Applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses

Abstract

The rebirth of modern analytical ultracentrifugation (AUC) began in 1990s. Since then many advanced AUC detectors have been developed that provide a vast range of versatile choices when characterizing the physical and chemical features of macromolecules. In addition, there have been remarkable advances in software that allow the analysis of AUC data using more sophisticated models, including quaternary structures, conformational changes, and biomolecular interactions. Here we report the application of AUC to protein size-and-shape distribution analysis and structure-and-function analysis in the presence of ligands or lipids. Using band-sedimentation velocity, quaternary structural changes and an enzyme's catalytic activity can be observed simultaneously. This provides direct insights into the correlation between quaternary structure and catalytic activity of the enzyme. On the other hand, also in this study, we have applied size-and-shape distribution analysis to a lipid-binding protein in either an aqueous or lipid environment. The sedimentation velocity data for the protein with or without lipid were evaluated using the c(s,f(r)) two-dimensional distribution model, which provides a precise and quantitative means of analyzing the protein's conformational changes.

Critical Assessment of the Important Residues Involved in the Dimerization and Catalysis of MERS Coronavirus Main Protease

“…It is unable to confirm that our observation is a substrate-induced or substrate/product-induced dimerization. To solve this, a modified AUC technique, AEC [ 33 ], was utilized to detect the quaternary structure change in the absence and presence of substrate ( Fig 4 ). Here the enzyme solution was put into the small well of band-forming centerpiece and then flowed into the substrate-containing channel when the centrifugation began.…”

“…Thus it can be concluded that, like the SARS-CoV M pro R298A mutant [ 25 ], the presence of substrate is able to induce the dimerization of MERS-CoV M pro . In addition, although the K d values of wild-type SARS-CoV M pro without or with substrates show no significant difference ( Table 2 ), it was possible to detect substrate-induced dimerization at a protein concentration of 1 μM by AEC [ 33 ].…”

“…To measure the substrate-induced dimerization, the active enzyme centrifugation (AEC) [ 33 ] was performed. Briefly, MERS-CoV M pro of 15 μl (1 mg/ml) was added into the small well of the band-forming centerpiece before the cell assembled.…”

Differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease

“…Multiple scans at different time intervals were then fitted to a continuous c(s, f/f 0 ) distribution model using the SEDFIT program [14,[21][22][23]. The protein concentrations of 13 lM in 50 mM phosphate buffer at various concentrations of urea.…”

Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein

“…The absorbance at 230 or 280 nm was monitored in continuous mode with a time interval of 300 s and a step size of 0.003 cm. Multiple scans at different time intervals were then fitted to a continuous c(M) distribution model using SEDFIT 37 38 .…”