Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties

Area of Science:

• Biophysics
• Structural Biology
• Fluorescence Microscopy

Background:

• Photoconvertible fluorescent proteins (PCFP) are crucial for advanced imaging techniques like photoactivatable localization microscopy (PALM).
• Complex photophysical behavior of PCFPs limits quantitative and single-particle-tracking PALM applications.
• The mEos4b protein is a widely used PCFP in biological research.

Purpose of the Study:

• To investigate the conformational heterogeneity of the mEos4b fluorescent protein in its Green state.
• To elucidate the role of conformational states in the photophysical and photochemical properties of mEos4b.
• To understand how conformational changes influence photoswitching and photoconversion.

Main Methods:

• Multidimensional Nuclear Magnetic Resonance (NMR) spectroscopy.
• Ensemble fluorescence measurements.
• Analysis of side-chain protonation and hydrogen-bond networks.

Main Results:

• mEos4b populates two distinct conformations (A and B) in its Green state, differing in protonation of E212 and H62.
• Conformational interconversion is accelerated by UV light, causing a population shift.
• State A exhibits more efficient photoswitching and photoconversion, while State B shows increased photobleaching.

Conclusions:

• Conformational heterogeneity plays a critical role in the photochemistry of fluorescent proteins.
• Understanding these states is essential for optimizing PCFP performance in advanced microscopy.
• This study provides insights into the molecular mechanisms governing PCFP behavior.