Solid-liquid phase boundaries of lens protein solutions.

Abstract

We report measurement of the solid-liquid phase boundary, or liquidus line, for aqueous solutions of three pure calf y6-crystallin proteins: y$I, yHIa, and yIHIb. We also studied the liquidus line for solutions of native yIV-crystallin calf lens protein, which consists of 85% yIVa/15% yIVb. In all four proteins the liquidus phase boundaries lie higher in temperature than the previously determined liquid-lquid coexistence curves. Thus, over the range of concentration and temperature for which liquid-liquid phase separation occurs, the coexistence of a protein crystal phase with a protein liquid solution phase is thermodynamically stable relative to the metastable separated liquid phases. The location ofthe liquidus lines clearly divides these four crystallin proteins into two groups: those in which liquidus lines flatten at temperatures >70rC: yMa and yIV, and those in which liquidus lines flatten at temperatures <500C: yll and -yIlb. We have analyzed the form of the liquidus lines by using specific choices for the structures of the Gibbs free energy in solution and solid phases. By applying the thermodynamic conditions for equilibrium between the two phases to the resulting chemical potentials, we can estimate the temperature-dependent free energy change upon binding of protein and water into the solid phase.Maintenance ofthe lens proteins in a single homogenous fluid phase is an essential condition for transparency of the eye lens (1, 2). Consequently, we previously investigated the location of the coexistence curve (3-5) for liquid-liquid phase separation for four pure calf y-crystallin protein solutions. In those studies, preliminary findings at a few points in the phase diagram suggested that the coexistence curve for solid-liquid phase equilibrium might be higher in temperature than the liquid-liquid coexistence curve (4, 5). We, therefore, undertook the present systematic investigation of the location of the solid-liquid coexistence curve for three pure lens crystallin proteins 'yII, yIIIa, and yIIIb, as well as for native 'yIV protein, which is a mixture of yIVa and yIVb in relative proportion of 85% to 15%, respectively, by number. We report here the measurement for each protein of the ascending limb of the solid-liquid coexistence curve. This limb is called the liquidus line; it is defined as the locus of points in the concentration (c) and temperature (T) plane that corresponds to equilibrium between protein crystals and an aqueous liquid solution of the same protein having concentration c. This locus can be designated by TL(c), or alternatively cL(T). At fixed temperature T, the concentration CL is the solubility of the protein in aqueous solution. The descending limb ofthe solid-liquid coexistence curve is called the solidus line c5(T), and it is the locus of points showing the protein concentration in the solid phase for each temperature T. For cL(T) < c < c,(T), the equilibrium state of the solution consists of a mixture of protein crystals of protein concentration c5(T) and aqueous liquid ...

Strong weak and metastable liquids structural and dynamical aspects of the liquid state

“…The first report of metastable liquid-liquid transitions in solutions of globular proteins dates back 20 years [18] and many similar observations have been made since [19][20][21][22][23][24][25][26]. Recently it was realized that the explanation for the metastability in these protein systems probably lies again in the fact that the interaction range is small compared to the protein size [19,[27][28][29][30][31].…”

Exploring Bovine Pancreatic Trypsin Inhibitor Phase Transitions

“…3 Protein phase separations can also be the cause of human diseases, such as cataract 4 or sickle cell disease. 5 Despite their fundamental importance, phase diagrams of only a few proteins have been studied: insulin, 6 fibrinogen, 7 γ-crystallin, 8 lysozyme, 4,9-12 concanavalin, 13 canavalin, 14 R-amylase, 15,16 collagenase, 17 bovine pancreatic trypsin inhibitor (BPTI), 16,18 apoferritin, 19 endoglucanase A, 20 and the photochemical reaction center from Rhodobacter sphaeroides Y. 21 Recently, there has been a renewed enthusiasm for phasetransition studies of colloids 22 and proteins [22][23][24][25][26][27] due to advances in experimental and computational technologies.…”

Altered phase diagram due to a single point mutation in human γD-crystallin

“…The protein crystals will dissolve until equilibrium has been reached. Because of the small number of crystals that grew, we determined the solubility of the protein crystal by starting from a supersaturated solution (19,20). In solutions in which crystals grew, we lowered the temperature until melting began to occur at the edges of the crystals.…”