Structural analyses of multicomponent protein complexes by analytical ultracentrifugation
Lead Research Organisation:
University College London
Department Name: Structural Molecular Biology
Abstract
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.
Publications
Abe Y
(2010)
Masking of the Fc region in human IgG4 by constrained X-ray scattering modelling: implications for antibody function and therapy.
in The Biochemical journal
Almogren A
(2009)
Functional and structural characterisation of human colostrum free secretory component.
in Molecular immunology
Bonner A
(2008)
Implications of the near-planar solution structure of human myeloma dimeric IgA1 for mucosal immunity and IgA nephropathy.
in Journal of immunology (Baltimore, Md. : 1950)
Bonner A
(2009)
The nonplanar secretory IgA2 and near planar secretory IgA1 solution structures rationalize their different mucosal immune responses.
in The Journal of biological chemistry
Bonner A
(2009)
Location of secretory component on the Fc edge of dimeric IgA1 reveals insight into the role of secretory IgA1 in mucosal immunity.
in Mucosal immunology
Furtado PB
(2008)
The partly folded back solution structure arrangement of the 30 SCR domains in human complement receptor type 1 (CR1) permits access to its C3b and C4b ligands.
in Journal of molecular biology
Islam M
(2013)
The concave face of decorin mediates reversible dimerization and collagen binding.
in The Journal of biological chemistry
Khan S
(2013)
The solution structure of heparan sulfate differs from that of heparin: implications for function.
in The Journal of biological chemistry
Khan S
(2010)
Semi-rigid solution structures of heparin by constrained X-ray scattering modelling: new insight into heparin-protein complexes.
in Journal of molecular biology
Khan S
(2011)
The Solution Structure of Heparan Sulfate Differs from That of Heparin
in Journal of Biological Chemistry
Khan S
(2013)
The solution structure of heparan sulfate differs from that of heparin: implications for function.
in The Journal of biological chemistry
Leonard PG
(2009)
Investigation of the self-association and hetero-association interactions of H-NS and StpA from Enterobacteria.
in Molecular microbiology
Leonard PG
(2010)
The absence of inorganic salt is required for the crystallization of the complete oligomerization domain of Salmonella typhimurium histone-like nucleoid-structuring protein.
in Acta crystallographica. Section F, Structural biology and crystallization communications
Li K
(2008)
Solution Structure of the Complex Formed between Human Complement C3d and Full-length Complement Receptor Type 2
in Journal of Molecular Biology
Li K
(2012)
Solution structure of TT30, a novel complement therapeutic agent, provides insight into its joint binding to complement C3b and C3d.
in Journal of molecular biology
Li K
(2010)
Self-association and domain rearrangements between complement C3 and C3u provide insight into the activation mechanism of C3.
in The Biochemical journal
Miller A
(2012)
Near-planar solution structures of mannose-binding lectin oligomers provide insight on activation of lectin pathway of complement.
in The Journal of biological chemistry
Nan R
(2008)
Uncontrolled zinc- and copper-induced oligomerisation of the human complement regulator factor H and its possible implications for function and disease.
in Journal of molecular biology
Nan R
(2011)
Zinc binding to the Tyr402 and His402 allotypes of complement factor H: possible implications for age-related macular degeneration.
in Journal of molecular biology
Nan R
(2013)
Zinc-induced self-association of complement C3b and Factor H: implications for inflammation and age-related macular degeneration.
in The Journal of biological chemistry
Nan R
(2008)
Implications of the progressive self-association of wild-type human factor H for complement regulation and disease.
in Journal of molecular biology
Niewiarowski A
(2010)
Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes.
in The Biochemical journal
Okemefuna A
(2010)
Complement Factor H Binds at Two Independent Sites to C-reactive Protein in Acute Phase Concentrations*
in Journal of Biological Chemistry
Okemefuna AI
(2009)
Multimeric interactions between complement factor H and its C3d ligand provide new insight on complement regulation.
in Journal of molecular biology
Okemefuna AI
(2010)
C-reactive protein exists in an NaCl concentration-dependent pentamer-decamer equilibrium in physiological buffer.
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 |