Abstract
α-Synuclein’s physiology and pathology have been linked by numerous reports to its ability to bind and remodel membranes, especially at synaptic terminals. It is therefore critical for researchers investigating the determinants of these interactions to rely on methods capable of providing an accurate and complete physicochemical snapshot of the binding events. Circular dichroism (CD) and isothermal titration calorimetry (ITC) are established techniques for the study of binding equilibria in biological systems and, especially when used in combination, allow a thorough characterization of the protein-lipid interplay.
Here we provide general guidelines and describe some common pitfalls of these experiments. This protocol describes the preparation of small unilamellar vesicles (SUVs), mimicking the curved bilayers α-synuclein normally interacts with, the CD-monitored titration of α-synuclein with SUVs, the ITC (lipid-into-protein) experiment, and the subsequent data analysis using an n independent binding site model.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Davidson WS, Jonas A, Clayton DF, George JM (1998) Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem 273:9443–9449. https://doi.org/10.1074/jbc.273.16.9443
Takamori S, Holt M, Stenius K et al (2006) Molecular anatomy of a trafficking organelle. Cell 127:831–846. https://doi.org/10.1016/j.cell.2006.10.030
George JM, Jin H, Woods WS, Clayton DF (1995) Characterization of a novel protein regulated during the critical period for song learning in the zebra finch. Neuron 15:361–372. https://doi.org/10.1016/0896-6273(95)90040-3
Jao CC, Hegde BG, Chen J et al (2008) Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement. Proc Natl Acad Sci USA 105:19666–19671. https://doi.org/10.1073/pnas.0807826105
Chandra S, Chen X, Rizo J et al (2003) A broken alpha-helix in folded alpha-Synuclein. J Biol Chem 278:15313–15318. https://doi.org/10.1074/jbc.M213128200
Ulmer TS, Bax A, Cole NB, Nussbaum RL (2005) Structure and dynamics of micelle-bound human alpha-synuclein. J Biol Chem 280:9595–9603. https://doi.org/10.1074/jbc.M411805200
Lautenschläger J, Kaminski CF, Kaminski Schierle GS (2017) α-Synuclein—regulator of exocytosis, endocytosis, or both? Trends Cell Biol 27:468–479. https://doi.org/10.1016/j.tcb.2017.02.002
Jo E, Fuller N, Rand RP et al (2002) Defective membrane interactions of familial Parkinson’s disease mutant A30P alpha-synuclein. J Mol Biol 315:799–807. https://doi.org/10.1006/jmbi.2001.5269
Choi W, Zibaee S, Jakes R et al (2004) Mutation E46K increases phospholipid binding and assembly into filaments of human alpha-synuclein. FEBS Lett 576:363–368. https://doi.org/10.1016/j.febslet.2004.09.038
Blume A, Garidel P (1999) Lipid model membranes and biomembranes. In: Kemp R (ed) Handbook of thermal analysis and calorimetry, From macromolecules to man, vol 4. Elsevier Science B.V, Amsterdam, pp 109–173
Nuscher B, Kamp F, Mehnert T et al (2004) Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study. J Biol Chem 279:21966–21975. https://doi.org/10.1074/jbc.M401076200
Bartels T, Kim NC, Luth ES, Selkoe DJ (2014) N-alpha-acetylation of α-synuclein increases its helical folding propensity, GM1 binding specificity and resistance to aggregation. PLoS One 9:1–10. https://doi.org/10.1371/journal.pone.0103727
Fasman GD (1996) Circular dichroism and the conformational analysis of biomolecules. Plenum Press, New York, NY
Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751:119–139. https://doi.org/10.1016/j.bbapap.2005.06.005
Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1:2876–2890. https://doi.org/10.1038/nprot.2006.202
Wiseman T, Williston S, Brandts JF, Lin LN (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137. https://doi.org/10.1016/0003-2697(89)90213-3
Freire E, Mayorga OL, Straume M (1990) Isothermal titration calorimetry. Anal Chem 62:950A–959A. https://doi.org/10.1021/ac00217a002
Fisher HF, Singh N (1995) Calorimetric methods for interpreting protein-ligand interactions. Methods Enzymol 259:194–221. https://doi.org/10.1016/0076-6879(95)59045-5
Indyk L, Fisher HF (1998) Theoretical aspects of isothermal titration calorimetry. Methods Enzymol 295:350–364. https://doi.org/10.1016/S0076-6879(98)95048-0
Noble JE, Bailey MJA (2009) Quantitation of protein. Methods Enzymol 463:73–95. https://doi.org/10.1016/S0076-6879(09)63008-1
Pace CN, Vajdos F, Fee L et al (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423. https://doi.org/10.1002/pro.5560041120
Szoka F, Papahadjopoulos D (1980) Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu Rev Biophys Bioeng 9:467–508. https://doi.org/10.1146/annurev.bb.09.060180.002343
MacDonald RC, MacDonald RI, Menco BP et al (1991) Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta 1061:297–303. https://doi.org/10.1016/0005-2736(91)90295-J
Bangham A, Hill M, Miller N (1974) Preparation and use of liposomes as models of biological membranes. In: Korn ED (ed) Methods in membrane biology, vol 1. Plenum Press, New York, NY, pp 1–68
Galvagnion C, Buell AK, Meisl G et al (2015) Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation. Nat Chem Biol 11:229–234. https://doi.org/10.1038/nchembio.1750
MicroCal ITC analysis software using Origin™ MAN0577-02-EN-00 (May 2015)
Cantor CR, Schimmel PR (1980) Biophysical chemistry, Part III: The behavior of biological macromolecules. W.H. Freeman & Co., New York, NY
Wieprecht T, Seelig J (2002) Isothermal titration calorimetry for studying interactions between peptides and lipid membranes. Curr Top Membr 52:31–56
Bartels T, Ahlstrom LS, Leftin A et al (2010) The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding. Biophys J 99:2116–2124. https://doi.org/10.1016/j.bpj.2010.06.035
Ferreon ACM, Deniz AA (2007) Alpha-synuclein multistate folding thermodynamics: implications for protein misfolding and aggregation. Biochemistry 46:4499–4509. https://doi.org/10.1021/bi602461y
Chen PS, Toribara TY, Huber W (1956) Microdetermination of phosphorus. Anal Chem 28:1756–1758. https://doi.org/10.1021/ac60068a036
Marsh D (2013) Handbook of lipid bilayers, 2nd edn. CRC Press, Boca Raton, FL
Litman BJ (1972) Effect of light scattering on the circular dichroism of biological membranes. Biochemistry 11:3243–3247. https://doi.org/10.1021/bi00767a018
Mao D, Wallace BA (1984) Differential light scattering and absorption flattening optical effects are minimal in the circular dichroism spectra of small unilamellar vesicles. Biochemistry 23:2667–2673. https://doi.org/10.1021/bi00307a020
Acknowledgments
M.R. thanks Kelly L. Arnett and the Center for Macromolecular Interactions at the Harvard Medical School Department of Biological Chemistry and Molecular Pharmacology for assisting with CD and ITC measurements, revising this manuscript, and many helpful discussions; Alex E. Powers for helping with data collection and analysis; and Martina Astrid Rodda for editing and revising the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rovere, M. (2019). Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes. In: Bartels, T. (eds) Alpha-Synuclein. Methods in Molecular Biology, vol 1948. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9124-2_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9124-2_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9123-5
Online ISBN: 978-1-4939-9124-2
eBook Packages: Springer Protocols