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Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling
Chapitres
- 00:05Titre
- 01:32Protein Expression Cysteine Mutation and mRNA Synthesis
- 02:07Oocyte Removal
- 03:36Oocyte Defoculation and mRNA Injection
- 05:05Site-specific Fluorescence Labeling
- 06:07Electrophysiology
- 07:26Anisotropy Measurements and Determination of Distance Constraints
- 09:17Relative Movement of Protein Subunits
- 10:07Results: Current and Fluorescence Changes with Site Specific Fluorophore Labelings
- 11:38Conclusion
We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.
Tags
Conformational DynamicsMembrane ProteinsSite-directed Fluorescence LabelingTwo Electrode Voltage Clamp Electrophysiology (TEVC)Ion TransportIon ChannelsIon PumpsTransportersSite-specific Fluorophore LabelingConformational DynamicsVoltage-clamp FluorometryCysteine ReplacementSite-directed Fluorophore LabelingMolecular MotionDistance ConstraintsXenopus Leavis OocytesFunctional ExpressionFluorescence Signal
