Metastable states such as liquid-liquid phase separation, aggregation and gelation can affect protein crystallization but their positive or negative effects are only partially understood. In this work, mixtures of PEG (MW 10 kDa) and a large model protein, glucose isomerase (MW 173 kDa), have been studied to characterize the effect of a metastable liquid-liquid phase separation on protein crystallization. Fluorescence labeling allowed confocal fluorescence microscopy observations and quantification of the partitioning of the protein and PEG between the liquid phases and showed two steps in the crystallization process. Two crystallization mechanisms within the liquid domain were revealed, yielding two different polymorphs. With one polymorph, few crystals nucleated and grew droplet-by-droplet in the dispersed concentrated liquid phase, while for the other homogeneous crystal nucleation and growth occurred independently and simultaneously in numerous droplets of the concentrated phase. The results demonstrate the substantial possible complexity of crystallization behavior, as well as its sensitivity to the location of the conditions on the phase diagram and to the physicochemical properties of the system.

This paper reports on the microstructures formed in aqueous solutions containing mixtures of sodium dodecyl sulfate (SDS) and a photosensitive, bolaform surfactant, bis(trimethylammoniumhexyloxy)azobenzene dibromide (BTHA). By using quasi-elastic light scattering and small-angle neutron scattering, we determined that aqueous solutions containing SDS and the trans isomer of BTHA (0.1 wt % total surfactant, 15 mol % BTHA, 85 mol % SDS) form vesicles with average hydrodynamic diameters of 1350 +/- 50 angstroms and bilayer thicknesses of 35 +/- 2 angstroms. The measured bilayer thickness is consistent with a model of the vesicle bilayer in which the trans isomer of BTHA spans the bilayer. Upon illumination with UV light, the BTHA underwent photoisomerization to produce a cis-rich photostationary state (80% cis isomer). We measured this photoisomerization to drive the reorganization of vesicles into cylindrical aggregates with cross-sectional radii of 19 +/- 3 angstroms and average hydrodynamic diameters of 240 +/- 50 angstroms. Equilibration of the cis-rich solution in the dark at 25 degrees C for 12 h or illumination of the solution with visible light leads to the recovery of the trans-rich photostationary state of the solution and the reformation of vesicles, thus demonstrating the potential utility of this system as the basis of a tunable fluid.

Addition of the hydrotropic salt sodium tosylate (TSNa) to solutions of a polymerizable cationic surfactant, methacryloyloxyundecyltrimethylammonium bromide (MUTB), leads to a transition from spherical to wormlike micelles about 40 nm in length. The wormlike micelles were successfully polymerized to yield stable, single-phase solutions of polymerized micelles. The polymerized wormlike micelles were several hundred nanometers long, although their cross-sectional radius remained unchanged (∼2 nm). The spherical to wormlike micelle transition, and the wormlike micelles polymerized at different molar ratios of TSNa to MUTB, were characterized using quasi-elastic and static light scattering, small-angle neutron scattering, NMR, and cryogenic transmission electron microscopy.

The interactions leading to crystallization of the integral membrane protein bacteriorhodopsin solubilized in n-octyl-beta-D-glucoside were investigated. Osmotic second virial coefficients were measured by self-interaction chromatography in the presence of sodium malonate, sodium formate and ammonium sulfate. Attractive protein-detergent complex (PDC) interactions were observed as the surfactant cloud-point temperature was approached for each salt, suggesting that surfactant interactions may play an important role in promoting PDC crystallization. Dynamic light scattering and tensiometry measurements show that the interaction trends are strongly influenced by micelle structure and surfactant phase behavior, both of which are sensitive to salt and surfactant concentration. Overall, detailed investigations using a combination of experimental techniques can provide insight into the complex nature of PDC interactions, which is essential to developing rational approaches to membrane-protein crystallization.

Thermoresponsive, pH-sensitive fibrous structures and gels are formed in aqueous mixtures of the amino acid lysine with oppositely charged sodium alkyl sulfate surfactants. The formation of these assemblies depends on the chain length of the surfactant, which is varied between 8 and 16, the chirality and degree of protonation of the amino acid, and the molar ratio of these species. Self-assembly of the fibers occurs when specific lysine enantiomers are in solution and for pH conditions in which the majority of the amine groups are protonated (i.e., at near-equimolar amounts of HCl and lysine). Racemic mixtures of lysine do not form fibers with sodium dodecyl sulfate. Micelles are the fiber precursors, and the fibers, which can be hundreds of micrometers long, entangle to form gels. With increasing temperature, the gels melt, the fibers dissolve, and a single micellar phase forms. The micelles elongate with decreasing pH when the acid concentration is greater than equimolar with respect to lysine, and they shrink with increasing temperature.

Polymer solutions in the vicinity of the theta-point are known to undergo shear-induced turbidity or phase separation. The present study shows that a similar phenomenon also occurs for certain wormlike micellar solutions. Wormlike micelles are the self-assembled counterparts of polymers and are characterized by their ability to reversibly break and recombine. In the system of interest, the micelles are formed by the cationic surfactant erucyl bis(hydroxyethyl)methylammonium chloride (EHAC), in conjunction with a salt such as sodium chloride (NaCl) or sodium salicylate (NaSal). Micellar samples that become turbid under shear show evidence of critical concentration fluctuations and may contain predominantly branched micelles. The shear-induced turbidity in these samples correlates with the appearance of flow-dichroism in rheooptic experiments and with an increase in low-q scattering in small-angle light scattering under flow (flow-SALS) experiments. The characteristic "butterfly" pattern, with enhanced scattering in the flow direction and a dark streak perpendicular to the flow direction, is typically observed in flow-SALS. The results suggest that the turbidity is due to a shear-induced growth of concentration fluctuations, which in turn manifests as large anisotropic domains, typically oriented along the vorticity axis.

The formation of multilamellar vesicles (MLVs) from the lamellar phase of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in brine can be controlled by steady, oscillatory shearing. Increasing stress amplitude or decreasingfrequency leads to more rapid onion formation. The formation kinetics can be scaled onto constant controlled shear stress experiments; this scaling demonstrates that a minimum strain amplitude of ∼14 that is independent of frequency is required for onion formation. Further, the ratio of deformation driving onion formation to the reversible, elastic deformation can be predicted from the loss tangent. The final size of the MLVs decreases with the stress amplitude, independent of frequency, in quantitative agreement with MLVs formed under steady, applied shear stress.