April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Identification Of The Binding Site Of Mini-alphaA Crystallin On Human GammaD-crystallin
Author Affiliations & Notes
  • Ajay Pande
    Chemistry, University at Albany-SUNY, Albany, New York
  • Priya R. Banerjee
    Chemistry, University at Albany-SUNY, Albany, New York
  • Shadakshara S. Puttamadappa
    Chemistry, University at Albany-SUNY, Albany, New York
  • Alexander Shekhtman
    Chemistry, University at Albany-SUNY, Albany, New York
  • Jayanti Pande
    Chemistry, University at Albany-SUNY, Albany, New York
  • Footnotes
    Commercial Relationships  Ajay Pande, None; Priya R. Banerjee, None; Shadakshara S. Puttamadappa, None; Alexander Shekhtman, None; Jayanti Pande, None
  • Footnotes
    Support  NIH Grants EY10535 (JP), GM085006 (AS)
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1614. doi:
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      Ajay Pande, Priya R. Banerjee, Shadakshara S. Puttamadappa, Alexander Shekhtman, Jayanti Pande; Identification Of The Binding Site Of Mini-alphaA Crystallin On Human GammaD-crystallin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1614.

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

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Purpose: : Mini αA-crystallin, a 19-residue peptide (DFVIFLDVKHFSPEDLTVK) of αA-crystallin has chaperone activity (Sreelakshmi, Y and Sharma, K, J. Protein Chemistry, Feb. 2001). However, it is not known where the mini-chaperone binds to γ-crystallin, one of the natural substrates of αA-crystallin in the lens. Here we use high-resolution NMR to identify the binding locus of mini-αA on human γD crystallin (HGD), a major γ-crystallin in the human lens.

Methods: : Recombinant HGD and synthetic mini-α (Genscript, 98% pure) were used in this study. The synthetic peptide was extensively dialyzed against water for 2 days and lyophilized to remove contaminating, residual solvents. The lyophilized protein was dissolved in 5 mM phosphate buffer and showed a far-UV circular dichroic (CD) spectrum typical of a predominantly β-sheet-containing peptide. The peptide structure did not change until 60 oC, the maximum temperature tested. At 55 oC HGD begins to show minor changes in the near-UV CD, which is the first indication of a structural perturbation. Therefore, HGD and mini-αA were kept at 55 oC for 10 min., then mixed in a 1:1 (w/w) ratio. CD of this mixture was compared with that at room temperature. For the NMR measurements, the same procedure was used with 15N-labeled HGD. HSQC (heteronuclear single-quantum coherence) data were obtained, and compared with that of unbound HGD alone.

Results: : CD data show that binding of mini-αA does not alter the structure of HGD. However, it does lead to a change in the chemical shift of a number of HGD residues as observed in the HSQC data. Affected residues in the C-terminal domain of HGD are H88, E96, F105, S110, R115, L127, N138, I171, and D172, and those from the N-terminal domain are T4, Q12, S19, H22, G40, I81, and H83. A model of HGD bound to 2 molecules of mini-αA was generated using the AUTODOCK program. This model accounts for nearly all the chemical shift data and shows that one molecule of mini-αA binds in the C-terminal end close to Trp131, while the other binds most of the affected N-terminal residues, and includes I171 and D172 from the C-terminal.

Conclusions: : The chaperone activity of mini-αA has been known for some time. Here we provide the first model based on high-resolution NMR data, which identifies the region on HGD to which the mini-chaperone binds.

Keywords: chaperones • crystallins • cataract 

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