Optima Analytical Ultracentrifuge for mechanistic insights into complex protein binding events

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.

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.