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Structures of biologically important multidomain proteins by analytical ultracentrifugation: a facility upgrade

Reference	BB/C51173X/1
Principal Investigator / Supervisor	Professor Stephen Perkins
Co-Investigators / Co-Supervisors
Institution	University College London
Department	Structural Molecular Biology
Funding type	Research
Value (£)	30,680
Status	Completed
Type	Research Grant
Start date	01/12/2004
End date	30/11/2005
Duration	12 months

Abstract

An existing Beckman XL-I analytical ultracentrifuge (AUC) in the Darwin Building of the Biochemistry Department at UCL will be upgraded to digital capture technology to permit high data acquisition rates and access lower sample concentrations. Using the AUC, sedimentation velocity experiments yield sedimentation coefficients to provide structural information. Sedimentation equilibrium experiments give molecular weights, leading to determinations of the oligomeric state of proteins in solution that is otherwise not measurable, and to equilibrium constants for the association of protein oligomers, and for protein-protein and protein-DNA complexes, etc. We have developed new procedures in which the joint analysis of AUC data in conjunction with our neutron (ISIS) and X-ray (ESRF) solution scattering data is most informative. From these data, medium resolution molecular structures are determined by modelling, using constraints from known crystal and NMR structures. The resulting structures are deposited in the Protein Data Bank. We have an established track record based on AUC and scattering. In recent years, we have analysed association equilibriums for a therapeutically-important single-chain Fv antibody, RuvA (involved in homologous recombination), and DDAH (involved in NO metabolism), and have determined solution structures for IgA1 and IgA2 antibodies, the complement proteins factor H and Crry, the complement CR2-C3d complex, and an Ig fold in muscle. The requested AUC upgrade will enable us to develop new AUC applications to proteins involved with the immune response. The solution properties of the SCR domain in large multi-SCR proteins are poorly understood. This will be deciphered by the study of carefully selected SCR-containing proteins, in particular to study the complexes formed between SCR proteins and their protein ligands. The diverse nature of hinge regions in the different antibody classes is crucial for antibody function. We will determine solution structures for IgA polymers and IgD and IgE antibodies, enabling us to appreciate the role of the hinge. Solution structures for antibody-antigen complexes and with ligands bound to the Fc regions will likewise be studied. The upgrade will facilitate other AUC projects for which there is demand. These are exemplified by projects involving viral glycoproteins, protein-heparin complexes, pentraxin-drug complexes, chromatin-Df31 complexes and muscle proteins. The AUC will be made available to other groups at UCL and Birkbeck, and to external users. The outcome of these studies will clarify structural and functional properties of these important macromolecules, and ultimately lead to new industrial compounds and applications. The construction of the new Diamond synchroton and the Second Target Station at ISIS will augment the importance of these methods, especially for solution structure determinations of very large proteins and their complexes that cannot be crystallised. Potentially, structural genomics strategies for characterising protein solution structures at high rates may be developed.

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 2004 (RE4) [2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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