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Novel state of the art 17O solid state NMR for the biosciences: from molecular architecture to functional enzymology

Reference	BB/C000471/1
Principal Investigator / Supervisor	Professor Mark Smith
Co-Investigators / Co-Supervisors	Professor Steven Brown, Professor Timothy Bugg, Professor Ray Dupree, Dr Andreas Kukol
Institution	University of Warwick
Department	Physics
Funding type	Research
Value (£)	268,486
Status	Completed
Type	Research Grant
Start date	21/02/2005
End date	20/02/2008
Duration	36 months

Abstract

17O solid state NMR data will be collected for some biomolecular problems by applying a range of known approaches with state of the art hardware and through the development of novel techniques to probe the local oxygen site geometry. A unique, state of the art 800 MHz double angle rotation (DOR) NMR probe, that will be the first computer controlled version optimised for 17O (with 1H decoupling) will provide a means for obtaining very high resolution 17O spectra (linewidths 1 ppm). To define the local geometry of oxygen-centred hydrogen bonds 3 measurements of either distance, angle or a combination are needed, (for O and H), and samples will be selectively either singly or doubly 17O labelled. Determining dipolar and scalar couplings that contain information about the local bonding (e.g. O-H distance) in the presence of large quadrupolar interactions is at the heart of the methodology to be developed. Synthetic chemistry for selective 17O-isotopic enrichment will be used, and pathways for enrichment have already been devised for all the biochemical problems to be tacked here. The structural implications of the NMR data will be extracted by computation since the NMR parameters, such as the electric field gradient and chemical shift, depend on the positions of the different atoms, such as where the proton is located in O-H---O bonds. Quantum calculation using the periodic pseudopotential code Paratec will allow general elucidation of hydrogen bonding in these systems. Hence a comprehensive 17O NMR approach will be adopted for each of the biomolecular problems to be tackled. 1. 17O NMR should be able to clearly distinguish antiparallel and parallel beta-sheet secondary structures b-amyloid fibrils since the geometry of hydrogen bonding is somewhat different in these two conformations. 170 NMR might also provide insight into the importance of hydrogen bonds in misfolding processes and the structure of intermediate states. 2. For WALP23 effects of mismatch between protein hydrophobic length and membrane hydrophobic thickness can be investigated which have particular relevance for the packing of helices in polytopic proteins. Very little is known about hydrogen bonds within the hydrophobic core, including their length, formation mechanism and resulting secondary structure and this will be elucidated here. 3. Phospholemman (PLM) is the predominant substrate for camp dependent protein kinases A and C in the heart and is found at high density in the cardiac plasma membrane, so that its structure and function has importance in understanding a whole family of membrane proteins with a single transmembrane domain. Samples either singly of doubly labelled will provide site specific structural information. Based on the geometry of the ¿C=17O---H-N- hydrogen bond it would be possible to differentiate between different types of a-helix and measure local bends in the helix structure. All this information will provide structural constrains, which can be used in combination with molecular dynamics simulations and IR to obtain an atomic model of PLM. 4. C-C hydrolase MhpC catalyses a mechanistically unusual reaction, namely the hydrolytic cleavage of a C-C bond. The enzyme contains a Ser-His-A catalytic triad at its active site, yet the precise role of Ser-110 is unclear. It appears to be a general mechanism involving attack of water on the reactive carbonyl centre, rather than attack of Ser-110. The interaction of the C-4 carbonyl with the catalytic triad, and Ser-110 in particular, is therefore of considerable mechanistic interest and will be studied using the proposed NMR methods.

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	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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