Modern analytical ultracentrifugation in protein science: a tutorial review

Abstract

Analytical ultracentrifugation (AU) is reemerging as a versatile tool for the study of proteins. Monitoring the sedimentation of macromolecules in the centrifugal field allows their hydrodynamic and thermodynamic characterization in solution, without interaction with any matrix or surface. The combination of new instrumentation and powerful computational software for data analysis has led to major advances in the characterization of proteins and protein complexes. The pace of new advancements makes it difficult for protein scientists to gain sufficient expertise to apply modern AU to their research problems. To address this problem, this review builds from the basic concepts to advanced approaches for the characterization of protein systems, and key computational and internet resources are provided. We will first explore the characterization of proteins by sedimentation velocity (SV). Determination of sedimentation coefficients allows for the modeling of the hydrodynamic shape of proteins and protein complexes. The computational treatment of SV data to resolve sedimenting components has been achieved. Hence, SV can be very useful in the identification of the oligomeric state and the stoichiometry of heterogeneous interactions. The second major part of the review covers sedimentation equilibrium (SE) of proteins, including membrane proteins and glycoproteins. This is the method of choice for molar mass determinations and the study of self-association and heterogeneous interactions, such as protein-protein, protein-nucleic acid, and protein-small molecule binding.

Fig. 1.

SV data of a bovine serum albumin sample. Shown are the concentration versus radius distributions at different times after start of the sedimentation at 50,000 rpm. Concentrations are in units of fringe displacement in the interference optical system, which corresponds to ∼0.3 mg/mL per fringe.

Fig. 2.

Sedimentation coefficient distributions calculated from a SV experiment of bovine serum albumin. Shown are the distribution of Lamm equation solution c(s) (solid line), the apparent sedimentation coefficient distribution g(s*) (dotted line), and the integral sedimentation coefficient distribution G(s), scaled to the loading concentration. The differential distributions c(s) and g(s*) are in units of fringes per svedberg, and the integral distribution G(s) is in units of loading concentration. The inset shows a chromatogram of BSA in size-exclusion HPLC (Toso Haas, TSK-gel Super SW3000, 4.6 mm × 30 cm).