A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Single-molecule Förster resonance energy transfer (smFRET) is crucial for quantitative biomolecular studies.
• Two-color smFRET offers limited insight into complex biological systems with multiple components.
• Understanding multicomponent systems requires correlating conformational changes with binding partner interactions.

Purpose of the Study:

• To demonstrate the capabilities and limitations of multicolor smFRET experiments.
• To present a multicolor smFRET setup and data analysis methodology.
• To apply multicolor smFRET to elucidate the function of complex biological systems.

Main Methods:

• Development and description of a multicolor smFRET setup.
• Presentation of a general analytical procedure for multicolor smFRET data.
• Application of 3D ensemble hidden Markov analysis for quantifying correlated dynamics.

Main Results:

• Multicolor smFRET enables simultaneous detection of conformational dynamics and nucleotide binding.
• Microscopic states within transient complexes were identified in the heat shock protein 90 system.
• Correlated dynamics and interactions were quantified, even out of equilibrium.

Conclusions:

• Multicolor smFRET provides unprecedented mechanistic insight into complex protein machines.
• This technique overcomes limitations of two-color smFRET for studying multicomponent biological systems.
• Multicolor smFRET is well-suited for sophisticated protein systems, advancing biological research.