Weichun Pan

Nucleation of ordered solid phases of proteins via a disordered high-density state: phenomenological approach.

Nucleation of ordered solid phases of proteins triggers numerous phenomena in laboratory, industry, and in healthy and sick organisms. Recent simulations and experiments with protein crystals suggest that the formation of an ordered crystalline nucleus is preceded by a disordered high-density cluster, akin to a droplet of high-density liquid that has been observed with some proteins; this mechanism allowed a qualitative explanation of recorded complex nucleation kinetics curves. Here, we present a simple phenomenological theory that takes into account intermediate high-density metastable states in the nucleation process. Nucleation rate data at varying temperature and protein concentration are reproduced with high fidelity using literature values of the thermodynamic and kinetic parameters of the system. Our calculations show that the growth rate of the near-critical and supercritical ordered clusters within the dense intermediate is a major factor for the overall nucleation rate. This highlights the role of viscosity within the dense intermediate for the formation of the ordered nucleus. The model provides an understanding of the action of additives that delay or accelerate nucleation and presents a framework within which the nucleation of other ordered protein solid phases, e.g., the sickle cell hemoglobin polymers, can be analyzed.

Origin of Anomalous Mesoscopic Phases in Protein Solutions

Long-living mesoscopic clusters of a dense protein liquid are a necessary kinetic intermediate for the formation of solid aggregates of native and misfolded protein molecules; in turn, these aggregates underlie physiological and pathological processes and laboratory and industrial procedures. We argue that the clusters consist of a nonequilibrium mixture of single protein molecules and long-lived complexes of proteins. The puzzling mesoscopic size of the clusters is determined by the lifetime and diffusivity of these complexes. We predict and observe a crossover of cluster dynamics to critical-like density fluctuations at high protein concentrations. We predict and experimentally confirm that cluster dynamics obey a universal, diffusion-like scaling with time and wave vector, including in the critical-like regime. Nontrivial dependencies of the cluster size and volume fraction on the protein concentration are established. Possible mechanisms of complex formation include domain swapping, hydration forces, dispersive interactions, and other, system-specific, interactions. We highlight the significance of the hydration interaction and domain swapping with regard to the ubiquity of the clusters and their sensitivity to the chemical composition of the solvent. Our findings suggest novel ways to control protein aggregation.

Nucleation of Protein Crystals under the Influence of Solution Shear Flow

Abstract:  Several recent theories and simulations have predicted that shear flow could enhance, or, conversely, suppress the nucleation of crystals from solution. Such modulations would offer a pathway for nucleation control and provide a novel explanation for numerous mysteries in nucleation research. For experimental tests of the effects of shear flow on protein crystal nucleation, we found that if a protein solution droplet of ∼ 5 μL (2–3 mm diameter at base) is held on a hydrophobic substrate in an enclosed environment and in a quasi‐uniform constant electric field of 2 to 6 kV cm−1, a rotational flow with a maximum rate at the droplet top of ∼ 10 μm s−1 is induced. The shear rate varies from 10−3 to 10−1 s−1. The likely mechanism of the rotational flow involves adsorption of the protein and amphiphylic buffer molecules on the air–water interface and their redistribution in the electric field, leading to nonuniform surface tension of the droplet and surface tension‐driven flow. Observations of the number of nucleated crystals in 24‐ and 72‐h experiments with the proteins ferritin, apoferritin, and lysozyme revealed that the crystals are typically nucleated at a certain radius of the droplet, that is, at a preferred shear rate. Variations of the rotational flow velocity resulted in suppression or enhancement of the total number of nucleated crystals of ferritin and apoferritin, while all solution flow rates were found to enhance lysozyme crystal nucleation. These observations show that shear flow may strongly affect nucleation, and that for some systems, an optimal flow velocity, leading to fastest nucleation, exists. Comparison with the predictions of theories and simulations suggest that the formation of ordered nuclei in a “normal” protein solution cannot be affected by such low shear rates. We conclude that the flow acts by helping or suppressing the formation of ordered nuclei within mesoscopic metastable dense liquid clusters. Such clusters were recently shown to exist in protein solutions and to constitute the first step in the nucleation mechanism of many protein and nonprotein systems.

Free Heme and the Polymerization of Sickle Cell Hemoglobin

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that the concentration of free heme in HbS solutions typically used in the laboratory is 0.02-0.04 mole heme/mole HbS. We show that dialysis of small molecules out of HbS solutions arrests HbS polymerization. The addition of 100-260 μM of free heme to dialyzed HbS solutions leads to rates of nucleation and polymer fiber growth faster by two orders of magnitude than before dialysis. Toward an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at a concentration of 66 μM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that spikes of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a significant factor in the complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell therapy.

Does Solution Viscosity Scale the Rate of Aggregation of Folded Proteins

Viscosity effects on the kinetics of complex solution processes have proven hard to predict. To test the viscosity effects on protein aggregation, we use the crystallization of the protein glucose isomerase (gluci) as a model and employ scanning confocal and atomic force microscopies at molecular resolution, dynamic and static light scattering, and rheometry. We add glycerol to vary solvent viscosity and demonstrate that glycerol effects on the activation barrier for attachment of molecules to the crystal growth sites are minimal. We separate the effects of glycerol on crystallization thermodynamics from those on the rate constant for molecular attachment. We establish that the rate constant is proportional to the reciprocal viscosity and to the protein diffusivity. This finding refutes the prevailing crystal growth paradigm and illustrates the application of fundamental kinetics laws to solution crystallization.

Free heme in micromolar amounts enhances the attraction between sickle cell hemoglobin molecules.

We probe the role of free heme in the interactions between sickle cell hemoglobin (HbS) molecules in simulated physiological solutions: polymerization of deoxy‐HbS is the primary pathogenic event of sickle cell anemia, and HbS releases heme after autoxidation more readily than normal adult hemoglobin. We characterize these interactions in terms of osmotic virial coefficients, which we determine by static light scattering. We analyze the results in the heme‐hemoglobin system using the Kirkwood‐Goldberg model. We show that in the absence of heme, the HbS molecules weakly attract and the attraction is not due to the lowered—as a result of the sickle cell mutation—molecular charge. We show that the part of the interface between the two αβ dimers, exposed in the deoxy‐state, plays a crucial role in this attraction. We show that heme at micromolar concentrations induces strong attraction between the hemoglobin molecules. We show that the high efficacy of the heme results from the statistics of electrostatic and hydrophobic interactions between the heme and hemoglobin molecules.