Anomalous Diffusion of Proteins Due to Molecular Crowding

Abstract

We have studied the diffusion of tracer proteins in highly concentrated random-coil polymer and globular protein solutions imitating the crowded conditions encountered in cellular environments. Using fluorescence correlation spectroscopy, we measured the anomalous diffusion exponent alpha characterizing the dependence of the mean-square displacement of the tracer proteins on time, r(2)(t) approximately t(alpha). We observed that the diffusion of proteins in dextran solutions with concentrations up to 400 g/l is subdiffusive (alpha < 1) even at low obstacle concentration. The anomalous diffusion exponent alpha decreases continuously with increasing obstacle concentration and molecular weight, but does not depend on buffer ionic strength, and neither does it depend strongly on solution temperature. At very high random-coil polymer concentrations, alpha reaches a limit value of alpha(l) approximately 3/4, which we take to be the signature of a coupling between the motions of the tracer proteins and the segments of the dextran chains. A similar, although less pronounced, subdiffusive behavior is observed for the diffusion of streptavidin in concentrated globular protein solutions. These observations indicate that protein diffusion in the cell cytoplasm and nucleus should be anomalous as well, with consequences for measurements of solute diffusion coefficients in cells and for the modeling of cellular processes relying on diffusion.

New insights into diffusion in 3D crowded media by Monte Carlo simulations: effect of size, mobility and spatial distribution of obstacles

“…In crowded media, typically in in vivo and in a great number of in vitro processes, the existence of different macromolecular species, proteins, nucleic acids, organelles, etc., hinders the diffusion process. In these cases, eqn (1) must be generalized to deal with a more complex process, known as anomalous diffusion 16,31,[55][56] which can be described by: (2) where a is defined as the anomalous diffusion exponent (0 < a < 1 is the case of subdiffusion and a > 1 holds for the case of superdiffusion) and G is a generalized transport coefficient, also known as anomalous diffusion coefficient, of units …”

“…Experimental and theoretical data 14,23,25,[27][28]31,[39][40][41] reveal that, in crowded media, there is a succession of diffusion behaviours that can be identified with the three distinct regions observed in the log(<r 2 >/t) versus log(t) plots:…”

“…A great deal of information about motion of molecules in living cells has been obtained from intracellular measurements using different experimental techniques 13,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and from simulations 14,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] . Experimental data are usually obtained by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) techniques.…”

“…In order to give only a few examples, FRAP experiments have revealed anomalous diffusion of dextrans [17][18]23 , of proteins in cytoplasm 19,[21][22]25,33 and of proteins in dextrans 35 . FCS experiments have also shown anomalous diffusion of nanoparticles with different sizes in agarose gel 26 , of dextrans in HeLa cell cytoplasm 28 , of proteins in three-dimensional crowded media 27,31 , of Alexa488 lightemitting particles in extracellular matrices 32 , respective of the inert gold nanoparticles in the cytoplasm and nucleoplasm under various stress conditions 34 .…”

“…Moreover, from several experimental works, it is well known that the anomalous diffusion emerges on cytoplasmatic macromolecules and it depends on the size and conformation of the tracer particle and on the total protein concentration of the solution 28 . There are even experimental studies that show anomalous protein diffusion in in vitro, with the anomalous diffusion exponent decreasing continuously with increasing obstacle concentration and molecular weight 31,35 .…”