Tracer diffusion of proteins in DNA solutions. 2. Green fluorescent protein in crowded DNA solutions

Proteins diffuse to their sites of action within cells in a crowded, strongly interacting environment of nucleic acids and other macromolecules. To investigate the dynamics of a typical globular protein in such an environment, we used fluorescence photobleaching recovery to measure the probe diffusion of green fluorescent protein (GFP) in dilute to highly concentrated aqueous solutions of glycerol, Ficoll 70, and persistence-length calf thymus DNA. In glycerol, GFP accurately obeyed the Stokes-Einstein equation that relates diffusion coefficient to solution viscosity. In concentrated Ficoll 70, GFP diffused moderately faster than predicted from viscosity, demonstrating the phenomenon of microviscosity in a molecularly heterogeneous solution. In DNA, the diffusion coefficient of GFP was markedly greater than predicted from the Stokes-Einstein equation, with deviations increasing at lower ionic strength. This behavior reflects microviscosity, Coulombic interactions, and the dynamics of probe diffusion in DNA solutions that had undergone the ordinary-extraordinary transition, which we demonstrated by dynamic Light scattering.