May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Probing Folding and Ultraviolet Absorption in Human gammaD Crystallin Using Tryptophan>Phenylalanine Mutants
Author Affiliations & Notes
  • J.A. King
    Biology, MIT, Cambridge, MA
  • M. Kosinski–Collins
    Biology, MIT, Cambridge, MA
  • Footnotes
    Commercial Relationships  J.A. King, None; M. Kosinski–Collins, None.
  • Footnotes
    Support  NIH GM17980
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3971. doi:
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      J.A. King, M. Kosinski–Collins; Probing Folding and Ultraviolet Absorption in Human gammaD Crystallin Using Tryptophan>Phenylalanine Mutants . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3971.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose:Human gD crystallin (HgD–Crys), a major component of the human eye lens, is a 173 residue primarily b–sheet protein that is found in juvenile and mature–onset cataracts. HgD–Crys has four tryptophans that are conserved throughout the g–crystallin family, with two in each of the homologous Greek key domains forming the native two–domain structure. HgD–Crys exhibits native state quenching of the UV–induced fluorescence of one or more of these tryptophans, despite the absence of ligands or cofactors. The tryptophan absorption and fluorescence quenching may be part of the mechanism of protecting the retina and lens from ambient ultraviolet damage. Covalent damage leading to partial unfolding may contribute to crystallin aggregation. Methods:Triple mutants each containing three tryptophan to phenylalanine substitutions with one native tryptophan have been constructed, expressed and characterized with respect to their UV absorption, fluorescence emission, stability, refolding and aggregation. Results:The two mutant proteins containing Trp42–only and Trp130–only exhibited a reversal of the inverse quenching between native and denatured states. This indicated that the local environments around Trp68 (domain I) and Trp156 (domain II) are responsible for the native state quenching of the Human gammaD crystallin fluorescence emission. The 3–D structure of HgD–Crys shows two Tyr–Tyr–His aromatic cages surrounding Trp68 and Trp156 that may be the source of the native–state quenching. During equilibrium refolding/unfolding the fluorescence signals indicated that tryptophans in domain I (W42–only and W68–only) unfolded at lower concentrations of GdnHCl than tryptophans in domain II (W130–only and W156–only). Kinetic analysis of the unfolding and refolding of the tryptophan mutants identified an intermediate along the HgD–Crys folding pathway with domain I unfolded and domain II intact. Conclusions:The aromatic environments surrounding the buried core Trp68 and Trp156 residues may protect HgD–crys from UV damage by quenching UV–induced excited states. The partially unfolded intermediate identified both kinetically and thermodynamically is a candidate for the precursor in the in vitro aggregation pathway of HgD–Crys. This may offer an in vitro model for cataract formation.

Keywords: crystallins • cataract • protein structure/function 
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