Size-distribution Analysis of Proteins by Analytical Ultracentrifugation: Strategies and Application to Model Systems

Biophys J. 2002 Feb;82(2):1096-111. doi: 10.1016/S0006-3495(02)75469-6.

Abstract

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.

MeSH terms

  • Animals
  • Biophysical Phenomena
  • Biophysics
  • Capsid / chemistry
  • Capsid Proteins
  • Centrifugation, Density Gradient
  • Immunoglobulin G / chemistry
  • Light
  • Lipids / chemistry
  • Models, Statistical
  • Protein Conformation
  • Reproducibility of Results
  • Scattering, Radiation
  • Simplexvirus / metabolism
  • Time Factors
  • Ultracentrifugation / methods*

Substances

  • Capsid Proteins
  • Immunoglobulin G
  • Lipids