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Hematin crystallization, which is an essential component of the physiology of malaria parasites and the most successful target for antimalarial drugs, proceeds in mixed organic-aqueous solvents both in vivo and in vitro. Here we employ molecular dynamics simulations to examine the structuring and dynamics of a water-normal octanol mixture (a solvent that mimics the environment hosting hematin crystallization in vivo) in the vicinity of the typical faces in the habit of a hematin crystal. The simulations reveal that the properties of the solvent in the layer adjacent to the crystal are strongly impacted by the distinct chemical and topological features presented by each crystal face. The solvent organizes into at least three distinct layers. We also show that structuring of the solvent near the different faces of β-hematin strongly impacts the interfacial dynamics. The relaxation time of n-octanol molecules is longest in the contact layers and correlates with the degree of structural ordering at the respective face. We show that the macroscopically homogeneous water-octanol solution holds clusters of water and n-octanol connected by hydrogen bonds that entrap the majority of the water but are mostly smaller than 30 water molecules. Near the crystal surface the clusters anchor on hematin carboxyl groups. These results provide a direct example that solvent structuring is not restricted to aqueous and other hydrogen-bonded solutions. Our findings illuminate two fundamental features of the mechanisms of hematin crystallization: the elongated shapes of natural and synthetic hematin crystals and the stabilization of charged groups of hematin and antimalarials by encasing in water clusters. In addition, these findings suggest that hematin crystallization may be controlled by additives that disrupt or reinforce solvent structuring.

Protein condensates play essential roles in physiological processes and pathological conditions. Recently discovered mesoscopic protein-rich clusters may act as crucial precursors for the nucleation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge settled paradigms of protein condensation as the constituent protein molecules present features characteristic of both partially misfolded and native proteins. Here we employ the antimicrobial enzyme lysozyme and examine the similarities between mesoscopic clusters, amyloid structures, and disordered aggregates consisting of chemically modified protein. We show that the mesoscopic clusters are distinct from the other two classes of aggregates. Whereas cluster formation and amyloid oligomerization are both reversible, aggregation triggered by reduction of the intramolecular S-S bonds is permanent. In contrast to the amyloid structures, protein molecules in the clusters retain their enzymatic activity. Furthermore, an essential feature of the mesoscopic clusters is their constant radius of less than 50 nm. The amyloid and disordered aggregates are significantly larger and rapidly grow. These findings demonstrate that the clusters are a product of limited protein structural flexibility. In view of the role of the clusters in the nucleation of ordered protein solids, our results suggest that fine-tuning the degree of protein conformational stability is a powerful tool to control and direct the pathways of protein condensation.

We develop a simple technique for the generation of droplets of a high-concentration protein solution of attoliter volume and a diameter ofor=500 nm, containing several thousand protein molecules, and their accurate deposition on micron-size electrodes, standalone or in an array. The technique is based on liquid-liquid phase separation in the homogeneous region of the phase diagram, induced by a nonuniform weak electric field, which also accurately directs the generated droplets toward the electrode. We show that the protein molecules are not chemically modified and retain their integrity, conformation, and activity post deposition. The technique is applicable to the manufacture of various types of protein/microelectrode arrays and is tunable, scalable, and applicable to a broad range of proteins.

Dense liquid phases, metastable with respect to a solid phase, but stable with respect to the solution, have been known to form in solutions of proteins and small-molecule substances. Here, with the protein lumazine synthase as a test system, using dynamic and static light scattering and atomic force microscopy, we demonstrate submicron size clusters of dense liquid. In contrast to the macroscopic dense liquid, these clusters are metastable not only with respect to the crystals, but also with respect to the low-concentration solution: the characteristic cluster lifetime is limited to approximately 10 s, after which they decay. The cluster population is detectable only if they occupy >10(-6) of the solution volume and have a number density >105 cm-3 for 3 to 11% of the monitored time. The cluster volume fraction varies within wide limits and reaches up to 10(-3). Increasing protein concentration increases the frequency of cluster detection but does not affect the ranges of the cluster sizes, suggesting that a preferred cluster size exists. A simple Monte Carlo model with protein-like potentials reproduces the metastable clusters of dense liquid with limited lifetimes and variable sizes and suggests that the mean cluster size is determined by the kinetics of growth and decay and not by thermodynamics.

For insight into the thermodynamics and phase behavior in concentrated protein solutions, we study the liquid−liquid phase separation with lysozyme. We determine independently the binodal and spinodal lines, and the second virial coefficient of the protein at 275 < T < 295 K. From these data, we determine the protein chemical potential and osmotic pressure for concentrations as high as 320 mg mL-1 in the above temperature range. We find that for this protein, the enthalpy of the liquid−liquid separation vanishes at the critical temperature, Tc, and is comparable to and may exceed the crystallization enthalpy (∼65 kJ mol-1) at lower Ts. The enthalpy of the pair interactions averaged over all polar angles is significantly lower; this comparison suggests structuring of the dense liquid. We propose that the pair of parameters (molecular volume, second virial coefficient) may be an adequate predictor of the phase behavior of solutions of proteins with relatively simple interaction potentials.

We aim at insight into the unsteady kinetics and the formation of spatiotemporal patterns of steps during the crystal growth in systems, in which the growth rate is controlled by the rate of supply...

We have developed a novel method for direct determination of the steady-state rates of homogeneous nucleation. The method is applicable to studies of crystallization, aggregation, and similar first-order phase transitions in solutions of proteins or other soluble slow-growing materials with temperature-dependent solubility. Temperature T control of the solution supersaturation allows fast supersaturation changes from a level inductive of nucleation to a level where no nucleation occurs, but existing crystals grow to detectable dimensions. In this way, nucleation takes place only at the first T setting at a constant supersaturation before solution depletion due to crystal growth becomes significant. We use inert oil to cover nucleating solutions and suppress nucleation on the solution−air interface. To obtain reproducible statistical characteristics of the intrinsically random nucleation process, a large number of simultaneous trials take place under identical conditions. First data for the nucleation of t...

High-resolution optical interferometry was used to investigate the effects of forced solution convection on the crystal growth kinetics of the model protein lysozyme. Most experiments were conducted with 99.99% pure protein solutions. To study impurity effects, approx. 1% of lysozyme dimer (covalently bound) was added in some cases. We show that the unsteady kinetics, corresponding to bunching of growth steps, can be characterized by the Fourier components of time traces of the growth rate. Specific Fourier spectra are uniquely determined by the solution conditions (composition, temperature, and flow rate) and the growth layer source activity. We found that the average step velocity and growth rate increase by approx. I0% with increasing flow rate, as a result of the enhanced solute supply to the interface. More importantly, faster convective transport results in lower fluctuation amplitudes. This observation supports our rationale for system-dependent effects of transport on the structural perfection of protein crystals. We also found that solution flow rates greater than 500 microns/s result in stronger fluctuations while the average growth rate is decreased. This can lead to growth cessation at low supersaturations. With the intentionally contaminated solutions, these undesirable phenomena occurred at about half the flow rates required in pure solutions. Thus, we conclude that they are due to enhanced convective supply of impurities that are incorporated into the crystal during growth. Furthermore, we found that the impurity effects are reduced at higher crystal growth rates. Since the exposure time of terraces is inversely proportional to the growth rate, this observation suggests that the increased kinetics instability results from impurity adsorption on the interface. Finally, we provide evidence relating earlier observations of "slow protein crystal growth kinetics" to step bunch formation in response to nonsteady step generation.