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

The P23T mutant of human γD-crystallin (HGD) is associated with cataract. We have previously investigated the solution properties of this mutant, as well as those of the closely related P23V and P23S mutants, and shown that although mutations at site 23 of HGD do not produce a significant structural change in the protein, they nevertheless profoundly alter the solubility of the protein. Remarkably, the solubility of the mutants decreases with increasing temperature, in sharp contrast to the behavior of the native protein. This inverted solubility corresponds to a strong increase in the binding energy with temperature. Here we have investigated the liquid–liquid coexistence curve and the diffusivity of the P23V mutant and find that these solution properties are unaffected by the mutation. This means that the chemical potentials in the solution phase are essentially unaltered. The apparent discrepancy between the interaction energies in the solution phase, as compared with the solid phase, is explicable in terms of highly anisotropic interprotein interactions, which are averaged out in the solution phase but are fully engaged in the solid phase

Oligomerization and phase separation in globular protein solutions. Biophys Chem 75(3):213–227

Abstract We have chemically crosslinked a globular protein, ␥ -crystallin, to produce a system of well-defined oligomers: IIIb monomers, dimers, trimers and a mixture of higher n-mers. Gel electrophoresis, size exclusion chromatography, quasielastic light scattering spectroscopy, and electrospray ionization mass spectrometry were used to characterize the oligomers formed. The liquid᎐liquid phase separation boundaries of the various oligomers were measured. We find that at a given concentration the phase separation temperature strongly increases with the molecular weight of the oligomers. This phase behavior is very similar to previous findings for ␥ -crystallin, for which oxidation-induced II oligomerization is accompanied by an increase in the phase separation temperature. These findings imply that for phase separation, the detailed changes of the surface properties of the proteins are less important than the purely steric effects of oligomerization. ᮊ 1998 Elsevier Science B.V. All rights reserved. Keywords: Liquid᎐liquid phase separation; ␥-Crystallins; Oligomerization; Crosslinking U Corresponding author. Tel.: q1 617 2534828; fax: q1 617 2252585; e-mail: [email protected] Abbre¨iations: BMH, bismaleimidohexane; CAT-HPLC, cation exchange high performance liquid chromatography; DTNB, 5,5Ј-di-Ž . thiobis 2-nitrobenzoic acid ; DTT, dithiothreitol; ESIMS, electrospray ionization mass spectrometry; GU, guanidine hydrochloride; HPLC, high performance liquid chromatography; NMR, nuclear magnetic resonance; PTC, phenylisothiocyanate; QLS, quasielastic light scattering spectroscopy; SX-HPLC, size exclusion high performance liquid chromatography 0301-4622r98r$ -see front matter ᮊ 1998 Elsevier Science B.V. All rights reserved. Ž . P I I S 0 3 0 1 -4 6 2 2 9 8 0 0 2 0 8 -7 ( ) N. Asherie et al. r Biophysical Chemistry 75 1998 213᎐227 21