Isolation, folding and structural investigations of the amino acid transporter OEP16
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.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1035740
- Journal Information:
- Protein Expression and Purification, Vol. 80, Issue 2
- Country of Publication:
- United States
- Language:
- English
Similar Records
High-resolution sup 13 C NMR study of the topography and dynamics of methionine residues in detergent-solubilized bacteriorhodopsin
Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex
Related Subjects
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