(R4873) Crystallin Proteins from Aquatic Organisms: Stability and Function in the Lens and Beyond

Rachel Martin (University of California, Irvine)| Marc Sprague-Piercy (University of California, Irvine)| Megha Unhelkar (University of California, Irvine)| Brenna Norton-Baker (University of California, Irvine)| Megan Rocha (University of California, Irvine)| Jessica Kelz (University of California, Irvine)| Chelsea Anorma (University of California, Irvine)

The crystallins are the major structural and refractive proteins of the eye lens. They are extremely long-lived proteins that maintain their stability for years or decades in the near-absence of protein turnover in the lens. In vertebrates, the structural crystallins are characterized by a common structural motif, the double-Greek key βγ-crystallin fold that originated as a Ca2+-binding protein, a function that is still maintained by microbial crystallins. Ca2+-binding βγ-crystallins are characterized by a conserved N/D-N/D-#-I-S/T-S double-clamp metal ion  binding motif located in a loop region. Despite the conservation of the overall fold, the ubiquitous vertebrate structural crystallins have mostly lost their cation-binding activity in favor of stabilization via other structural means.  The βγ-crystallin from the tunicate Ciona intestinalis provides a fascinating example of a protein that is intermediate in function: it tightly binds two Ca2+ ions, and is greatly stabilized by doing so. At the same time, it is expressed in the sensory organs of the larval tunicate, and earlier work by our group has shown that its refractive index increment is higher than would be expected based on its amino acid sequence alone, suggesting an additional refractive function.  Structurally, the tunicate crystallin resembles one half of a vertebrate βγ-crystallin, which evolved from a common ancestor of these proteins via gene duplication and selection for new functionality in the lens.

Recent work from my laboratory has focused on investigating the structure and dynamics of the tunicate crystallin in the presence and absence of Ca2+ using solution-state NMR spectroscopy. Although a crystal structure (PDB ID:2BV2) was apreviously solved by Shimeld et al., NMR data enable us to probe the molecular motions on a site-by-site basis. Identification of protein sidechains or regions of the backbone with lower-amplitude dynamics in the holo form compared to the apo form highlight features that increase the stability of the protein upon Ca2+ binding. Comparison of these sequence and structural factors with those of the vertebrate structural crystallins point to features that are needed to provide stability in the absence of cation binding as well as higher refractivity in the vertebrate lens. I will present NMR spectra and other spectroscopic data illustrating the structural and motional differences between Ca2+-bound and apo tunicate crystallin, as well as interactions with the solvent. Finally, I will discuss similarities differences between the vertebrate structural crystallins and an evolutionarily unrelated protein that serves the same function, the J2-crystallin of the box jelly Tripedalia cystophora. Unlike C. intestinalis and most other non-cephalopod invertebrates, T. cystophora has camera-type eyes with a lens and a retina. J2-crystallin has a completely different fold than the vertebrate proteins, yet it must perform the same function. I will present structural and biophysical data for this protein and discuss general functional characteristics of structural and refractive lens proteins.  

Support or Funding Information

NSF BMAT DMR-2002837, NIH R01EY021514



(A) The βγ-crystallin domain consists of a double Greek key motif. A single  Greek key is boxed. They stack to form wedge like domains as shown in B and C. (B) The  Ciona intestinalis (tunicate) crystallin  (PDB ID:2BV2) has a single βγ-crystallin domain and two Ca2+ binding sites (circled). (C) The human lens γS-crystallin (PDB ID:2M3T) comprises two βγ-crystallin domains.