Structural analyses of multicomponent protein complexes by analytical ultracentrifugation

Lead Research Organisation: University College London
Department Name: Structural Molecular Biology


Analytical ultracentrifugation (AUC) is a powerful method that enables protein association or degradation in solution to be studied in detail. Protein samples are inserted into a cell assembly with windows at the top and bottom. The cells are placed into a titanium rotor which is then spun at speeds up to 50,000 revs per min inside the analytical ultracentrifuge. Optical systems are used to observe the protein in either high-speed or low-speed experiments in which the protein slowly moves to the outside of the rotor, being continuously observed as it moves. Up to 200-500 scans are recorded during the experiment. The high speed 'velocity' experiments measure how quickly the protein moves to the bottom of the cell, from which we learn about the shape of the protein, and how many different species exist in the sample. This is especially useful for analysing complexes formed between different proteins, or discovering how many different types of proteins are present in the sample, and whether they are associated or cleaved. The low speed 'equilibrium' experiments balance the tendency of the protein to diffuse in the cell with that to sediment to the bottom of the cell. This data tells us about the size of the protein in solution and the strength of any associative behaviour in the sample. Modern AUC instrumentation provides a wealth of new information on proteins that can be deciphered using new powerful software packages. For example, all the velocity scans can be inputted into software such as SEDFIT, as the result of which all the macromolecular species present in the solution can be identified, even the minor ones. We can then dissect the formation of protein complexes in detail, including determining the association constants for their formation, or follow protein degradation or cleavage in other cases. Other software such as SEDANAL or SEDPHAT analyses equilibrium scans in detail. Hence the modern AUC makes possible new types of experiments in which protein complexes can be studied as a function of many biologically important variables such as cofactors and inhibitors in order to clarify the mechanisms responsible for activity and function. The requested AUC will be applied to key problems. In the complement immune defence system of the body, we will analyse the multiple interactions made by an abundant regulator of complement activation called Factor H with its targets. The biology of Factor H is important as this has been implicated in inflammatory disorders related to blindness and kidney failure, so the ability to control its behaviour has great advantages. Antibodies are also important in immunology. We can use AUC data to understand better the way in which antibodies recognise foreign material that invades the body and how antibodies bind to cell surface receptors to control the immune response. Enzymes are important in many industrial applications, so it becomes essential to discover novel ways of creating more robust versions that will perform their chemical reactions. The AUC will help us identify enzymes that have been re-engineered to be more stable. We will use the AUC to study how specialised human proteins called TIP48 and TIP49 associate with each other and how this is modified by small molecules. Both proteins use chemical energy to perform their role in large nuclear complexes. Oligomerisation is crucial to couple ATP hydrolysis to the molecular action of these proteins. A heat-stable form of TIP49 in archaeal organisms will be studied to discover both the importance of these small molecules for association processes and also the effect of deleting part of TIP49 on its subunit organisation. A different set of proteins are involved in mitosis, the process of cell division. The AUC will be invaluable for defining how these mitotic complexes are formed and their stability, and this work is crucial to lead to more detailed molecular structures that will be determined by other methods.

Technical Summary

New analytical ultracentrifugation (AUC) methods result in detailed shape and compositional information for proteins and their complexes. This strongly complements techniques such as solution scattering, NMR and crystallography, with the advantage that AUC samples are studied in physiologically relevant solution conditions. Our requested AUC instrumentation will give us the capability to study a broad range of complexes. The following AUC projects will be undertaken. (1) The regulation of the alternative pathway of the complement cascade of immune defence is essential to control inflammation. Factor H is a key regulator. We will study the interactions of Factor H with its known physiological ligands (including activated C3 and heparin) in order to explain how Factor H regulates complement activation. (2) The conformational properties of antibody hinge regions between the Fab and Fc fragments when antibodies bind to antigens or receptors are poorly characterized. Our AUC data on antibody complexes with antigen and receptor analogues will show whether movements between the Fab and Fc fragments are integral to the immune response. (3) The generation of catalytic activities under a wide range of conditions are required to fully explore the potential of highly specific and selective enzymes. More robust forms of the enzyme transketolase will be purified and validated using the AUC. (4) Human TIP48 and TIP49 are closely related, highly conserved and essential eukaryotic AAA+ proteins. Their ability to form homo- and hetero-oligomers will be elucidated by AUC, focusing on dodecamer formation. (5) We will characterize archaeal TIP49 by AUC in its wild type and domain-deleted variants in order to define more closely the assembly of TIP oligomers. (6) In order to study key macromolecular complexes important for coordinating mitosis, we will employ a battery of structural techniques, in which AUC data are essential to show that these complexes are formed.


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Islam M (2013) The concave face of decorin mediates reversible dimerization and collagen binding. in The Journal of biological chemistry

Description The identification of the sedimentation behaviour of macromolecules defines one of their basic features that is useful for understanding its properties and function. A broad range of applications has been developed that has resulted in 30 publications so far.
Exploitation Route The identification of the sedimentation behaviour of macromolecules defines one of their basic features that is useful for understanding its properties and function. As such, this can be used by others. This includes complementary studies performed by X-ray and neutron scattering
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description The AUC equipment continues to be used for a broad range of applications in characterising biological macromolecules and their interactions with each other. It is covered by maintenance contracts and in regular use both as a research facility as well as by collaborators.
First Year Of Impact 2014
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Description British Biophysical Society meeting, Warwick University, July 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk sparked questions and discussion afterwards

The talk on modelling of the complement MASP protein encouraged attendees to consider taking up CCP-SAS software
Year(s) Of Engagement Activity 2014
Description CCP-SAS meeting, Diamond, September 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Presented out molecular modelling of MASP. Talk sparked questions and discussion afterwards

After the talk, it became possible to review how well our software worked in the modelling of the complement MASP protein.
Year(s) Of Engagement Activity 2014