GFP as potential cellular viscosimeter

Abstract

The molecular dimensions of proteins such as green fluorescent protein (GFP) are large as compared to the ones of solvents like water or glycerol. The microscopic viscosity, which determines the resistance to diffusion of, e.g. GFP, is then the same as that determined from the resistance of the solvent to flow, which is known as macroscopic viscosity. GFP in water/glycerol mixtures senses this macroscopic viscosity, because the translational and rotational diffusion coefficients are proportional to the reciprocal value of the viscosity as predicted by the Stokes-Einstein equations. To test this hypothesis, we have performed time-resolved fluorescence anisotropy (reporting on rotational diffusion) and fluorescence correlation spectroscopy (reporting on translational diffusion) experiments of GFP in water/glycerol mixtures. When the solvent also contains macromolecules of similar or larger dimensions as GFP, the microscopic and macroscopic viscosities can be markedly different and the Stokes-Einstein relations must be adapted. It was established from previous dynamic fluorescence spectroscopy observations of diffusing proteins with dextran polysaccharides as co-solvents (Lavalette et al 2006 Eur. Biophys. J. 35 517-22), that rotation and translation sense a different microscopic viscosity, in which the one arising from rotation is always less than that from translation. A microscopic viscosity parameter is defined that depends on scaling factors between GFP and its immediate environment. The direct consequence is discussed for two reported diffusion coefficients of GFP in living cells.

Time-Resolved Fluorescence Anisotropy and Molecular Dynamics Analysis of a Novel GFP Homo-FRET Dimer

“…6 a , as fluorescence depolarization only occurs through Brownian rotational motion. For eGFP in PBS, we found a rotational correlation time of 16.46 ± 0.20 ns, which agrees well with previous studies ( 18 , 37 , 61 , 80 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 ). As glycerol was added, the Brownian rotational motion slowed and the rotational correlation time increased, up to 123 ± 6 ns in 50% glycerol.…”

“…1 ), the actual rotational correlation time of the GFP monomer in water can be recalculated by multiplying the result of the simulation by a scaling factor 1/0.321, which yields 14.3 ± 0.1 ns. This scaled value is in reasonable agreement with the experimentally measured rotational correlation time, θ = 16.5 ± 0.2 ns ( 18 , 85 , 86 , 87 ).…”

“…The eGFPs are considered to be motionless during the timescale of the fluorescence decay because the fluorescence lifetime of eGFP is much shorter (∼2.5 ns ( 11 , 78 , 79 , 80 ); Table S1 ) than its rotational correlation time (∼15 ns in water ( 18 , 37 , 61 , 80 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 ); Table S2 ). Therefore, the fluorescence anisotropy is assumed to be exclusively determined by the process of energy transfer.…”

“…In fluorescence anisotropy studies, the rotational mobility of the fluorophore is described by the rotational correlation time, which accounts for the time it takes the fluorophore to rotate by 1 radian ( 17 ). This property is sensitive to viscosity ( 18 ), which can be imaged via time-resolved fluorescence anisotropy imaging ( 19 ).…”

Universal guidelines for the conversion of proteins and dyes into functional nanothermometers

“…This guide will also be useful in the case that the fluorescent protein can be bioengineered to have a desired size and FL. For example, previous publications have reported green fluorescent protein to be an excellent ABNT in solution and intracellularly (cancer cells and live organisms) . Approximating a fluorescent protein Stokes radius of 2.4 nm (for the monomer) and their FL, which typically ranges from 2 to 5.5 ns , the temperature sensitivity of different fluorescent proteins can be estimated from Figure E.…”