Crystallins are highly stable eye lens proteins that maintain solubility at high concentrations, up to 400 mg/mL in humans and 1000 mg/mL in fish, over the entire lifetime of an organism. Here, we investigate γS1-crystallin (DMγS1) from the Antarctic toothfish, Dissostichus mawsoni. The focus is the effect of deamidation on the biophysical properties and structure of this cold-tolerant, aggregation-resistant protein. Deamidation, the conversion of Asp/Glu to Asn/Iso-Asn/Gln, is a post-translational modification commonly found in cataractous lenes. It has previously been shown that progressive deamidation can drastically affect the structure, stability, and dimerization/aggregation propensity in HγS crystallin by promoting both intra- and inter-molecular disulfide bonds. We made three deamidated variants of DMγS1 with 3-, 5-, and 7-deamidation sites. Progressive deamidation of DMγS1 crystallin does not change the global structure of each variant; however, there are significant local structural perturbations between the WT and 7SV proteins. Progressive deamidation of DMγS1 does change the mechanism of unfolding when exposed to chemical denaturant. WT and 3SV DMγS1 undergo a two-state unfolding mechanism while 5SV and 7SV undergo a three-state unfolding mechanism. Each variant has one intramolecular disulfide bond between the same two cysteines. We find that 7SV is the least aggregation resistant followed by 5SV, WT, and 3SV. The addition of reducing agent enables each variant to resist both aggregation and unfolding at higher temperatures. This suggests that the intra-molecular disulfide bond does not promote stability but instead locks the protein in an unfavorable configuration.
Sroge et al. (Sun,) studied this question.