a wide range in the ratios of the two partners to be explored and thus help to overcome problems associated with NMR-unfriendly exchange rates. It will also allow experiments to be accomplished with less material or in a shorter time, and with the high-field advantages of TROSY-based methods. These benefits will be critical given the rather large sizes of some complement components, and the growing number of interacting partners that we have expressed or received through collaborations. The resulting, atomic-level information will suggest hypotheses for the mechanism of the complement cascade and will help in design of complement-controlling therapeutics. PJS is involved in studies of the design and mechanism of action of metal-based drugs. Systems which will be studied by NMR spectroscopy include photoactivated platinum anticancer complexes, organometallic ruthenium arene anticancer complexes, macrocyclic antiHIV and stem-cell-mobilising agents, mammalian and bacterial zinc and iron transport proteins. The cryoprobe is a necessity because the highest sensitivity is required to allow both thermodynamic and kinetic studies of ligand exchange reactions of the complexes to be carried out at physiologically-relevant concentrations (micromolar). We will study adducts of the anticancer complexes with both single and double-stranded DNA in order to identify the metallation sites and induced structural distortions (which is likely to be correlated with activity). The metal macrocyles appear to have specific binding sites on membrane co-receptors involving direct metal binding to carbosylate side-chains (Asp, Glu), H-bonding and hydrophobic interactions with Trp residues. Tuning such interactions should lead to improvements in drug design. The metal transport proteins are large (human transferring 80 kDa, bacterial transferrin 34 kDa, serum albumin 66 kDa) and require the dispersion available at high frequency and high sensitivity for multidimensional multinuclear experiments.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Research Equipment Initiative 2005 (RE5) [2005]
Funding Scheme	X – not Funded via a specific Funding Scheme

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