GFP as potential cellular viscosimeter.

Area of Science:

• Biophysics
• Protein Dynamics
• Fluorescence Spectroscopy

Background:

• Proteins like green fluorescent protein (GFP) are much larger than solvent molecules (e.g., water, glycerol).
• Microscopic viscosity, affecting protein diffusion, is often assumed equal to macroscopic solvent viscosity.
• Stokes-Einstein equations predict diffusion coefficients are inversely proportional to viscosity.

Purpose of the Study:

• To investigate if green fluorescent protein (GFP) senses macroscopic viscosity in water/glycerol mixtures.
• To determine if Stokes-Einstein relations hold for GFP diffusion in varying solvent environments.
• To explore how co-solvents affect microscopic and macroscopic viscosity and their impact on protein diffusion.

Main Methods:

• Time-resolved fluorescence anisotropy measurements to study rotational diffusion of GFP.
• Fluorescence correlation spectroscopy (FCS) to analyze translational diffusion of GFP.
• Experiments conducted in water/glycerol mixtures with and without co-solvents.

Main Results:

• GFP diffusion coefficients in simple water/glycerol mixtures align with macroscopic viscosity predictions.
• When co-solvents (macromolecules) are present, microscopic and macroscopic viscosities differ significantly.
• Rotational and translational diffusion sense different microscopic viscosities, with rotation experiencing lower viscosity.

Conclusions:

• Stokes-Einstein relations require adaptation when large co-solvents alter the microenvironment around proteins.
• A microscopic viscosity parameter, dependent on scaling factors, is proposed.
• Findings have implications for understanding protein diffusion coefficients within living cells.