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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 a-helices. 15N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effectivemore » 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.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1035740
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Protein Expression and Purification
Additional Journal Information:
Journal Volume: 80; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ABUNDANCE; AFFINITY; AMINO ACIDS; CRYSTALLOGRAPHY; DETERGENTS; DICHROISM; ESCHERICHIA COLI; FLUORESCENCE; FLUORESCENCE SPECTROSCOPY; IN VITRO; LIPOSOMES; MEMBRANE PROTEINS; NMR SPECTRA; PROTEINS; SPECTRA; SPECTROSCOPY; STABILIZATION; TRYPTOPHAN; Environmental Molecular Sciences Laboratory

Citation Formats

Ni, Da Qun, Zook, James, Klewer, Douglas A, Nieman, Ronald A, Soll, J, and Fromme, Petra. Isolation, folding and structural investigations of the amino acid transporter OEP16. United States: N. p., 2011. Web. doi:10.1016/j.pep.2011.08.004.
Ni, Da Qun, Zook, James, Klewer, Douglas A, Nieman, Ronald A, Soll, J, & Fromme, Petra. Isolation, folding and structural investigations of the amino acid transporter OEP16. United States. https://doi.org/10.1016/j.pep.2011.08.004
Ni, Da Qun, Zook, James, Klewer, Douglas A, Nieman, Ronald A, Soll, J, and Fromme, Petra. 2011. "Isolation, folding and structural investigations of the amino acid transporter OEP16". United States. https://doi.org/10.1016/j.pep.2011.08.004.
@article{osti_1035740,
title = {Isolation, folding and structural investigations of the amino acid transporter OEP16},
author = {Ni, Da Qun and Zook, James and Klewer, Douglas A and Nieman, Ronald A and Soll, J and Fromme, Petra},
abstractNote = {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 a-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.},
doi = {10.1016/j.pep.2011.08.004},
url = {https://www.osti.gov/biblio/1035740}, journal = {Protein Expression and Purification},
number = 2,
volume = 80,
place = {United States},
year = {Thu Dec 01 00:00:00 EST 2011},
month = {Thu Dec 01 00:00:00 EST 2011}
}