Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS
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 wordsIntramolecular conformational dynamics Lipid interaction smFRET FRET-FCCS
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.
- 11.Schwille P (2001) Cross-correlation analysis in FCS. In: Rigler R, Elson ES (eds) Fluorescence correlationspectroscopy. Springer, New York, pp 360–378Google Scholar
- 15.Eid, J. S. (2002) Two-photon dual channel fluctuation correlation spectroscopy: theory and application. Ph.D. Dissertation, Urbana, IllinoisGoogle Scholar
- 16.Margittai M, Widengren J, Schweinberger E, Schroder GF, Felekyan S, Haustein E, Konig M, Fasshauer D, Grubmuller H, Jahn R, Seidel CA (2003) Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1. Proc Natl Acad Sci U S A 100:15516–15521CrossRefGoogle Scholar