Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes.
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.
['Rovere|Matteo|M|']
D002151:Calorimetry D002462:Cell Membrane D002942:Circular Dichroism D000078332:Data Analysis D008051:Lipid Bilayers D008081:Liposomes D011485:Protein Binding D013696:Temperature D051844:alpha-Synuclein
Circular dichroism Isothermal titration calorimetry α-Synuclein Small unilamellar vesicles