Abstract
Purpose: :
The Glu107 to Ala (E107A) mutant of human γD crystallin (HGD) is associated with nuclear cataract. To determine the molecular basis of light scattering and opacity due to this mutation, we compared the solution behavior of HGD and E107A.
Methods: :
Spectroscopic methods such CD, and tryptophan- and ANS- fluorescence were used to compare the secondary and tertiary structure and surface hydrophobicity of HGD and E107A. Isoelectric focusing was used to determine pI values. Thermal unfolding of the proteins was monitored using CD, to compare their stabilities. Liquid-liquid phase transition (LLPT) temperatures were determined using cloud-point measurements. Preliminary measurements of the LLPT temperatures of mixtures of these two individual proteins with alpha crystallin were made at several compositions.
Results: :
Our most striking observation is that the E107A mutation in HGD has no effect on the solubility or light scattering profile of the mutant protein relative to HGD, at physiological pH, even though the pI of E107A is about 1.5 pH units higher than that of HGD. Furthermore, all our spectroscopic studies show that E107A is comparable to HGD in secondary and tertiary structure as well as thermal stability. In experiments with alpha-gamma crystallin mixtures, we observe an increase in the LLPT temperatures of both mixtures (namely HGD-alpha and E107A-alpha), as expected (1) at protein concentrations comparable to that in the human lens. Moreover, the differential increment in LLPT temperature appears to maximize when the mixture composition is close to 50% (w:w) alpha crystallin: The HGD-alpha mixture has a significantly higher LLPT temperature than the E107A-alpha mixture. Stradner et al (2) have suggested that an increase in the normal attractive interactions between gamma- and alpha-crystallin would lead to increased light scattering and altered LLPT. The significantly higher pI of E107A than HGD is therefore likely to lead to increased attractive interactions with alpha crystallin, and consequently to increased light scattering and altered LLPT in the E107A-alpha crystallin mixtures.
Conclusions: :
The cataract-associated mutant protein E107A does not show any individual property that could be implicated as a likely cause of light scattering in cataract. However, altered interactions of the mutant protein with alpha crystallin may be responsible for the increased light scattering and opacity observed in vivo.1. Thurston G.M. (2006) J. Chem. Phys. 124(13):134909.2. Stradner A, Foffi G, Dorsaz N, Thurston G, Schurtenberger P. (2007) Phys Rev Lett. 99(19):198103.
Keywords: crystallins • cataract • protein structure/function