June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Structure and function of the aa108-126 mobile loop of RPE65
Author Affiliations & Notes
  • T. Michael Redmond
    LRCMB, NEI, NIH, Bethesda, Maryland, United States
  • Sheetal Uppal
    LRCMB, NEI, NIH, Bethesda, Maryland, United States
  • Tingting Liu
    LRCMB, NEI, NIH, Bethesda, Maryland, United States
  • Susan Gentleman
    LRCMB, NEI, NIH, Bethesda, Maryland, United States
  • Eugenia Poliakov
    LRCMB, NEI, NIH, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   T. Michael Redmond, None; Sheetal Uppal, None; Tingting Liu, None; Susan Gentleman, None; Eugenia Poliakov, None
  • Footnotes
    Support  Supported by the Intramural Research Program of the National Eye Institute, NIH.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3584. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      T. Michael Redmond, Sheetal Uppal, Tingting Liu, Susan Gentleman, Eugenia Poliakov; Structure and function of the aa108-126 mobile loop of RPE65. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3584.

      Download citation file:

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

  • Supplements

Purpose : While the solved structures of RPE65 retinol isomerase have provided invaluable insights into its catalytic mechanism and endoplasmic reticulum (ER) membrane interaction, none of the structures to date provide high resolution of the critical loop containing aa110-126 of RPE65. This putatively mobile loop, containing a PDPCK motif highly conserved in the carotenoid oxygenase family, in RPE65 is thought to interact with the ER membrane, in part via the putatively palmitoylated C112 residue. We used in vitro biochemical approaches to study the role of this loop.

Methods : Secondary structure, helical wheel projections and other modeling were carried out using web-based tools. The RPE65 ORF, cloned into the pVIT2 expression vector, was individually mutated by site-directed mutagenesis (SDM) to alanines at residues aa108-126. In addition, selected residues were mutated to other residues as required. Resultant purified mutant plasmids were assayed for isomerase activity in a minimal visual cycle assay by transfection into HEK293F cells.

Results : Secondary structure analysis of the aa108-126 loop of RPE65 predicts an amphipathic alpha helical structure with the key C112 found in the hydrophobic face, spatially flanked by highly hydrophobic Phe residues. In contrast, the hydrophilic face is overall basic in charge. The relatively high magnitude (7.36) of the calculated hydrophobic moment reflects the contrast between the hydrophobic and hydrophilic faces of the predicted helix. The effect on the retinol isomerase activity seen in the panel of alanine mutants varies from little or no change to loss of activity. About one-third of these 19 residues when mutated to alanine have severe or total loss of activity, and include ones on both hydrophobic and hydrophilic aspects of the predicted helix. Significantly, SDM of a number of these residues involving nominally conservative changes (e.g., Asp to Glu, or Asn to Gln) were often as damaging as change to Ala, indicating the importance not only of residue polarity but also of residue side-chain length.

Conclusions : Our analysis of the putatively mobile aa108-126 loop of RPE65 indicates an important role in RPE65 function. We conclude that the contrast between hydrophobic and hydrophilic faces of this putative amphipathic helix is important to its function. We propose that the hydrophobic face embeds into the ER membrane; the precise role of the charged hydrophilic face, however, remains unclear.

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.