George M. Thurston

Phenomenological theory of equilibrium thermodynamic properties and phase separation of micellar solutions

A detailed description and generalization of a recently developed theory, which provides analytic representations of the distribution of micellar species and the equilibrium thermodynamic properties of amphiphile–water solutions that exhibit phase separation and critical phenomena, is presented. We propose a form for the structure of the Gibbs free energy which accurately describes the essential physical factors responsible for micellization and phase separation. These are: the free‐energy advantage associated with the formation of individual micellar species,the entropy of mixing of the extended micelles and the water molecules, and the free energy of interaction between each member of the micellar size distribution. By applying to this Gibbs free energy the conditions of multiple chemical equilibrium and thermodynamic stability, all the relevant statistical and thermodynamic equilibrium properties of the micellar solution can be calculated. These properties include the location of the critical concentra...

THEORETICAL AND EXPERIMENTAL BASIS FOR THE INHIBITION OF CATARACT

Aggregation of the lens proteins to form high molecular weight clusters is a major contributing factor in age-onset nuclear cataract [Benedek, G. B. (1971) Theory of transparency of the eye. Appl. Optics, 10, 459-473]. This aggregation occurs continually throughout life and contributes to an exponential increase, as a function of age, in the intensity of the light backscattered out of the lens. The time constant deltaT for this exponential increase in human populations is a valuable index, helpful for conducting clinical trials. In-vitro studies have identified reagents capable of inhibiting high molecular weight aggregate formation, as well as the non-covalent interprotein interactions responsible for phase separation. These reagents are also found experimentally to be effective cataract inhibitors in animal model systems in vivo. We believe that the stage is now set for human clinical trials of putative cataract inhibitors. We present rough quantitative estimates of the trial parameters needed to assure an unambiguous determination of efficacy in a trial population. Such a trial simply requires a measurement of the time constant deltaT in the treated population relative to the untreated population. A successful outcome of the trial is indicated if deltaT increases by 20% over that found for the untreated population. Our estimates suggest efficacy could be determined in a two year trial involving about 300 subjects in the treated group.

Light Scattering and Phase Behavior of Lysozyme-Poly(Ethylene Glycol) Mixtures

Measurements of liquid-liquid phase transition temperatures (cloud points) of mixtures of a protein (lysozyme) and a polymer, poly(ethylene glycol) (PEG) show that the addition of low molecular weight PEG stabilizes the mixture whereas high molecular weight PEG was destabilizing. We demonstrate that this behavior is inconsistent with an entropic lysozyme-PEG depletion interaction and suggest that an energetic lysozyme-PEG attraction is responsible. In order to independently characterize the lysozyme-PEG interactions, light scattering experiments on the same mixtures were performed to measure second and third virial coefficients. These measurements indicate that PEG induces repulsion between lysozyme molecules, contrary to the depletion prediction. Furthermore, it is shown that third virial terms must be included in the mixtures free energy in order to qualitatively capture our data.

New insight into cataract formation : Enhanced stability through mutual attraction

Small-angle neutron scattering experiments and molecular dynamics simulations combined with an application of concepts from soft matter physics to complex protein mixtures provide new insight into the stability of eye lens protein mixtures. Exploring this colloid-protein analogy we demonstrate that weak attractions between unlike proteins help to maintain lens transparency in an extremely sensitive and nonmonotonic manner. These results not only represent an important step towards a better understanding of protein condensation diseases such as cataract formation, but provide general guidelines for tuning the stability of colloid mixtures, a topic relevant for soft matter physics and industrial applications.

Cataract-associated mutant E107A of human γD-crystallin shows increased attraction to α-crystallin and enhanced light scattering

Several point mutations in human γD-crystallin (HGD) are now known to be associated with cataract. So far, the in vitro studies of individual mutants of HGD alone have been sufficient in providing plausible molecular mechanisms for the associated cataract in vivo. Nearly all the mutant proteins in solution showed compromised solubility and enhanced light scattering due to altered homologous γ–γ crystallin interactions. In sharp contrast, here we present an intriguing case of a human nuclear cataract-associated mutant of HGD—namely Glu107 to Ala (E107A), which is nearly identical to the wild type in structure, stability, and solubility properties, with one exception: Its pI is higher by nearly one pH unit. This increase dramatically alters its interaction with α-crystallin. There is a striking difference in the liquid–liquid phase separation behavior of E107A–α-crystallin mixtures compared to HGD–α-crystallin mixtures, and the light-scattering intensities are significantly higher for the former. The data show that the two coexisting phases in the E107A–α mixtures differ much more in protein density than those that occur in HGD–α mixtures, as the proportion of α-crystallin approaches that in the lens nucleus. Thus in HGD–α mixtures, the demixing of phases occurs primarily by protein type while in E107A–α mixtures it is increasingly governed by protein density. Analysis of these results suggests that the cataract due to the E107A mutation could result from the instability caused by the altered attractive interactions between dissimilar proteins—i.e., heterologous γ–α crystallin interactions—primarily due to the change in surface electrostatic potential in the mutant protein.

Theory of thermodynamic properties and phase separation of micellar solutions with lower consolute points

A recently developed thermodynamic theory is extended to study the single‐phase properties and the phase separation of micellar solutions which exhibit lower consolute points. Analytical results for the location of the critical point, shape of the coexistence curve, micellar size distribution, and osmotic compressibility along the critical isochore are presented. These results are in excellent agreement with experimental findings in micellar solutions of the nonionic amphiphile n‐dodecyl hexaoxyethylene glycol monoether and water.

Liquid-liquid phase separation and static light scattering of concentrated ternary mixtures of bovine α and γB crystallins

We have used light scattering, turbidimetry, and thermodynamic analysis to study the phase diagram of concentrated aqueous mixtures of the bovine lens proteins, γB crystallin, and α crystallin. We find that dilute α crystallin raises the phase separation temperature of concentrated γB crystallin, while more concentrated α crystallin suppresses phase separation. Very concentrated α∕γB mixtures can reversibly cloud above 37°C, even though γB alone phase separates only below temperatures near 0°C, and α does not phase separate. At the scattering vector magnitude used, high-concentration α∕γB mixtures scatter less light than the weighted average of their component α and γB solutions, while low-concentration α∕γB mixtures scatter more than such a weighted average. We use a mean-field thermodynamic analysis of such ternary mixtures to show that the observed light scattering and phase boundaries of α and γB crystallin mixtures give evidence for prominent local fluctuations of relative protein composition. In the...

Colloidal characterization and thermodynamic stability of binary eye lens protein mixtures.

We present a study of binary mixtures of eye lens crystallin proteins. A coarse-grained model of aqueous alpha- and gamma-crystallin mixtures based on molecular dynamics simulations and SANS experiments is proposed. Thermodynamic perturbation theory is implemented to obtain the stability boundaries, or spinodal surface, of the binary mixture in the full parameter space. The stability of these high-concentration crystallin mixtures was found to depend on the alpha-gamma attraction in a manner that is both extremely sensitive and nonmonotonic; stronger or weaker attraction resulted in a spectacularly enhanced instability. The relevance of these mechanisms as possible sources of the alteration of the spatial distribution of the lens proteins encountered in cataract disease is discussed.

Hard sphere-like glass transition in eye lens α-crystallin solutions.

Significance Normal vision and accommodation rely on the clarity and softness of the eye lens. Hardening of the lens has been linked with presbyopia, the loss of accommodative capability with age, and lens clarity is disrupted in cataract, a leading cause of blindness worldwide. Here, realistically concentrated solutions of a prevalent eye lens structural protein, α-crystallin, which exhibits short-range order needed for lens transparency, are found in addition to show high-concentration dynamical slowing down similar to that of hard-sphere glass transitions. This suggests that analogous investigation of concentrated crystallin mixtures, like those in the living lens, may help to advance understanding of the molecular basis of both presbyopia and cataract. We study the equilibrium liquid structure and dynamics of dilute and concentrated bovine eye lens α-crystallin solutions, using small-angle X-ray scattering, static and dynamic light scattering, viscometry, molecular dynamics simulations, and mode-coupling theory. We find that a polydisperse Percus–Yevick hard-sphere liquid-structure model accurately reproduces both static light scattering data and small-angle X-ray scattering liquid structure data from α-crystallin solutions over an extended range of protein concentrations up to 290 mg/mL or 49% vol fraction and up to ca. 330 mg/mL for static light scattering. The measured dynamic light scattering and viscosity properties are also consistent with those of hard-sphere colloids and show power laws characteristic of an approach toward a glass transition at α-crystallin volume fractions near 58%. Dynamic light scattering at a volume fraction beyond the glass transition indicates formation of an arrested state. We further perform event-driven molecular dynamics simulations of polydisperse hard-sphere systems and use mode-coupling theory to compare the measured dynamic power laws with those of hard-sphere models. The static and dynamic data, simulations, and analysis show that aqueous eye lens α-crystallin solutions exhibit a glass transition at high concentrations that is similar to those found in hard-sphere colloidal systems. The α-crystallin glass transition could have implications for the molecular basis of presbyopia and the kinetics of molecular change during cataractogenesis.

The effect of salt identity and concentration on liquid-liquid phase separation in aqueous micellar solutions of C8-lecithin

We report measurements of the location and shape of the coexistence curve for binary liquid–phase separation in aqueous micellar solutions of C8 ‐lecithin, as a function of the concentrations of KI, NaI, LiI, KCl, NaCl, and LiCl. The salts containing iodide dramatically lower the critical temperature for phase separation Tc as the salt concentration is increased. The chloride salts, however, first lower and then raise Tc as the salt concentration is increased. The identity of the added cations used has a comparatively minor effect on Tc . Potassium ions are more effective in changing Tc than sodium ions, which in turn are more effective than lithium ions. We have used our data to deduce the dependence on salt identity and concentration of the parameters C and Δμ, which reflect the strength of intermicellar interactions and the extent of micellar growth, respectively, within a Gibbs free energy model for micellar solutions. The theoretical coexistence curves calculated using the deduced parameters agree we...