June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
The cataract-associated Tyr66Asn mutant of human gammaS-crystallin has a folding defect due to a compromised tyrosine corner
Author Affiliations & Notes
  • Jayanti Pande
    Chemistry, University at Albany, SUNY, Albany, New York, United States
  • Wenjuan Hou
    Chemistry, University at Albany, SUNY, Albany, New York, United States
  • Alexander Shekhtman
    Chemistry, University at Albany, SUNY, Albany, New York, United States
  • Ajay Kumar Pande
    Chemistry, University at Albany, SUNY, Albany, New York, United States
  • Footnotes
    Commercial Relationships   Jayanti Pande, None; Wenjuan Hou, None; Alexander Shekhtman, None; Ajay Pande, None
  • Footnotes
    Support  NIH Grant EY010535
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3474. doi:
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    • Get Citation

      Jayanti Pande, Wenjuan Hou, Alexander Shekhtman, Ajay Kumar Pande; The cataract-associated Tyr66Asn mutant of human gammaS-crystallin has a folding defect due to a compromised tyrosine corner. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3474.

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

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Abstract

Purpose :
The Tyr66Asn (Y66N) mutation in human gammaS-crystallin (HGS) leads to nuclear cataract. Despite the unique nature of this mutation (Y66 is invariant and an integral part of the “tyrosine corner”), the mechanism for the observed light scattering and lens opacity is not yet established. Here we provide a plausible mechanism

Methods :
Recombinant wild-type (WT)-HGS, and Y66N, and model mutants Y66F and Y66D were expressed in E.coli and purified using size-exclusion and ion-exchange chromatography. Purity was checked with electrospray-ionization mass spectrometry. Structural characterizations were performed using Circular Dichroism, tryptophan fluorescence and solution Nuclear Magnetic Resonance. Protein refolding experiments were conducted with the Guanidine Hydrochloride-unfolded, soluble form of Y66N. Molecular Dynamics (MD) simulations were performed with GROMACS 2019.3.

Results :
Unlike WT-HGS, Y66N partitions into soluble and insoluble forms when expressed in E.coli at 37°C. Expression of insoluble Y66N shows temperature dependence: At 37°C, Y66N partitions almost equally into soluble and insoluble phases, but at 4°C, Y66N is mainly in the soluble phase. A van’t Hoff-type plot of the data is linear, suggesting that the two forms are in a temperature-dependent equilibrium. NMR data show that replacement of Y66 with N occurs without a significant change in overall protein structure. Protein refolding experiments show a complex kinetic profile relative to those of WT-HGS. Folding in the presence of polyethylene glycol (PEG), a cell crowding mimic, can lead to precipitation of Y66N, in contrast to WT-HGS. The solution behaviors of Y66F, and WT-HGS are closely similar, but Y66D resembles Y66N, suggesting that the phenyl ring of Y in the “tyrosine corner” (but not the –OH), is essential for structural integrity. MD simulations for unfolding show that the N-terminal domain (NTD) in Y66N unfolds more readily than in HGS.

Conclusions :
Elimination of the “tyrosine corner” – a key nucleation site for folding – leads to a folding defect in the NTD of Y66N. Two temperature-dependent, interconvertible forms are produced at physiological temperature. We suggest that the insoluble form is responsible for light scattering and lens opacity due to this mutation.

This is a 2020 ARVO Annual Meeting abstract.

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