Protein self-diffusion in crowded solutions

Abstract

Macromolecular crowding in biological media is an essential factor for cellular function. The interplay of intermolecular interactions at multiple time and length scales governs a fine-tuned system of reaction and transport processes, including particularly protein diffusion as a limiting or driving factor. Using quasielastic neutron backscattering, we probe the protein self-diffusion in crowded aqueous solutions of bovine serum albumin on nanosecond time and nanometer length scales employing the same protein as crowding agent. The measured diffusion coefficient DðφÞ strongly decreases with increasing protein volume fraction φ explored within 7% ≤ φ ≤ 30%. With an ellipsoidal protein model and an analytical framework involving colloid diffusion theory, we separate the rotational D r ðφÞ and translational D t ðφÞ contributions to DðφÞ. The resulting D t ðφÞ is described by short-time self-diffusion of effective spheres. Protein self-diffusion at biological volume fractions is found to be slowed down to 20% of the dilute limit solely due to hydrodynamic interactions. macromolecular crowding | quasi-elastic neutron scattering | globular proteins T he interior of biological cells is a medium with a macromolecular volume fraction of up to 40%. This crowding crucially affects reaction kinetics and equilibria in the cell (1, 2). Cellular function and structure thus cannot be understood without a systematic understanding of both phase behavior and transport processes in crowded media. Diffusion is the main transport process for systems at low Reynolds numbers, governing many dynamic processes in nature (3). From the perspective of a single tracer molecule, all other molecules act as obstacles. In vivo diffusion coefficients for globular proteins in living cells (4-7) are strongly decreased compared to the in vitro diffusion coefficient in dilute buffer solutions. Systematic measurements of the tracer diffusion of proteins dissolved in concentrated suspensions of crowding agents, i.e., other proteins or polymers, reveal a complex dependence of the slowing down on the combination of tracer molecule and crowding agent (8-10). Furthermore, macromolecular crowding is found to induce subdiffusive behavior in several cases (11,12), being suggested as a slower but more reliable diffusive search process inside the cell (13). This anomalous diffusion process has been found also in theory and simulations (12-15) suggesting a crossover from subdiffusive behavior at small times to diffusive behavior at larger times.Proteins are macromolecules generally with a nonspherical shape and a nonhomogeneous surface charge, showing specific interactions with ligands. Furthermore, proteins not only show global motions like translational and rotational diffusion but also internal and interdomain motions. Therefore, proteins pose a challenge to colloid theory (16,17). In a recent simulation study Ando and Skolnick (4) revealed that using an equivalent-sphere model for macromolecules is a reasonable approximation to describe diffusion. Moreover, ...

Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding

“…S6). These results are also in good agreement with previous measurements of the effect of molecular crowding on diffusion (28,31,32) and indicate that severe osmotic compression decreases the mobility of Hog1p.…”

“…Increasing the protein concentration increases the probability of protein interactions and protein association rates and may lead to more rapid biochemical kinetics. However, the diffusion of proteins may be reduced in an overcrowded cytoplasm (31,32), thus slowing down biochemical kinetics.…”

Multiscale simulation of molecular processes in cellular environments

“…Experiments have probed a reduction of protein mobility by about one order of magnitude when the fraction of occupied volume approaches the level of the in vivo cellular environment (about 30% in volume). This reduced mobility depends on the fold and size of the proteins as well as on the nature of the crowders [8,[36][37][38]. It is debated whether solvent-mediated interactions play a key role on top of excluded volume interactions.…”

“…Hydrodynamic interactions are therefore key to understanding both the motion of proteins in dilute and in crowding conditions. This has been pointed out in seminal theoretical and experimental work [7][8][9][10].…”

Freezing lines of colloidal Yukawa spheres. I. A Rogers-Young integral equation study

“…Well-studied experimental systems which fall into this category are (sulfonate) polystyrene latex spheres in water 8,9 or in an ethanol-water mixture, 3,4 silica and polymethylacrylate (PMMA) spheres in an organic solvent (mixture), 6,10,11 and to some extent also charged globular proteins in water such as apoferritin 12,13 or bovine serum albumin (BSA). 14,15 The pair potential in Eq. (1) depends on four dimensionless parameter groups {L B /σ , Z , C s σ 3 , φ}, which can be controlled experimentally to a larger extent.…”