Analytical Ultracentrifugation as a Matrix-Free Probe for the Study of Kinase Related Cellular and Bacterial Membrane Proteins and Glycans

Abstract

Analytical ultracentrifugation is a versatile approach for analysing the molecular mass, molecular integrity (degradation/aggregation), oligomeric state and association/dissociation constants for self-association, and assay of ligand binding of kinase related membrane proteins and glycans. It has the great property of being matrix free—providing separation and analysis of macromolecular species without the need of a separation matrix or membrane or immobilisation onto a surface. This short review—designed for the non-hydrodynamic expert—examines the potential of modern sedimentation velocity and sedimentation equilibrium and the challenges posed for these molecules particularly those which have significant cytoplasmic or extracellular domains in addition to the transmembrane region. These different regions can generate different optimal requirements in terms of choice of the appropriate solvent (aqueous/detergent). We compare how analytical ultracentrifugation has contributed to our understanding of two kinase related cellular or bacterial protein/glycan systems (i) the membrane erythrocyte band 3 protein system—studied in aqueous and detergent based solvent systems—and (ii) what it has contributed so far to our understanding of the enterococcal VanS, the glycan ligand vancomycin and interactions of vancomycin with mucins from the gastrointestinal tract.

Keywords

• sedimentation velocity
• sedimentation equilibrium
• molecular mass
• oligomeric state
• ligand binding
• detergent binding