title: Isolation, folding and structural investigations of the amino acid transporter OEP16.
abstract: Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. ¹⁵N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA.
authors: Da Qun
authors: da Q
authors: Ni
authors: James
authors: J
authors: Zook
authors: Douglas A
authors: DA
authors: Klewer
authors: Ronald A
authors: RA
authors: Nieman
authors: J
authors: J
authors: Soll
authors: Petra
authors: P
authors: Fromme
mesh_terms: D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002736:Chloroplasts / Q000737:chemistry / Q000235:genetics; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D017343:Genes, Plant; D002479:Inclusion Bodies / Q000737:chemistry; D007425:Intracellular Membranes / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008823:Micelles; D017551:Microdialysis / Q000379:methods; D018532:Pisum sativum / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012995:Solubility; D014364:Tryptophan / Q000737:chemistry; D014462:Ultrafiltration / Q000379:methods
chemical_list: D026905:Amino Acid Transport Systems; D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins; D008823:Micelles; D010940:Plant Proteins; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D014364:Tryptophan