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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2017)

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.

    Cover page: Investigating the Structural and Metal Binding Properties of an Ancestral Eye Lens Crystallin
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2016)
    The tunicate (Ciona intestinalis) βγ-crystallin represents an intermediate case between the calcium-binding proteins ancestral to the vertebrate βγ-crystallin fold and the vertebrate structural crystallins. Unlike the structural βγ-crystallins in the vertebrate eye lens, this βγ-crystallin strongly binds Ca2+. Furthermore, Ca2+ binding greatly stabilizes the protein, an effect that has previously been observed in microbial βγ-crystallins but not in those of vertebrates. This relationship between binding and protein stabilization makes the tunicate βγ-crystallin an interesting model for studying the evolution of the human βγ-crystallin. We also compare and contrast the binding sites of tunicate βγ-crystallin with those of other βγ-crystallins to develop hypotheses about the functional origin of the lack of Ca2+-binding sites in human crystallins.
      Cover page: Calcium Binding Dramatically Stabilizes an Ancestral Crystallin Fold in Tunicate βγ-Crystallin
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2016)
      Many nucleic acids and proteins require divalent metal ions such as Mg(2+) and Ca(2+) for folding and function. The lipophilic alignment media frequently used as membrane mimetics also bind these divalent metals. Here we demonstrate the use of (31)P NMR spectrum of a metal ion chelator (deoxycytidine diphosphate) to measure the bound [Mg(2+)] and [Ca(2+)] in situ for several biological model systems at relatively high divalent ion concentrations (1-10 mM). This method represents a general approach to measuring divalent metal ion binding in NMR samples where the amount and type of metal ion added to the system is known.
      • 1 supplemental ZIP
      Cover page: In situ NMR measurement of macromolecule-bound metal ion concentrations
      • Article
      • Peer Reviewed
      LBL Publications (2018)
      The refractive index gradient of the eye lens is controlled by the concentration and distribution of its component crystallin proteins, which are highly enriched in polarizable amino acids. The current understanding of the refractive index increment ([Formula: see text]) of proteins is described using an additive model wherein the refractivity and specific volume of each amino acid type contributes according to abundance in the primary sequence. Here we present experimental measurements of [Formula: see text] for crystallins from the human lens and those of aquatic animals under uniform solvent conditions. In all cases, the measured values are much higher than those predicted from primary sequence alone, suggesting that structural factors also contribute to protein refractive index.
        Cover page: Protein refractive index increment is determined by conformation as well as composition
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