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J. Mc Manus, A. Pande, A. Lomakin, O. Ogun, G. Benedek, J. Pande; Modulation of the Phase Behavior of the Cataractogenic Mutant P23T of Human D–Crystallin by Replacement of Thr23 by Val . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2006.
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To investigate the phase behavior of the protein containing the Thr23 to Val substitution in the cataract–producing mutant P23T of human γD–crystallin (HGD).
We expressed recombinant proteins (native γD, its P23V and P23T mutants) in E.coli and investigated the phase diagram of P23V in–vitro using cloud point measurements and optical microscopy. We also examined the kinetics of aggregation using dynamic light scattering (DLS).
The replacement of Thr23 with a Ser or a Val residue restores some of the solubility of the P23T mutant, with the P23V mutant having solubility close to that of the native protein . P23V exhibits aggregation, crystallization and liquid–liquid phase separation, under specific conditions of solution concentration and temperature. Interestingly, the formation of condensed phases of P23V occurs at higher temperatures, compared with other gamma crystallins. Melting of the condensed phase is observed when the temperature is lowered. The native–like solubility of P23V coupled with the tendency to form condensed phases (like those of mutant P23T) over a wide concentration range, and within a workable temperature region , provided us with a wide range of experimental conditions to examine the phase behavior of this mutant in detail. DLS measurements have shown that in the soluble region the diffusion coefficient of P23V is consistent with monomeric protein and no aggregation is observed if the temperature remains constant. On increasing temperature, the formation of large aggregates is observed after a lag time which appears to vary with concentration. We are examining the kinetics of formation of the condensed phases using DLS.
The P23T mutation in human γD–crystallin is associated with several different cataract phenotypes. A detailed phase diagram of this mutant has not been determined due to its low solubility. P23V displays similar behavior to P23T, but solutions of P23V mutant exhibit phase–transitions over a wider temperature range. We have observed thermodynamic phase transitions such as aggregation, crystallization and liquid–liquid phase separation in P23V solutions. These transitions can be induced by temperature changes and are reversible. This tunable system exhibits rich phase behavior which makes it an excellent model system for understanding the general mechanisms of cataract formation due to specific mutations at site 23 in human γD–crystallin.  A. Pande, O. Anunziata, N. Asherie, O. Ogun, G.B. Benedek, J. Pande, Biochemistry 44, 2491–2500 (2005).
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