Analytical ultracentrifugation combined with X-ray and neutron scattering: Experiment and modelling.
Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data. The constrained modelling of ultracentrifugation and scattering data is important to confirm the experimental data analysis and yields families of best-fit molecular models for comparison with crystallography and NMR structures. This modelling of ultracentrifugation and scattering data is described in terms of starting models, their conformational randomisation in trial-and-error fits, and the identification of the final best-fit models. Seven applications of these methods are described to illustrate the current state-of-the-art. These include the determination of antibody solution structures (the human IgG4 subclass, and oligomeric forms of human IgA and its secretory component), the solution structures of the complement proteins of innate immunity (Factor H and C3/C3u) and their interactions with macromolecular ligands (C-reactive protein), and anionic polysaccharides (heparin). Complementary features of joint ultracentrifugation and scattering experiments facilitate an improved understanding of crystal structures (illustrated for C3/C3u, C-reactive protein and heparin). If a large protein or its complex cannot be crystallised, the joint ultracentrifugation-scattering approach provides a means to obtain an overall macromolecular structure.
Department of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK. s.perkins@ucl.ac.uk
Stephen J
SJ
Perkins
Ruodan
R
Nan
Keying
K
Li
Sanaullah
S
Khan
Yuki
Y
Abe
D002097:C-Reactive Protein / Q000737:chemistry; D017242:Complement Factor H / Q000737:chemistry; D018360:Crystallography, X-Ray; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D009502:Neutrons; D011134:Polysaccharides / Q000737:chemistry; D012542:Scattering, Radiation; D014461:Ultracentrifugation / Q000295:instrumentation / Q000379:methods*
C493495:CFH protein, human; D007074:Immunoglobulin G; D011134:Polysaccharides; D017242:Complement Factor H; D006493:Heparin; D002097:C-Reactive Protein