17ALERT bid: A new multi-wavelength analytical ultracentrifuge for the study of biomolecular interactions

Lead Research Organisation: University of Nottingham
Department Name: Sch of Biosciences

Abstract

An analytical ultracentrifuge (AUC) is a machine capable of observing macromolecules sediment under gravity when a rotor is spun at a speeds from 1000 to 60 000 rpm. We are able to observe their sedimentation properties due to optical data capture systems mounted on the instrument. To date, these have be limited to single wavelength capture and an allied Rayleigh Interference optical system. A new AUC released by Beckman Coulter is now capable of true multi-wavelength capture so that more complex biomolecular systems can be analysed by following each component due to its unique optical signature. Where there is no unique optical signature, it is possible to label a compound either with a coloured probe, or incorporate into a protein using analogues of natural amino acids. As such, the instrument is capable of measuring a very wide range of biomolecular interactions and characterising them rigorously. In addition, the instrument will be maintained by a permanent technician, data analysis will be provided by the PI who is an expert in AUC, and new software methods will be provided through workshops. The latter is provided by a grant from the Fullbright Fund, which will allow a week long workshop to be undertaken. Given the wide range of systems that can be worked on to give information about size, shape and stoichiometry of biomolecules, this application ranges widely across BBSRC priorities and strategies.

Technical Summary

We wish to purchase the new Beckman Coulter OPTIMA analytical ultracentrifuge. This has true multi-wavelength capacity and a much improved interference camera capable of 20 fringe capture compared with the regular 4 on the existing Beckman XL-I. This means that we are able to deconvolute the sedimentation of complex macromolecular interactions where each of the components has a different optical spectrum. Where two components have a similar optical spectrum we are able to use both intrinsic and extrinsic labels to distinguish them in the mix. The obvious applications of this system are in the characterisation of complex protein/protein and protein/nucleic acid mixtures. With new analysis methods we will be able to determine size and stoichiometry of the interactions using only sedimentation velocity runs. We also will be able to make the instrument available to UK scientists by locating it within the Research Complex at Harwell, where access will be managed by a dedicated technician. Training to the UK user base will be through a series of workshops targeted at academic scientists, BBSRC DTP students and industrial users. The UK has a vibrant AUC community with more than 40 machines in the UK, and as such we will be able to provide access to the new instrument to a wide range of UK research groups, many of whom hold or wish to apply for BBSRC funding.

Planned Impact

The summary of the impact of the project is as follows:
1) Scientific impact: the provision to the UK biosciences community of a new analytical ultracentrifugation instrument that will provide a better characterisation of many complex samples. In addition, it will be maintained by a permanent technician and expert data analysis and software support will be provided. This will impact across a wide range of BBSRC funded projects, of which exemplars are given in the case for support.
2) Economic Impact: better characterised samples leads to a better knowledge of their properties and hence a higher confidence in their efficacy. This has an obvious economic impact as it shortens lead time from development to saleable product. Given the wide range of sample that can be studied with an AUC, it touches many important economic areas such as biofuels, food, plant materials and function, biopharamceuticals and studies underpinning a life long healthy life style: these are all project areas that fall within the BBSRC remit.
3) Training impact: there is a sizeable AUC community in the UK based around 40 machines in universities and industry. We will be able to have a very positive impact on training both existing and future users in AUC, which will have a large positive impact on their project areas due to the addition of a rigorous and quantitative method of measurement.
4) Societal Impact and public policy impact: Better economic impacts and better healthcare solutions have an obvious positive societal impact, and this leads to changes in public policy to better enable these positive outcomes.

Publications

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Umar AA (2020) Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended ß-sheet conformation. in Biochimica et biophysica acta. General subjects

 
Description The purpose of this grant was to purchase, install and establish a new multi-wavelength analytical ultracentrifuge in the Research Complex at Harwell as part of a multi-university consortium led by the University of Nottingham. The equipment arrived in Oct 2018, and after set up and commissioning, it has been used extensively by groups from Nottingham, Glasgow, Warwick, Imperial College, Birmingham and Oxford.
Exploitation Route Data has been processed and we have 5 papers published, as well as numerous data sets used as part of protein characterisation in the complex. The work carried out primarily is from Oct 2018-March 2020. The instrument has been used during lockdown when access to the RCaH was possible, and samples have been processed for several groups. We are working with them through the lockdown period in order to maximise data production. We are preparing this data for submission to journals and grant committees.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Splitting STAT Dimers to Understand Interferon Balance: A Strategy to Dissociate Beneficial and Detrimental Interferon Effects in Infection?
Amount £543,000 (GBP)
Funding ID BB/V004824/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2021 
End 04/2024