Abstract
The size of intrinsically disordered proteins (IDPs) is large compared to their molecular mass and the resulting mass-to-size ratio is unusual. The sedimentation coefficient, which can be obtained from sedimentation velocity (SV) analytical ultracentrifugation (AUC), is directly related to this ratio and can be easily interpreted in terms of frictional ratio. This chapter is a step-by-step protocol for setting up, executing and analyzing SV experiments in the context of the characterization of IDPs, based on a real case study of the partially folded C-terminal domain of Sendai virus nucleoprotein.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Receveur-Brechot V, Bourhis JM, Uversky VN et al (2006) Assessing protein disorder and induced folding. Proteins 62:24–45
Uversky VN (2002) What does it mean to be natively unfolded? Eur J Biochem 269:2–12
Ebel C (2007) Analytical ultracentrifugation. State of the art and perspectives. In: Uversky VN, Permyakov EA (eds) Protein structures: methods in protein structure and stability analysis. Nova, New York
Lebowitz J, Lewis MS, Schuck P (2002) Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci 11:2067–2079
Ebel C (2004) Analytical ultracentrifugation for the study of biological macromolecules. Prog Colloid Polym Sci 127:73–82
Howlett GJ, Minton AP, Rivas G (2006) Analytical ultracentrifugation for the study of protein association and assembly. Curr Opin Chem Biol 10:430–436
Manon F, Ebel C (2010) Analytical ultracentrifugation, a useful tool to probe intrinsically disordered proteins. In: Uversky VN, Longhi S (eds) Instrumental analysis of intrinsically disordered proteins: assessing structure and conformation. Wiley, Hoboken
Demeler B, Behlke J, Ristau O (2000) Molecular parameters from sedimentation velocity experiments: whole boundary fitting using approximate and numerical solutions of Lamm equation. Methods Enzymol 321:38–66
Schuck P (1998) Sedimentation analysis of noninteracting and self-associating solutes using numerical solutions to the Lamm equation. Biophys J 75:1503–1512
Stafford WF, Sherwood PJ (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
Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J 78:1606–1619
Uversky VN (2007) Size-exclusion chromatography in protein structure analysis. In: Uversky VN, Permyakov EA (eds) Protein structures: methods in protein structure and stability analysis. Nova, New York
Uversky VN (2010) Analysing intrinsically disordered proteins by size-exclusion chromatography. In: Uversky VN, Longhi S (eds) Instrumental analysis of intrinsically disordered proteins: assessing structure and conformation. Wiley, Hoboken
Arnheiter H, Davis NL, Wertz G et al (1985) Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis. Cell 41:259–267
Jensen MR, Houben K, Lescop E et al (2008) Quantitative conformational analysis of partially folded proteins from residual dipolar couplings: application to the molecular recognition element of Sendai virus nucleoprotein. J Am Chem Soc 130:8055–8061
Balbo A, Zhao H, Brown PH et al (2009) Assembly, loading, and alignment of an analytical ultracentrifuge sample cell. J Vis Exp 33:1530
Saluja A, Fesinmeyer RM, Hogan S et al (2010) Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation. Biophys J 99:2657–2665
Balbo A, Minor KH, Velikovsky CA et al (2005) Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation. Proc Natl Acad Sci U S A 102:81–86
le Maire M, Arnou B, Olesen C et al (2008) Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+-ATPase as an example. Nat Protoc 3:1782–1795
Brown PH, Schuck P (2006) Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys J 90:4651–4661
Buisson M, Valette E, Hernandez JF et al (2001) Functional determinants of the Epstein-Barr virus protease. J Mol Biol 311:217–228
Schuck P (2003) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem 320:104–124
Solovyova A, Schuck P, Costenaro L et al (2001) Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents. Biophys J 81:1868–1880
Tcherkasskaya O, Uversky VN (2001) Denatured collapsed states in protein folding: example of apomyoglobin. Proteins 44:244–254
Cantor CR, Schimmel PR (1980) Techniques for the study of biological structure and function. Biophysical chemistry. W. H. Freeman, San Franscisco
Acknowledgments
We thank the IBS platform of the Partnership for Structural Biology and the Institut de Biologie Structurale in Grenoble (PSB/IBS), for the assistance and access to the instrument of AUC.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
Salvay, A.G., Communie, G., Ebel, C. (2012). Sedimentation Velocity Analytical Ultracentrifugation for Intrinsically Disordered Proteins. In: Uversky, V., Dunker, A. (eds) Intrinsically Disordered Protein Analysis. Methods in Molecular Biology, vol 896. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-3704-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-4614-3704-8_6
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4614-3703-1
Online ISBN: 978-1-4614-3704-8
eBook Packages: Springer Protocols