title: Cloning, expression, and preliminary structural characterization of RTN-1C.
abstract: Reticulons (RTNs) are endoplasmic reticulum-associated proteins widely distributed in plants, yeast, and animals. They are characterized by unique N-terminal parts and a common 200 amino acid C-terminal domain containing two long hydrophobic sequences. Despite their implication in many cellular processes, their molecular structure and function are still largely unknown. In this study, the reticulon family member RTN-1C has been expressed and purified in Escherichia coli and its molecular structure has been analysed by fluorescence and CD spectroscopy in different detergents in order to obtain a good solubility and a relative stability. The isotopically enriched protein has been also produced to perform structural studies by NMR spectroscopy. The preliminary results obtained showed that RTN-1C protein possesses helical transmembrane segments when a membrane-like environment is produced by detergents. Moreover, fluorescence experiments indicated the exposure of tryptophan side chains as predicted by structure prediction programs. We also produced the isotopically labelled protein and the procedure adopted allowed us to plan future NMR studies to investigate the biochemical behaviour of reticulon-1C and of its peptides spanning out from the membrane.
authors.affiliation: Department of Science and Chemical Technologies, University of Rome Tor Vergata, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Fazi
authors.forename: Sonia
authors.initials: S
authors.lastname: Melino
authors.forename: Federica
authors.initials: F
authors.lastname: Di Sano
authors.forename: Daniel O
authors.initials: DO
authors.lastname: Cicero
authors.forename: Mauro
authors.initials: M
authors.lastname: Piacentini
authors.forename: Maurizio
authors.initials: M
authors.lastname: Paci
mesh_terms: D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D009419:Nerve Tissue Proteins; C081781:RTN1 protein, human; D011993:Recombinant Fusion Proteins