Crystallohydrodynamics of protein assemblies: Combining sedimentation, viscometry and x-ray scattering

¹ University of Nottingham
² University of Murcia
³ SRS Daresbury Laboratory
⁴ University of Oslo

^* To whom correspondence should be addressed. E-mail: steve.harding{at}nottingham.ac.uk.

Submitted on February 16, 2006
Revised on April 17, 2006
Accepted on 24 May 2006

	Abstract

Crystallohydrodynamics describes the domain orientation in solution of antibodies and other multi-domain 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.

Key Words: bead-shell model, hydration, immunoglobulin, uniqueness

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