Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology.
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.
Vos
Werner Louwrens
WL
The Marie Curie Laboratory for Membrane Proteins, National University of Ireland Maynooth, County Kildare, Ireland.
Vaughan
Sebastian
S
Lall
Patrick Y
PY
McCaffrey
John G
JG
Wysocka-Kapcinska
Monika
M
Findlay
John B C
JB
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002942:Circular Dichroism; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D007381:Intermediate Filament Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D064531:Peripherins; D010843:Pichia / Q000235:genetics; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D055214:Retinal Photoreceptor Cell Outer Segment / Q000378:metabolism*; D013057:Spectrum Analysis
D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C578994:PRPH protein, human; C578997:PRPH2 protein, human; D064531:Peripherins