Methods for the Design and Analysis of Sedimentation Velocity and Sedimentation Equilibrium Experiments with Proteins

Borries Demeler1

1 Department of Computer Science, The University of Texas at San Antonio, San Antonio, Texas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.13
DOI:  10.1002/0471140864.ps0713s60
Online Posting Date:  April, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Analytical ultracentrifugation experiments play an integral role in the solution phase characterization of recombinant proteins and other biological macromolecules. This unit discusses the design of sedimentation velocity and sedimentation equilibrium experiments performed with a Beckman Optima XL‐A or XL‐I analytical ultracentrifuge. Optimal instrument settings and experimental design considerations are explained, and strategies for the analysis of experimental data with the UltraScan data analysis software package are presented. Special attention is paid to the strengths and weaknesses of the available detectors, and guidance is provided on how to extract maximum information from analytical ultracentrifugation experiments. Curr. Protoc. Protein Sci. 60:7.13.1‐7.13.24. © 2010 by John Wiley & Sons, Inc.

Keywords: analytical ultracentrifugation; solution studies; sedimentation velocity; sedimentation equilibrium; UltraScan; 2‐dimensional spectrum analysis; absorbance optics; intensity measurements

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Background
  • Experimental Design
  • Tips for Data Analysis
  • Data Management
  • Checklists
  • Experimental Analysis Flowchart
  • Conclusion
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bhattacharyya, S.K., Maciejewska, P., Börger, L., Stadler, M., Gülsün, A.M., Cicek, H.B., and Cölfen, H. 2006. Development of fast fiber based UV‐Vis multiwavelength detector for an ultracentrifuge. Prog. Colloid Polym. Sci. 131:9‐22.
   Brookes, E. and Demeler, B. 2007. Parsimonious regularization using genetic algorithms applied to the analysis of analytical ultracentrifugation experiments. GECCO Proceedings ACM. pp. 361‐368.
   Brookes, E. and Demeler, B. 2008. Parallel computational techniques for the analysis of sedimentation velocity experiments in UltraScan. Prog. Colloid Polym. Sci. 286:138‐148.
   Brookes, E., Cao, W., and Demeler, B. 2009. A two‐dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur. Biophys. J. In press.
   Cao, W. and Demeler, B. 2005. Modeling analytical ultracentrifugation experiments with an adaptive space‐time finite element solution of the Lamm equation. Biophys J. 89:1589‐1602.
   Cao, W. and Demeler, B. 2008. Modeling analytical ultracentrifugation experiments with an adaptive space‐time finite element solution for multi‐component reacting systems. Biophys. J. 95:54‐65.
   Cölfen, H., Laue, T.M., Wohlleben, W., Schilling, K., Karabudak, E., Langhorst, B.W., Brookes, E., Dubbs, B., Zollars, D., Rocco, M., and Demeler, B. 2009. The Open AUC Project. Eur. Biophys. J. In press.
   Demeler, B. 2005. UltraScan‐a comprehensive data analysis software package for analytical ultracentrifugation experiments. In Modern Analytical Ultracentrifugation: Techniques and Methods (D.J. Scott, S.E. Harding, and A.J. Rowe, eds.) pp. 210‐229. Royal Society of Chemistry, London.
   Demeler, B. 2009. UltraScan Version 9.9 ‐ A multi‐platform analytical ultracentrifugation data analysis software package: http://www.ultrascan.uthscsa.edu.
   Demeler, B. and van Holde, K.E. 2004. Sedimentation velocity analysis of highly heterogeneous systems. Anal. Biochem. 335:279‐288.
   Demeler, B. and Brookes, E. 2008. Monte Carlo analysis of sedimentation experiments. Prog. Colloid Polym. Sci. 286:129‐137.
   Demeler, B., Saber, H., and Hansen, J.C. 1997. Identification and interpretation of complexity in sedimentation velocity boundaries. Biophys. J. 72:397‐407.
   Demeler, B., Brookes, E., Wang, R., Shirf, V., and Kim, C.A. 2010. Characterization of reversible associations by sedimentation velocity with UltraScan. Macromol. Biosci. In press.
   Giebeler, R. 1992. The Optima XL‐A: A new analytical ultracentrifuge with a novel precision absorption optical system. In Analytical Ultracentrifugation in Biochemistry and Polymer Science (S.E. Harding, A.J. Rowe, and J.C. Horton, eds.) pp. 16‐25. Royal Society of Chemistry, Cambridge.
   Johnson, M.L., Correia, J.J., Yphantis, D.A., and Halvorson, H.R. 1981. Analysis of data from the analytical ultracentrifuge by nonlinear least squares techniques. Biophys. J. 36:575‐588.
   Kar, S.R., Kingsbury, J.S., Lewis, M.S., Laue, T.M., and Schuck, P. 2000. Analysis of transport experiments using pseudo‐absorbance data. Anal. Biochem. 285:135‐142.
   Kingsbury, J.S., Klimtchuk, E.S., Laue, T.M., Théberge, R., Costello, C.E., and Connors, L.H. 2008. The modulation of transthyretin tetramer stability by cysteine‐10 adducts and the drug diflunisal: Direct analysis by fluorescence‐detected analytical ultracentrifugation. J. Biol. Chem. 283:11887‐11896.
   Kroe, R.R. and Laue, T.M. 2009. NUTS and BOLTS: Applications of fluorescence detected sedimentation. Anal. Biochem. (In press).
   Kumar, D. 2006. Absorbance spectra for common buffer systems. http://uslims.uthscsa.edu/cauma/buffer2.php
   Lamm, O. 1929. Die Differentialgleichung der Ultrazentrifugierung. Ark. Mat. Astr. Fys. 21B:1‐4.
   Laue, T.M. 1996. Choosing which optical system of the Optima XL‐I analytical ultracentrifuge to use. Beckman Publication: http://www.beckmancoulter.com/literature/Bioresearch/1821a(a).pdf
   MacGregor, I.K., Anderson, A.L., and Laue, T.M. 2004. Fluorescence detection for the XLI Ultracentrifuge. Biophys. Chem. 108:165‐185.
   Schirf, V. and Planken, K.L. 2008. Analytical Ultracentrifuge User Guide, Volume 1: Hardware. (http://wiki.bcf.uthscsa.edu/aucmanual/)
   Schuck, P. 1998. Sedimentation analysis of noninteracting and self‐associating solutes using numerical solutions to the Lamm equation. Biophys. J. 75:1503‐1512.
   Schuck, P. and Demeler, B. 1999. Direct sedimentation analysis of interference optical data in analytical ultracentrifugation. Biophys. J. 76:2288‐2296.
   Stafford, W. 1992. Boundary analysis in sedimentation transport experiments: A procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal. Biochem. 203:295‐301.
   Stafford, W. 2009. Sedanal AUC data analysis software. http://rasmb.bbri.org/rasmb/windows/sedanal‐stafford/.
   Stafford, W.F. and Sherwood, P.J. 2004. Analysis of heterologous interacting systems by sedimentation velocity: Curve fitting algorithms for estimation of sedimentation coefficients, equilibrium and kinetic constants. Biophys. Chem. 108:231‐243.
   Vistica, J., Dam, J., Balbo, A., Yikilmaz, E., Mariuzza, R.A., Rouault, T.A., and Schuck, P. 2004. Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal. Biochem. 326:234‐256.
   Yphantis, D.A. 1964. Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3:297‐317.
   Yphantis, D.A., Lary, J.W., Stafford, W.F., Liu, S., Olsen, P.H., Hayes, D.B., Moody, T.P., Ridgeway, T.M., Lyons, D.A., and Laue, T.M. 1994. On‐line data acquisition for the Rayleigh interference optical system of the analytical ultracentrifuge. In Modern Analytical Ultracentrifugation (T.M. Schuster and T.M. Laue, eds.) pp. 209‐226. Birkhäuser, Boston, Mass.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library