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Ajay Pande, Jayanti Pande; Self-interaction and stability of human lens GammaC-crystallin. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5308.
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© ARVO (1962-2015); The Authors (2016-present)
GammaC crystallin (HGC) is one of the three major gamma crystallins in the human lens. The other two – namely – GammaD (HGD) and GammaS (HGS) crystallins, show net attractive and repulsive self-interactions respectively. Here we report the self-interaction data for GammaC-crystallin.
Protein self-interaction was measured using polyethylene glycol (PEG)–induced liquid-liquid phase separation (LLPS). We compared the LLPS data for HGS and HGC solutions at identical protein concentrations as a function of PEG (Mol. wt. 8 KDa) concentrations (10-20% range). Static and Dynamic light scattering measurements (SLS and DLS respectively) were carried out for HGC in the temperature range 5-25°C. The 2nd Virial Coefficient, B22, was determined using SLS and the diffusion interaction parameter, kD, using DLS. Both parameters provide quantitative measures of the magnitude of net attractive or repulsive self-interactions in proteins. SLS and DLS were also measured as a function of NaCl concentrations (2-8%) to determine the effect of salt concentration on protein self-interaction and stability.
HGC shows no LLPS at pH 7 even at high concentrations (~100 mg/mL), which is similar to HGS but unlike human GammaD-crystallin (HGD). However, HGC shows LLPS at PEG concentrations lower than that needed for HGS. Consistent with this behavior, HGC shows a negative B22 value compared to that for HGS: B22 (HGC) = –3.2 X10-4 mLmolg-2, whereas B22 (HGS) = +8.9 X 10-4 mLmolg-2. Surprisingly, kD was found to increase from –0.6 mL/g to −6.0 mL/g in the range 5°C–25°C for HGC, suggesting that the self-interactions in HGC become more attractive with increasing temperature. Similarly a 2-8% range in NaCl concentration changes kD from +0.9 mL/g to –2.1 mL/g, suggesting that self-interactions in HGC become marginally more attractive with increasing salt.
HGC displays stronger net attractive self-interactions compared to HGS in which the net interactions are repulsive. However the attractive interactions in HGC are weaker than those of HGD. Our data suggest that the self-interactions in HGC become more attractive with increasing temperature and marginally attractive with increasing salt concentration, which should help set the crystallization conditions for HGC. These data strengthen the argument that optimally-tuned protein interactions minimize instability in mixtures and suppress protein aggregation thus maintaining lens transparency.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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