Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems.
Strategies for the deconvolution of diffusion in the determination of size-distributions from sedimentation velocity experiments were examined and developed. On the basis of four different model systems, we studied the differential apparent sedimentation coefficient distributions by the time-derivative method, g(s*), and by least-squares direct boundary modeling, ls-g*(s), the integral sedimentation coefficient distribution by the van Holde-Weischet method, G(s), and the previously introduced differential distribution of Lamm equation solutions, c(s). It is shown that the least-squares approach ls-g*(s) can be extrapolated to infinite time by considering area divisions analogous to boundary divisions in the van Holde-Weischet method, thus allowing the transformation of interference optical data into an integral sedimentation coefficient distribution G(s). However, despite the model-free approach of G(s), for the systems considered, the direct boundary modeling with a distribution of Lamm equation solutions c(s) exhibited the highest resolution and sensitivity. The c(s) approach requires an estimate for the size-dependent diffusion coefficients D(s), which is usually incorporated in the form of a weight-average frictional ratio of all species, or in the form of prior knowledge of the molar mass of the main species. We studied the influence of the weight-average frictional ratio on the quality of the fit, and found that it is well-determined by the data. As a direct boundary model, the calculated c(s) distribution can be combined with a nonlinear regression to optimize distribution parameters, such as the exact meniscus position, and the weight-average frictional ratio. Although c(s) is computationally the most complex, it has the potential for the highest resolution and sensitivity of the methods described.
Schuck
Peter
P
Division of Bioengineering and Physical Science, Office of Research Services, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892, USA. pschuck@helix.nih.gov
Perugini
Matthew A
MA
Gonzales
Noreen R
NR
Howlett
Geoffrey J
GJ
Schubert
Dieter
D
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins; D002499:Centrifugation, Density Gradient; D007074:Immunoglobulin G / Q000737:chemistry; D008027:Light; D008055:Lipids / Q000737:chemistry; D015233:Models, Statistical; D011487:Protein Conformation; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D018139:Simplexvirus / Q000378:metabolism; D013997:Time Factors; D014461:Ultracentrifugation / Q000379:methods*
D036022:Capsid Proteins; D007074:Immunoglobulin G; D008055:Lipids