Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation

Abstract

Significant progress in the interpretation of analytical ultracentrifugation (AUC) data in the last decade has led to profound changes in the practice of AUC, both for sedimentation velocity (SV) and sedimentation equilibrium (SE). Modern computational strategies have allowed for the direct modeling of the sedimentation process of heterogeneous mixtures, resulting in SV size-distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of AUC, such as the study of protein self- and hetero-association, the study of membrane proteins, and applications in biotechnology. New global multi-signal modeling and mass conservation approaches in SV and SE, in conjunction with the effective-particle framework for interpreting the sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multi-protein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current commentary is to supplement previous AUC protocols, Current Protocols in Protein Science 20.3 (1999) and 20.7 (2003), and 7.12 (2008), and provide an update describing the current tools for the study of soluble proteins, detergent-solubilized membrane proteins and their interactions by SV and SE.

Human Herpesvirus 7 U21 Tetramerizes To Associate with Class I Major Histocompatibility Complex Molecules

“…This would in principle be consistent with the dimer and tetramer not being in a reversible equilibrium and instead the result of persistent structural or chemical differences, even though both the dimer and tetramer are functional in binding HLA-A2 (see below). Alternatively, the hydrodynamic separation of species at virtually concentration-independent sedimentation coefficients and populations would be consistent with species that are in a very slow equilibrium on the time scale of days (15). Assuming the latter, the approximately equal abundance of dimers and tetramers in samples equilibrating for several days at low micromolar concentrations would suggest in a back-of-the-envelope estimate an equilibrium dissociation constant (K D ) for the dimertetramer association in the low micromolar range.…”

“…Sedimentation equilibrium (SE) and sedimentation velocity (SV) experiments were carried out as described previously (15) in Optima XL-I analytical ultracentrifuges equipped with the ProteomeLabTM XL-A/XL-I graphical user interface acquisition software version 6.0 (firmware version 5.06) (Beckman Coulter, Indianapolis, IN).…”

Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

“…Analytical Ultracentrifugation-Experiments were conducted in a ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter, Indianapolis, IN) following standard protocols unless mentioned otherwise (19). All samples in a buffer containing 20 mM Tris, pH 7.5, 200 mM NaCl were loaded into a cell assembly composed of a double sector charcoal-filled centerpiece with a 12-mm path length and either quartz or sapphire windows.…”

“…The model was expressed in terms of a macroscopic association constant K a, 1 for the first binding event and the ratio K a, 2 /K a, 1 for the second binding event. Global least squares modeling was performed at multiple rotor speeds with the software SEDPHAT (24,25), using the reversible two-site heterogeneous association model as well as the two discrete species model (19).…”

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

“…Analytical ultracentrifugation experiments were conducted in an XL-I analytical ultracentrifuge (Beckman Coulter) with an An50Ti rotor, following previously described protocols (44). Protein samples were prepared by dilution of concentrated stocks with the working buffer (PBS).…”