Binary-liquid phase separation of lens protein solutions.

Abstract

We have determined the coexistence curves (plots of phase-separation temperature T versus protein concentration C) for aqueous solutions of purified calf lens proteins. The proteins studied, calf y'Ila-, yIlb-, and yIVacrystallin, have very similar amino acid sequences and threedimensional structures. Both ascending and descending limbs of the coexistence curves were measured. We find that the coexistence curves for each of these proteins and for yIcrystallin can be fit, near the critical point, to the function W(Cc -C)/CJI = A[(Tc -T)/TCJ, where fi = 0.325, Cc is the critical protein concentration in mg/ml, T, is the critical temperature for phase separation in K, and A is a parameter that characterizes the width of the coexistence curve. We find that A andCc are approximately the same for all four coexistence curves (A = 2.6 +-0.1, Cc = 289 ± 20 mg/ml), but that Tc is not the same. For yIH-and yIIIb-crystallin, Tc7-5°C, whereas for yIIa-and yIVa-crystallin, Tc 38C. By comparing the published protein sequences for calf, rat, and human y-crystallins, we postulate that a few key amino acid residues account for the division of y-crystallins into low-Tc and high-Tc groups.The y-crystallins constitute a family of highly homologous mammalian lens proteins (1-4). Concentrated aqueous solutions of y-crystallins (5-8) exhibit the phenomena of binaryliquid-phase separation (9-11), also known as coacervation (12). These solutions separate into two coexisting liquid phases of unequal protein concentration at temperatures less than the critical temperature for phase separation Tc. From previous studies (5,7,8), it is known that location of the coexistence curve depends sensitively on the amino acid sequence of the crystallin molecule. Two distinct groups of y-crystallins have been identified in rat (7) and human (8) lenses: high-Tc crystallins and low-Tc crystallins. In these rat and human studies, the precise values of Tc for each crystallin, though inferred from the data, were not determined explicitly. For the high-Tc crystallins, only the ascending limb of the coexistence curves was measured. For the low-Tc crystallins, only an upper bound for the Tc values was established.In this paper, we report on measurements of coexistence curves for three purified calf y-crystallins-yIIIa, yIIIb, and yIVa-[in Table 2 we indicate the current nomenclature for mammalian y-crystallins (2)] and for the native calf y-crystallin mixture yIV. We have determined both the ascending and descending limbs of each coexistence curve. This information enables us to characterize the coexistence curves in detail and to determine explicitly the values of the critical concentration Cc and the critical temperature Tc for each protein. Such detailed analysis of y-crystallin phase separation, which requires gram quantities of purified protein, has been performed only for calf yII (6).We find that the purified calf y-crystallins, in accord with the purified rat and human y-crystallins, fall into two distinct groups: high-Tc (Tc > 350C) proteins, ...

How fluorescent labelling alters the solution behaviour of proteins

“…46 The degree to which T ph changes is, however, different for the two positions at which the protein was labelled. For the protein mixture containing protein modified with HiLyte 405 at the amine 2 position, it was possible to add up to x L = 0.01 of labelled protein before significant precipitation of protein occurred.…”

“…45 The volume fraction is calculated using the expression c = fn where c is the concentration in mg ml À1 , f is the volume fraction and n is the partial specific volume equal to 7.1 AE 0.1 Â 10 À4 ml mg À1 for the g crystallins. 46 Modification of Lys-2 and Cys-110 amino acid residues. Amine modification of Lys-2 in HGD was carried out using an AnaTag HiLyte 405 kit, purchased from AnaSpec (Freemont, CA, USA), as per supplied instructions via amine modification after purification.…”

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].…”

Colloidal Characterization and Thermodynamic Stability of Binary Eye Lens Protein Mixtures

“…19 While the exact location of the coexistence curve depends on the solvent and on the particular γ-crystallin being investigated, the solution properties have been found to scale with the reduced density and the reduced temperature (T -T c )/T c . 19,20,26,27 The first parameter that we shall set is the range for the γ-γ interaction. This is fixed to reproduce the critical density obtained by the experiments.…”