Recent Advances in the Application of Solution NMR Spectroscopy to Multi-Span Integral Membrane Proteins

Korea Polar Research Institute, Korea Ocean Research and Development Institute, Incheon, 406-840, Korea.
Progress in Nuclear Magnetic Resonance Spectroscopy (Impact Factor: 7.24). 11/2009; 55(4):335-360. DOI: 10.1016/j.pnmrs.2009.07.002
Source: PubMed


Integral membrane proteins (IMPs), which can only be released from the membrane by disruption of the membrane, perform a host of vital cellular functions as receptors, transporters, channels, electrical and photo-transducers. The aggregate molecular weight of an IMP-model membrane complex offers some insight into the challenge that a solution NMR structural effort will present under the most favorable circumstances. Cell-free systems have emerged as a promising alternative for preparing large quantities of isotopically labeled membrane proteins. Higher complexity eukaryotic hosts have also been used for producing protein samples for NMR characterization. These systems include baculovirus-infected insect cells and transfected mammalian cells. Successful NMR studies of IMPs have been carried out in organic solvent mixtures. A report from MacKenzie and co-workers has demonstrated that the addition of very modest amounts of phospholipids to micelles can result in the enhancements of NMR spectral quality for some integral membrane proteins.

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Available from: Hak Jun Kim, Jul 31, 2015
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    • "One disadvantage by using cell based expression is the inability to produce toxic proteins, as it was the case for TMD0 in our study. The E. coli based CF expression system is a perfect tool to overcome this problem, because it is independent of cellular integrity (Kim et al. 2009). Further advantages of this system are that many conditions can rapidly be screened Fig. 7 Comparison of NMR spectra of construct B and C. [ 15 N, 1 H]-TROSY NMR spectra of construct B (a–c) and C (d– f) in 1–2 % DHPC without (a, d) or with 75 mM NaCl (b, c, e, f) were recorded at 323 K in 25 mM Na-acetate pH 5.0. "
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    ABSTRACT: The ATP binding cassette transporter TAPL translocates cytosolic peptides into the lumen of lysosomes driven by the hydrolysis of ATP. Functionally, this transporter can be divided into coreTAPL, comprising the transport function, and an additional N-terminal transmembrane domain called TMD0, which is essential for lysosomal targeting and mediates the interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. To elucidate the structure of this unique domain, we developed protocols for the production of high quantities of cell-free expressed TMD0 by screening different N-terminal expression tags. Independently of the amino acid sequence, high expression was detected for AU-rich sequences in the first seven codons, decreasing the free energy of RNA secondary structure formation at translation initiation. Furthermore, avoiding NGG codons in the region of translation initiation demonstrated a positive effect on expression. For NMR studies, conditions were optimized for high solubilization efficiency, long-term stability, and high quality spectra. A most critical step was the careful exchange of the detergent used for solubilization by the detergent dihexanoylphosphatidylcholine. Several constructs of different size were tested in order to stabilize the fold of TMD0 as well as to reduce the conformation exchange. NMR spectra with sufficient resolution and homogeneity were finally obtained with a TMD0 derivative only modified by a C-terminal His10-tag and containing a codon optimized AT-rich sequence.
    Full-text · Article · Sep 2013 · Journal of Biomolecular NMR
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    • "For this reason, it is crucial to select a proper solution medium for NMR studies of membrane proteins. Choice of detergent is empirical and protein-specific, and has to be optimized during the sample preparation procedure [18]. The solid state NMR technique is suitable to proteins of higher molecular weight, because in contrast to the solution state, the coherence lifetimes in the solid state are not affected by molecular tumbling [19]. "
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    ABSTRACT: Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane.
    Full-text · Article · Jun 2012 · PLoS ONE
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    • "Chemically synthesizing a peptide corresponding to a TM domain of a protein is challenging due to the hydrophobic nature of the amino acids present in membrane proteins [9]. In addition, the peptide/micelle complex present as a protein with a high molecular weight, which makes isotopic label of the peptide necessary for structural determination using NMR spectroscopy. "
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    ABSTRACT: Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr). The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy. These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.
    Full-text · Article · Apr 2012 · Microbial Cell Factories
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