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SNAREs pp 345-359 | Cite as

Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS

  • Pei Li
  • Yawei Dai
  • Markus Seeger
  • Yan-Wen Tan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

Fӧrster-type resonance energy transfer (FRET) with fluorescence cross-correlation spectroscopy (FCCS) is a powerful combination for observing intramolecular conformational dynamics on the micro- to millisecond timescale. Owing to its sensitivity to various physical parameters, FRET-FCCS has also been used to detect the reagent effects on proteins dynamics. However, FRET-FCCS alone cannot acquire the exact measurements of rate constants. Moreover, this technique is highly model dependent and can be unreliable when determining too many parameters at once. On the contrary, single-molecular FRET (smFRET) can measure the conformational states and their populations directly, although it is extremely challenging for probing fast dynamics under 1 ms. In this chapter, we describe how to realize sub-millisecond conformational dynamics measurements of a SNARE protein Ykt6 under lipid environments by smFRET and FRET-FCCS. This protocol includes sample preparation, microscope designs, data acquisition, and analysis methodology.

Key words

Intramolecular conformational dynamics Lipid interaction smFRET FRET-FCCS 

Notes

Acknowledgments

We thank Song Song and Jian Chang for building the microscopes. This work was sponsored by National Natural Science Foundation of China (No. 11274076 and 21773039). The original experiments were conducted by Yawei Dai and Markus Seeger in Yan-Wen Tan’s lab at State Key Laboratory of Surface Physics and Department of Physics, Fudan University.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Pei Li
    • 1
  • Yawei Dai
    • 1
    • 2
  • Markus Seeger
    • 3
  • Yan-Wen Tan
    • 1
  1. 1.State Key Laboratory of Surface Physics and Department of PhysicsFudan UniversityShanghaiChina
  2. 2.Department of PhysicsThe University of Hong KongHong KongChina
  3. 3.Biological Imaging CenterTechnische Universität MünchenMünchenGermany

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