On the analysis of sedimentation velocity in the study of protein complexes.
Sedimentation velocity analytical ultracentrifugation has experienced a significant transformation, precipitated by the possibility of efficiently fitting Lamm equation solutions to the experimental data. The precision of this approach depends on the ability to account for the imperfections of the experiment, both regarding the sample and the instrument. In the present work, we explore in more detail the relationship between the sedimentation process, its detection, and the model used in the mathematical data analysis. We focus on configurations that produce steep and fast-moving sedimentation boundaries, such as frequently encountered when studying large multi-protein complexes. First, as a computational tool facilitating the analysis of heterogeneous samples, we introduce the strategy of partial boundary modeling. It can simplify the modeling by restricting the direct boundary analysis to species with sedimentation coefficients in a predefined range. Next, we examine factors related to the experimental detection, including the magnitude of optical aberrations generated by out-of-focus solution columns at high protein concentrations, the relationship between the experimentally recorded signature of the meniscus and the meniscus parameter in the data analysis, and the consequences of the limited radial and temporal resolution of the absorbance optical scanning system. Surprisingly, we find that large errors can be caused by the finite scanning speed of the commercial absorbance optics, exceeding the statistical errors in the measured sedimentation coefficients by more than an order of magnitude. We describe how these effects can be computationally accounted for in SEDFIT and SEDPHAT.
Brown
Patrick H
PH
Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892-5766, USA.
Balbo
Andrea
A
Schuck
Peter
P
D002498:Centrifugation / Q000379:methods*; D003198:Computer Simulation; D031521:Fractionation, Field Flow / Q000379:methods*; D008956:Models, Chemical; D009038:Motion; D046912:Multiprotein Complexes / Q000737:chemistry*
D046912:Multiprotein Complexes