Mesophase separation and probe dynamics in protein–polyelectrolyte coacervates

Abstract

Protein-polyelectrolyte coacervates are self-assembling macroscopically monophasic biomacromolecular fluids whose unique properties arise from transient heterogeneities. The structures of coacervates formed at different conditions of pH and ionic strength from poly(dimethyldiallylammonium chloride) and bovine serum albumin (BSA), were probed using fluorescence recovery after photobleaching. Measurements of self-diffusion in coacervates were carried out using fluorescein-tagged BSA, and similarly tagged Ficoll, a non-interacting branched polysaccharide with the same size as BSA. The results are best explained by temporal and spatial heterogeneities, also inferred from static light scattering and cryo-TEM, which indicate heterogeneous scattering centers of several hundred nm. Taken together with previous dynamic light scattering and rheology studies, the results are consistent with the presence of extensive dilute domains in which are embedded partially interconnected 50-700 nm dense domains. At short length scales, protein mobility is unobstructed by these clusters. At intermediate length scales, proteins are slowed down due to tortuosity effects within the blind alleys of the dense domains, and to adsorption at dense/dilute domain interfaces. Finally, at long length scales, obstructed diffusion is alleviated by the break-up of dense domains. These findings are discussed in terms of previously suggested models for protein-polyelectrolyte coacervates. Possible explanations for the origin of mesophase separation are offered.

From Water and Ions to Crowded Biomacromolecules:In VivoStructuring of a Prokaryotic Cell

“…The very fast picosecond dynamics of water fits well with the already well-established and surprisingly fast diffusion of metabolites and biomacromolecules in cells and organelles (and in corresponding systems in vitro), as recently reviewed (48,140), and is corroborated by similar results for diffusion in vitro in nanopores of complex polymeric coacervates, which is also slowed down by a factor of only about 10 compared to that of pure water (40,108,109).…”

Effect of Polyelectrolyte Structure on Protein−Polyelectrolyte Coacervates: Coacervates of Bovine Serum Albumin with Poly(diallyldimethylammonium chloride) versus Chitosan

“…This hypothesis is supported by a further decrease in that exponent with increasing protein charge (Table 3). Reduced connectivity at higher protein charge could also explain why the fast DLS mode for PDAD-MAC-BSA increases monotonically with pH, 23,24 an effect previously overlooked, which we can now attribute to less constrained dilute mesophase volumes when clusters shrink. The dynamic breakup of these clusters is slowed down, leading to more prominent DLS slow modes for PDADMAC-BSA coacervates at high pH and low I.…”

“…In coacervates, these aggregates must be elemental components of the mesophase dense domains previously deduced from DLS, rheology, and fluorescence recovery after photobleaching. 23,24 It is not known whether a different method of coacervate preparation (e.g., mixing of protein and polyelectrolyte solutions preadjusted to pH > pH φ ) would yield the same microstructure, but the repeatability of DLS spectra after months of storage and the reproducibility of data for samples prepared in different laboratories indicate the equilibrated nature of coacervates prepared by the present approach.…”

“…For chitosan-BSA coacervates, two fast modes are identified: F1 (identical to the fast mode typically seen for PDADMAC coacervates) and a second fast mode F2. In the PDADMAC-BSA coacervates, F1 has been attributed to the "free" BSA diffusion in the dilute domains, 23,24 but in this case it is not the dominant mode. F2 appears only in the chitosan-BSA coacervate and where it is the dominant mode (Table 1).…”

“…Because the term "coacervate" often refers to the metastable liquid-liquid suspension, it is important to point out that all of the measurements in here and in our previous work refer to the discontinuous phases, isolated by centrifugation as optically clear, viscous fluids, which, in contrast to their precursor suspensions, are true equilibrium systems, stable for months. 23,24 Because the unusual properties of protein-polyelectrolyte coacervates (e.g., the absence of aggregation at high protein concentration, unusually large protein diffusivities coupled with large bulk viscosity) appear to arise from mesophase heterogeneity, 24 the striking difference between coacervates prepared with chitosan vs PDADMAC appeared to offer clues about structure-property relations in proteinpolyelectrolyte coacervates. We report here results from rheology, dynamic light scattering, and small-angle neutron scattering that support the proposal that the structure and dynamics of equilibrium mesophase dense domains dominate the behavior of these fluids.…”