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Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time
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Bioquímica
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JoVE Journal Bioquímica
Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

DOI:
10.3791/62655-v

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07:56 min

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May 30, 2021

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1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J. Safra Campus,The Hebrew University of Jerusalem, 2The Center for Nanoscience and Nanotechnology,The Hebrew University of Jerusalem

Capítulos

  • 00:05Introduction
  • 00:48Single-Molecule Protein-Induced Fluorescence Enhancement (smPIFE) Sample Preparation and Data Acquisition
  • 02:19smPIFE Burst Analysis
  • 05:07Results: Estimation of Mean Fluorescence Lifetime and Burst Recurrence Analysis
  • 07:12Conclusion

Summary

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Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in α-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Tags

Keywords: Time-resolvedProtein-induced Fluorescence EnhancementSingle-moleculeAlpha-synucleinStructural SubpopulationsLocal ConformationsSulfo-Cy3BSAWater Immersion ObjectiveLaser FocusPhoton DetectionFRETBurstsPhoton-HDF5

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