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J. Mc Manus, A. Lomakin, O. Ogun, G. B. Benedek; Phase Separation in Multicomponent Aqueous Protein Solutions-A Study of Gamma and Beta Crystallin Mixtures. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2032. doi: https://doi.org/.
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To investigate liquid-liquid phase separation (LLPS) in mixtures of gamma and beta crystallin proteins.
We obtained native γD and Beta B1 crystallin by expression of the relevant DNA clones in E.coli. We determined the liquid-liquid coexistence curve using cloud point measurements and dynamic light scattering spectroscopy. The mass fraction (composition) of beta to gamma crystallin present was determined by HPLC.
We present measurements of the coexistence curve in ternary aqueous mixtures of human γD crystallin (HGD) and human ßB1 crystallin, as a function of total protein volume fraction (Φ) for a range of ßB1/γD compositions (α). Human gamma-D crystallin exhibits liquid-liquid phase separation (LLPS) and has a Tc of ~3°C. As the composition of ßB1 crystallin is increased, the temperature for LLPS near the critical point appears to decrease. This implies an effective reduction in the attraction between gamma crystallins. This is important in highlighting the importance of protein mixtures in maintaining transparency of the lens.
These experiments demonstrate that upon increasing the beta B1 fraction, the liquid-liquid phase separation phenomenon is retained, thereby permitting a deduction of the relative strengths of the beta-beta, and beta-gamma interaction potentials [1, 2] Liu C., Lomakin A., Thurston G. M., Hayden D., Pande A., Pande J., Ogun O., Asherie N., Benedek G.B., 1995, J. Phys. Chem, 99, 454-461 Liu C., Asherie N., Lomakin A., Pande J., Ogun O., Benedek G. B., 1996, Proc. Natl. Acad. Sci, 93, 377-382
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