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

Area of Science:

• Biophysics
• Molecular Dynamics
• Protein Science

Background:

• Förster-type resonance energy transfer (FRET) combined with fluorescence cross-correlation spectroscopy (FRET-FCCS) is valuable for studying protein dynamics but struggles with precise rate constant determination and model dependency.
• Single-molecule FRET (smFRET) directly measures conformational states and populations but faces challenges in probing dynamics faster than 1 millisecond.

Purpose of the Study:

• To develop and describe a protocol for measuring sub-millisecond conformational dynamics of proteins in lipid environments.
• To overcome the limitations of FRET-FCCS and smFRET for fast dynamic measurements using a combined approach.

Main Methods:

• Utilized a combination of single-molecule Förster resonance energy transfer (smFRET) and FRET with fluorescence cross-correlation spectroscopy (FRET-FCCS).
• Focused on the SNARE protein Ykt6 within lipid environments.
• Detailed sample preparation, microscope design, data acquisition, and analysis methodologies for sub-millisecond dynamics.

Main Results:

• Successfully demonstrated the capability to measure sub-millisecond conformational dynamics.
• Provided a comprehensive protocol applicable to studying protein dynamics in complex environments.

Conclusions:

• The integrated smFRET and FRET-FCCS approach enables robust measurement of fast protein conformational dynamics.
• This protocol offers a powerful tool for investigating protein behavior, such as the Ykt6 SNARE protein, in biologically relevant lipid environments.