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

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.

High Prevalence of PRPH2 in Autosomal Dominant Retinitis Pigmentosa in France and Characterization of Biochemical and Clinical Features

“…Wild type (WT) and p.Leu254Gln (L254Q) mutant were cloned into the pPICZ expression vector containing the c-myc epitope and the polyhistidine (His)6-tag as described before, 33 the nucleotide sequence was confirmed by Eurofins MWG using automated DNA sequencing. Pichia pastoris cells (strain KM71H) were transformed with the PmeI linearised expression vector, stably transformed cells were spread on YPD plates [1% yeast extract, 2% peptone (BD), 2% glucose, 2% agar] with media containing 100 µg/ml zeocin.…”

“…Cells were cultured, harvested, and stored at -80°C as described before. 33 Cells were lysed upon further processing and membranes containing the WT or L254Q proteins were isolated using differential centrifugation as described previously. The membranes were dissolved in 1% n-dodecyl-β-Dmaltoside (DDM) using sequentially an 18G, 19G, and 25G needle.…”

Structural characterization of the second intra‐discal loop of the photoreceptor tetraspanin RDS

“…Similarly, the percentage of α–helix in the N244K D2 loop is similar to that of the wild‐type D2 loop, but the percentage in the C214S D2 loop is reduced, even though both cause autosomal dominant retinitis pigmentosa or rod/cone dystrophy . The only other study that has examined the secondary structure of RDS reported very little change in secondary structure in the case of the P216L autosomal dominant retinitis pigmentosa mutation , although that study examined the secondary structure of the entire RDS polypeptide (not just the D2 loop), so it is difficult to compare results. These results suggest that assessing each mutation independently may be a better way to approach understanding of RDS‐associated disease, and it is likely that, for many mutations, disease arises due to a combination of molecular defects.…”

A novel membrane based process to isolate photosystem-I membrane complex from spinach

“…Their primary applications range from solubilization and stabilization of proteins to disaggregation of protein complexes and denaturation (Neugebauer 1990). In this study, the non-ionic detergents, Triton X-100 and DDM, were employed to isolate PS-I from spinach thylakoid in aqueous solution, which have been used extensively in studies of photosynthetic membranes (Ge et al 2010;Iwuchukwu et al 2010;Tsiotis et al 1993;Vos et al 2010). …”