Award details

A dual DLS and SEC-MALS instrumentation to characterize protein oligomerization for structural and mechanistic biology

Reference	BB/V01966X/1
Principal Investigator / Supervisor	Dr Julien Bergeron
Co-Investigators / Co-Supervisors	Dr Joseph Atherton, Dr Andrew Beavil, Professor Paula Booth, Prof. Maria R Conte, Dr James Garnett, Dr Rivka Isaacson, Professor Michael Malim, Dr Sarah Mizielinska, Dr Manuel Muller, Professor Annalisa Pastore, Dr Mark Pfuhl, Dr Argyris Politis, Dr Mark Sanderson, Professor Roberto Steiner, Dr Andrew Surman, Professor Brian Sutton
Institution	King's College London
Department	Randall Div of Cell and Molecular Biophy
Funding type	Research
Value (£)	205,223
Status	Completed
Type	Research Grant
Start date	03/11/2021
End date	02/11/2022
Duration	12 months

Abstract

Particles in solution diffract monochromatic light (i.e. lasers), and the resulting diffraction pattern is correlated to the dimensions of the particles. Because of this property, it is therefore possible to determine the accurate molecular weight, and/or level of aggregation, of purified protein samples, heterogeneous samples, and synthetic polymers, by analysing their diffraction pattern. Specifically, the requested equipment uses light scattering in two different ways: - By analyzing the temporal fluctuation of this spectrum (Dynamic Light Scattering, DLS), it is possible to identify the distribution of sizes of the particles present in a sample. This does not provide accurate molecular weight measurements, but is used as a tool to measure polymerization or aggregation, as well as for diagnostics measurement of sample homogeneity. In particular, measurements are very fast (~ 1 second), and can be performed in a high-throughput, time-resolved manner. This can be exploited for assembly inhibitor screens, or to identify conditions for stability. - When the sample is illuminated from multiple angles, the pattern of interference correlates with the radius of gyration of the particles in the sample. In turn, this can be coupled with a size-exclusion chromatography (SEC) line, which allows to separate the various molecule populations in the sample, permitting to determine their absolute molecular weight. DLS is used routinely to study protein aggregation, particularly in the context of filamentous assembly such as cytoskeletal proteins or amyloid fibrils. In particular, the possibility to use it in a high-throughput fashion has been exploited extensively for the screening of amyloid inhibitors. In contrast, SEC-MALS is mainly applied to determine the stoichiometry of macromolecular assemblies. This is particularly important for structural studies, where the determination of the exact stoichiometry is required for structure determination.

Summary

The physical characterization of proteins is critical for the fundamental understanding of biological processes, as well as for new drug development. In particular, the capacity to determine if proteins naturally assemble, interact with partners (other proteins, nucleic acids, lipids etc.), or is modified by other molecules such as lipids, sugars, etc.., is essential to decipher their function. We seek to acquire a device that exploits how solutions that contain proteins affect laser light, to precisely and accurately determine the "size" (molecular weight) of a protein. This is an essential information to elucidate its mechanism of action, how it interacts with other molecules, etc. In addition, this equipment allows to monitor how proteins stick to each other ("aggregate"), which can happen for their natural function, for example in proteins that forms filaments across the cell to deliver cargo (for example, actin or tubulin), but also can be a sign of pathology (amyloids). In particular, we will use this equipment extensively in our research efforts to understand proteins involved in bacterial virulence and antibiotic resistance, viral pathogenesis (including SARS-CoV-2), cancer and tumor pathogenesis, and neurodegenerative diseases such as Alzheimer's and Parkinson's diseases.

Committee	Not funded via Committee
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	Advanced Life Sciences Research Technology Initiative (ALERT) [2013-2014]
Funding Scheme	X – not Funded via a specific Funding Scheme

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