June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Role of the N-terminal in tuning the chaperone activity of vertebrate αB crystallin
Author Affiliations & Notes
  • Sanjay Mishra
    Chemical & Physical Biology Program, Vanderbilt University, Nashville, Tennessee, United States
  • Hassane S Mchaourab
    Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee, United States
  • Footnotes
    Commercial Relationships   Sanjay Mishra, None; Hassane Mchaourab, None
  • Footnotes
    Support  NIH Grant EY12018
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5599. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Sanjay Mishra, Hassane S Mchaourab; Role of the N-terminal in tuning the chaperone activity of vertebrate αB crystallin. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5599.

      Download citation file:

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

  • Supplements

Purpose : α-crystallins are canonical small heat shock proteins (sHSP) that are crucial to maintain the optical clarity of the lens. Mutants of αA-crystallin and αB-crystallin have been implicated in autosomal cataracts in humans. Zebrafish (Danio rerio) expresses two distinct homologs of αB crystallin – a lens predominant αB1, and a ubiquitous αB2. We have discovered that αB1 has order of magnitude higher basal chaperone activity than its mammalian homologs. To identify the sequence elements responsible for the activation of the zebrafish ortholog, we have carried out homolog scanning mutagenesis replacing stretches of Danio αB1 sequences with the corresponding sequences from human αB-crystallin.

Methods : We generated chimera by swapping N- and C-terminal residues flanking the alpha crystallin domain from human αB to Danio αB1. We purified the engineered proteins by cloning into bacterial systems, analyzed their molecular mass by light scattering gel filtration and analytical centrifugation. We assayed their chaperone activity against fluorescently labeled model substrate T4-lysozyme.

Results : In vitro expressed zebrafish αB1 assembles into higher order oligomers than human αB1. We can isolate two distinct oligomeric subpopulations: one averaging about 48 subunits and the other averaging about 96 subunits. Progressively swapping N-terminal sequence of Danio αB1 with the human sequence shifts the equilibrium away from the larger oligomer and reduces the chaperone activity of the 48-subunit oligomer. Swapping of the C-terminal sequences does not affect the chaperone activity and the oligomeric assembly.

Conclusions : Our preliminary results suggest that oligomeric architecture of sHsps is modulated by their N-terminal flanking region, which modulates differential chaperone activity. Detailed analyses on the observed effects are currently in progress. We surmise that this approach can be used to identify the role of sequence elements in the evolutionary tuning of the chaperone function of a sHsp.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.


This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.