European Biophysics Journal

, Volume 39, Issue 4, pp 679–688

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology

  • Werner Louwrens Vos
  • Sebastian Vaughan
  • Patrick Y. Lall
  • John G. McCaffrey
  • Monika Wysocka-Kapcinska
  • John B. C. Findlay
Original Paper

DOI: 10.1007/s00249-009-0553-7

Cite this article as:
Vos, W.L., Vaughan, S., Lall, P.Y. et al. Eur Biophys J (2010) 39: 679. doi:10.1007/s00249-009-0553-7

Abstract

Peripherin/RDS is a member of the tetraspanin family of integral membrane proteins and plays a major role in the morphology of photoreceptor outer segments. Peripherin/RDS has a long extracellular loop (hereafter referred to as the LEL domain), which is vital for its function. Point mutations in the LEL domain often lead to impaired photoreceptor formation and function, making peripherin/RDS an important drug target. Being a eukaryotic membrane protein, acquiring sufficient peripherin/RDS for biophysical characterisation represents a significant challenge. Here, we describe the expression and characterisation of peripherin/RDS in Drosophila melangolaster Schneider (S2) insect cells and in the methylotrophic yeast Pichia pastoris. The wild-type peripherin/RDS and the retinitis pigmentosa causing P216L mutant from S2 cells are characterised using circular dichroism (CD) spectroscopy. The structure of peripherin/RDS and of a pathogenic mutant is assessed spectroscopically for the first time. These findings are evaluated in relation to a three-dimensional model of the functionally important LEL domain obtained by protein threading.

Keywords

PichiaS2 cellsCircular dichroismProtein threadingPeripherin-2Retinitis pigmentosa

Abbreviations

LEL domain

Long extracellular loop domain

CD

Circular dichroism

p/RDS

Peripherin/retinal degeneration slow

NMR

Nuclear magnetic resonance

CD81

Cluster of differentiation 81

DDM

n-Dodecyl beta-d-maltoside

BCA

Bicinchoninic acid

LC–MS/MS

Liquid chromatography-mass spectrometry/mass spectrometry

ROM-1

Retinal outer segment membrane protein 1

FTIR

Fourier transform infrared

SDS

Sodium dodecyl sulfate

PAGE

Polyacrylamide gel electrophoresis

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • Werner Louwrens Vos
    • 1
  • Sebastian Vaughan
    • 3
  • Patrick Y. Lall
    • 2
  • John G. McCaffrey
    • 2
  • Monika Wysocka-Kapcinska
    • 1
  • John B. C. Findlay
    • 1
    • 3
  1. 1.The Marie Curie Laboratory for Membrane ProteinsNational University of Ireland MaynoothCounty KildareIreland
  2. 2.Department of ChemistryNational University of Ireland MaynoothCounty KildareIreland
  3. 3.Institute for Membrane and Systems Biology, Faculty of Biological SciencesUniversity of LeedsLeedsUnited Kingdom