Abstract
Here, we describe the single molecule force spectroscopy (SMFS)-based experimental protocol we have recently used to single out different classes of conformations in a chimeric multimodular protein containing an intrinsically disordered (human Alpha Synuclein) domain. Details are provided regarding cloning, expression and purification of the chimeric polyprotein constructs, optimal surface preparation, SMFS data collection and filtering. Although the specificity of the issue and the ensemble of nonstandard techniques needed to perform the described procedures render this a rather unorthodox protocol, it is relatively straightforward to adapt it to the study of other protein domains.
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References
Uversky VN (2003) Protein folding revisited. A polypeptide chain at the folding–misfolding–nonfolding cross-roads: which way to go? Cell Mol Life Sci 60:1852–1871
Fink AL (2005) Natively unfolded proteins. Curr Opin Struct Biol 15:35–41
Uversky VN, Dunker AK (2010) Understanding protein non-folding. Biochim Biophys Acta 1804:1231–1264
Uversky VN, Longhi S (eds) (2009) Instrumental analysis of intrinsically disordered proteins: assessing structure and conformation. Wiley, Hoboken, NJ
Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev 6:197–208
Uversky VN (2010) Multitude of binding modes attainable by intrinsically disordered proteins: a portrait gallery of disorder-based complexes. Chem Soc Rev 2011 Mar; 40(3):1623–34. Epub 2010 Nov 3
Uversky VN (2010) Targeting intrinsically disordered proteins in neurodegenerative and protein dysfunction diseases: another illustration of the D(2) concept. Expert Rev Proteomics 7:543–564
Ferreon AC, Moran CR, Gambin Y, Deniz AA (2010) Single-molecule fluorescence studies of intrinsically disordered proteins. Methods Enzymol 472:179–204. http://www.ncbi.nlm.nih.gov/pubmed/20580965
Eliezer D (2007) Characterizing residual structure in disordered protein states using nuclear magnetic resonance. Methods Mol Biol (Clifton, NJ) 350:49–67
Eliezer D (2009) Biophysical characterization of intrinsically disordered proteins. Curr Opin Struct Biol 19:23–30
Sandal M, Valle F, Tessari I, Mammi S, Bergantino E, Musiani F, Brucale M, Bubacco L, Samori B (2008) Conformational equilibria in monomeric alpha-synuclein at the single-molecule level. PLoS Biol 6:99–108
Brucale M, Sandal M, Di Maio S, Rampioni A, Tessari I, Tosatto L, Bisaglia M, Bubacco L, Samori B (2009) Pathogenic mutations shift the equilibria of alpha-synuclein single molecules towards structured conformers. Chembiochem 10:176–183
Steward A, Toca-Herrera JL, Clarke J (2002) Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy. Protein Sci 11:2179–2183
Sandal M, Benedetti F, Brucale M, Gomez-Casado A, Samori B (2009) Hooke: an open software platform for force spectroscopy. Bioinformatics (Oxford, England) 25:1428–1430
Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298
Wagner P, Hegner M, Guntherodt HJ, Semenza G (1995) Formation and in-situ modification of monolayers chemisorbed on ultraflat template-stripped gold surfaces. Langmuir 11:3867–3875
Acknowledgments
We warmly acknowledge Dr. Massimo Sandal, Dr. Francesco Valle, Dr. Fabrizio Benedetti, Dr. Francesco Musiani, Dr. Sabrina Bonuso, Dr. Laura Civiero, and Dr. Elisa Belluzzi for their contribution to constant refinement of the laboratory procedures during their respective stay in BS’s and LB’s groups.
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Brucale, M., Tessari, I., Bubacco, L., Samorì, B. (2012). Single-Molecule Force Spectroscopy of Chimeric Polyprotein Constructs Containing Intrinsically Disordered Domains. 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_3
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DOI: https://doi.org/10.1007/978-1-4614-3704-8_3
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