Optima Analytical Ultracentrifuge for mechanistic insights into complex protein binding events

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

Protein complexes that are made-up of multiple different proteins and often multiples of the same protein, are inherently difficult to study. Correct assembly of these proteins is essential for our immune response to pathogens, such as viruses, and for our tissues, such as kidneys to function correctly. Therefore, knowledge of the mechanism behind how these complexes are formed is fundamental to our understanding of key biological processes and why, when they don't form properly, this can lead to disease. The AUC is one of the only methods of studying the stoichiometry of multi-component complexes and the data that is generated is highly complementary to data gained from single-molecule electron microscopy and crystallography, which together allow us to interrogate the mechanism involved in complex formation at the molecular level and is vital to our ongoing research into fundamental biological mechanisms, drug discovery and synthetic biology.

The proposed equipment is the Optima Analytical Ultracentrifuge by Beckman. An AUC is a high-speed centrifuge with optical devices fitted that allows the user to follow the movement of particles in a centrifugal field, in our case the particles are predominantly protein molecules. The rate of movement of the particles through a liquid (sedimentation) is related to their mass, conformation and effects of the solute. Over the last few decades, AUC instruments have evolved to house different detection systems and improved data analysis has allowed separation of discrete species within a complex mixture. However, in older instruments, it is not possible to identify the different sedimenting species observed in a complex sample. For example, we cannot tell if there are dimers of one protein interacting with monomers of another or vice versa therefore severely limiting usability. The new Optima AUC has far greater signal-to-noise and faster scan-rates meaning multiple wavelengths can be scanned at once. For the first time, this allows the sedimentation of multiple species to be monitored in the same cell which opens up exciting possibilities for the investigation of protein complex formation.

The new AUC will allow us to monitor multiple absorbance profiles instantaneously, providing an unparalleled insight into complex assembly processes that we cannot currently do.

Information gained from the AUC has also been extremely beneficial to the university and the pharmaceutical-industry in understanding conformational changes induced by drug target interactions (see letter of support). It is in fact the only method that can separate differences in conformation and oligomerisation induced by drug binding. This application aims to provide continued access to analytical ultracentrifugation for the local and national pharmaceutical sector and for the N8 University partnership, for which this will be the only Optima AUC instrument. There are only 2 others currently in UK universities, with one at Harwell available for external use. The new Optima AUC will provide additional exciting capabilities to projects from the main users, who are co-applicants, and include the analysis of extracellular matrix proteins involved in cell signalling, tissue strength and inflammation; determining the structures of membrane proteins responsible for multidrug resistance and kidney function; investigation of enzyme mechanism, biocatalysis and protein formulation.

The instrument will also form part of the BACF training workshops and will be an integral part of hydrodynamics training for the next generation of biochemical scientists.

Technical Summary

The AUC underpins a significant number of research projects, generating over 50 publications in the last 10-years. Projects will include: supramolecular analysis of extracellular matrix components/complexes (e.g. in the context immune response to COVID-19); other inflammatory conditions (e.g., investigating mucus biology); kidney function during homeostasis and pathological conditions; investigation of mechanisms of biological catalysis; protein formulation (e.g., in concentrated solutions); and small molecule-protein interactions within drug discovery pipelines. The Optima AUC will provide unique mechanistic insights, both alone and in combination with data from other techniques such as cryo-EM and X-ray scattering. Importantly, it will form a vital link between surface and solution-phase biophysical techniques, allowing otherwise intractable data to be fully understood.

This application is for the new Optima AUC which is a re-designed analytical ultracentrifuge replacing Beckman's older analytical instruments the XLA and XLI. The most significant innovations are the wavelength reproducibility and faster scanning speed providing multi-wavelength experiments of up-to 20 different wavelengths simultaneously. This allows deconvolution of extremely complex solution-based multi-component interactions using the absorbance profiles of labelled molecules thereby identifying discrete components within complex sedimentation profiles. This provides stoichiometric information that is currently not possible and is vital in our understanding of fundamental biological mechanisms.

The Optima AUC will replace a 29-year-old Beckman XLA ultracentrifuge and 20-year-old XLI both with significant data acquisition problems, large maintenance costs and outdated computing requirements. The university has invested heavily in complementary biophysical techniques such as surface plasmon resonance and electron microscopy and is contributing 20% towards the cost of the new instrument.

Publications

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Description This award was for the purchase of an item of equipment that allowed researchers to determine the size, shape and stoichiometry of multi-component protein complexes. Researchers traditionally have found it difficult to characterise the formation of multi-component complexes which are made through recombinant techniques. The formation of these complexes is important in our understanding of dynamics, turnover and binding site availability which can lead to ways in which we can help or abrogate these complexes in disease situations. We have integrated the Optima analytical ultracentrifuge into our pipeline of protein production/purification and characterisation prior to structural analysis of the complexes through cryo-electron microscopy. This has already, within the 9-months we have had the instrument, led to the structure of PLA2R being published (DOI: 10.1073/pnas.2202209119) and to help our understanding of the multi-oligomeric states of the chemokine CXCL4 (doi: 10.1016/j.celrep.2022.111930).
Exploitation Route The instrumentation is still in its infancy with respect to the number of projects that are being used with it. The instrument is situated within the biophysics core facility within the University of Manchester and is being used extensively on projects defined within the application. The results of these will be published in the next few years. It is anticipated that the lifetime of this instrument is way beyond 10-years and therefore will provide a huge amount of important data and aid multiple labs.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Funding was used to purchase Optima analytical Ultracentrifuge, this is of interest to exernal industrial partners and has increased our standing as a biophysics centre of excellence within the UK.
First Year Of Impact 2018
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title Hydrodynamics at a 
Description The Optima is an excellent addition to the research infrastructure of the UoM Biophysics core facility. We have already published two papers using the instrument and it will go a long way into helping our research teams define complexes in ways that were previously extremely difficult. 
Type Of Material Improvements to research infrastructure 
Year Produced 2023 
Provided To Others? No  
Impact Defining the assembly of collagen triple helical peptides, assessing the stoichiometry of nanobody-scaffold chimeras, defining the oligomerisation and subsequent ways to abrogate assembly of chemokines and furthering our understanding of high concentration formulations. 
 
Title New tools for cryo-electron microscopy 
Description The Optima AUC is being used extensively for generation of novel tools to help in the determination of protein structures through cryo electron microscopy. The method briefly entails the development of well-defined complexes that are conjugated to nanobodies created through our nanobody screening library. These nanoobdies are raised against a specific target and therefore provide a large scaffold, importantly with symmetry, with which we can define accurate class averages. By using this technique we can start to gain high resolution structures of proteins that wold otherwise be too small for conventional electron microscopy techniques. The Optima AUC is vital in this regard for defining accurate complex formation. This cannot be done with any other technique. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? No  
Impact The instrument has only been with us for 9-months, so it is still early days for publication into protein structures using this technique, but it already we are generating structures using this technique which will be published in the next year.