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Crystallohydrodynamics of Protein Assemblies: Combining Sedimentation, Viscometry, and X-Ray Scattering

Yanling Lu ^*, Emma Longman ^*, Kenneth G. Davis ^*, Álvaro Ortega , J. Günter Grossmann , Terje E. Michaelsen , José García de la Torre  and Stephen E. Harding ^*

^* National Centre for Macromolecular Hydrodynamics, University of Nottingham, School of Biosciences, Sutton Bonington, England; Departamento de Quimica Fisica, Universidad de Murcia, Murcia, Spain; CCLRC Daresbury Laboratory, Synchrotron Radiation Department, Warrington, Cheshire, England; and Norwegian Institute of Public Health, Oslo, and Institute of Pharmacy, University of Oslo, Blindern, Oslo, Norway

Correspondence: Address reprint requests to Stephen E. Harding, NCMH Laboratory, University of Nottingham, School of Biosciences, Sutton Bonington, LE12 5RD, England. E-mail: steve.harding{at}nottingham.ac.uk.

Crystallohydrodynamics describes the domain orientation in solution of antibodies and other multidomain protein assemblies where the crystal structures may be known for the domains but not the intact structure. The approach removes the necessity for an ad hoc assumed value for protein hydration. Previous studies have involved only the sedimentation coefficient leading to considerable degeneracy or multiplicity of possible models for the conformation of a given protein assembly, all agreeing with the experimental data. This degeneracy can be considerably reduced by using additional solution parameters. Conformation charts are generated for the three universal (i.e., size-independent) shape parameters P (obtained from the sedimentation coefficient or translational diffusion coefficient), (from the intrinsic viscosity), and G (from the radius of gyration), and calculated for a wide range of plausible orientations of the domains (represented as bead-shell ellipsoidal models derived from their crystal structures) and after allowance for any linker or hinge regions. Matches are then sought with the set of functions P, , and G calculated from experimental data (allowing for experimental error). The number of solutions can be further reduced by the employment of the D_max parameter (maximum particle dimension) from x-ray scattering data. Using this approach we are able to reduce the degeneracy of possible solution models for IgG3 to a possible representative structure in which the Fab domains are directed away from the plane of the Fc domain, a structure in accord with the recognition that IgG3 is the most efficient complement activator among human IgG subclasses.

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