βγ-crystallins are refractive proteins that are essential for the visual system. These proteins evolved from ancient Ca 2+ binding proteins and then diverged into multiple vertebrate lens proteins. The progression of βγ-crystallin from metal cation-binding proteins to lens proteins is a central question for understanding evolution of the visual system. Ciona intestinalis βγ-crystallin (Ci-βγ) could be the key linkage to understanding the evolution of the visual system due to it maintaining cation binding properties and sharing the same structural motifs as current vertebrate proteins. Ci-βγ has higher thermal stability than current vertebrate lens proteins. It’s inferred that Ci-βγ’s unique structure of having both cation binding sites and a double greek motif contributes to its thermal and chemical stability. Currently, I am identifying structural motifs that are essential for Ci-βγ’s binding and stability. Comparing these motifs to structural features to current vertebrate lens proteins will provide insights into the evolutionary pathway that shaped the development of the lens. Identifying the moieties of crystallins that allow for protein stability will also contribute to understanding crystallin aggregation. Crystallin aggregation causes cataract to form, which is the leading cause of blindness. I have conducted a temperature ramp up 1 H- 15 N heteronuclear single quantum coherence (HSQC) to identify the intermolecular and intramolecular interactions that occur within Ci-βγ. I have identified 5 residues that switch from intermolecular to intramolecular interactions contributing to its increased stability. I have also conducted 1 H- 15 N HSQCs of Ci-βγ from apo to holo form and identified key residues that undergo strong/weak binding that may contribute to its stability. I have conducted 1H15N chemical saturation exchange transfer NMR experiments to further quantify/characterize Ci-βγ binding dynamics and T1/T2 relaxation experiments to characterize the proteins’ overall dynamics.
Jimenez et al. (Sun,) studied this question.