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Investigating the Structural and Metal Binding Properties of an Ancestral Eye Lens Crystallin

  • Author(s): Kozlyuk, Natalia
  • Advisor(s): Martin, Rachel W.
  • et al.
Abstract

βγ-crystallins are structural proteins in the eye lens that refract light to produce an image. These proteins are found at extremely high concentrations in the lens and must remain sol- uble and transparent. If their solubility is perturbed due to a mutation, chemical damage, or a disturbance in metal ion homeostasis, then cataract can develop and cloud the eye lens. In order to understand more about the evolutionary origin of the structure and aggrega- tion of these proteins in the human eye lens, the structure and metal binding properties of an ancestral Ca2+ binding βγ-crystallin found in the Ciona intestinalis tunicate were investigated using biophysical characterization techniques such as fluorescence, CD, ITC, DLS and solution-state NMR. It was found that the tunicate crystallin has a conformational change upon addition of Ca2+ and other divalent cations. This protein is highly stabilized both chemically and thermally upon binding to Ca2+ and it binds more strongly than has been previously reported for other Ca2+ binding βγ-crystallins. It was also found that this crystallin interacts with other divalent cations, all of which thermally stabilize the protein. Although divalent metal ions raise the melting temperature of the tunicate crystallin, aggre- gation is induced by the interaction with some of these metal ions well below the melting temperature. Several solution-state NMR techniques were developed in order to solve the structure of this tunicate βγ-crystallin and other related biomolecules in the presence of divalent metal ions. One of the methods uses DLPC/DHPC bicelles, a low-temperature alignment medium, to obtain long range distance and angular restraints of dynamic and heat sensitive biomolecules in the presence of divalent metal ions. These restraints are de- termined via measurement of residual dipolar couplings, where J-couplings of a biomolecule in isotropic solution are compared with the J+D values for the biomolecule in the presence of an alignment medium. If there is a change in this peak splitting, then alignment of the biomolecule is occuring. Another technique involves using a small molecular probe dCDP and 31P NMR to determine the macromolecule-free and macromolecule-bound divalent metal ion concentrations in NMR samples out of a total amount of divalent metal ions put into the system.

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