Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins

Kim, Soo Hyun; Howell, Stanley C.; Horn, Wade D. Van; Jeon, Young Ho; Sanders, Charles R.

doi:10.1016/j.pnmrs.2009.07.002

Cited by 142 publications

(110 citation statements)

References 420 publications

(501 reference statements)

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“…Significant peak overlap is apparent in all conditions tested, as is typically observed in α-helical membrane proteins (Kim et al 2009). The global conformation and/or local dielectric constant experienced by a given region of the protein appears to vary as a function of the solvent or micelle system employed, as indicated by some peak motion in the HSQC spectra ( Figure 7D).…”

Section: Solution-state Nmr Spectroscopysupporting

confidence: 58%

Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor

Pandey

Sarker

Liu

et al. 2014

Biochem. Cell Biol.

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G-protein coupled receptors (GPCRs) are inherently dynamic membrane protein modulators of various important cellular signaling cascades. The apelin receptor (AR or APJ) is a class A GPCR involved in numerous physiological processes, implicated in angiogenesis during tumour formation and as a CD4 co-receptor for entry of human immunodeficiency virus type 1 (HIV-1) to cells. Due to the lack of efficient methods to produce full-length GPCRs enriched with nuclear magnetic resonance (NMR) active 15 N, 13 C and/or 2 H isotopes, small GPCR fragments typically comprising 1-2 transmembrane segments are frequently studied using NMR spectroscopy. Here, we report successful overexpression of transmembrane segments 1-3 of AR (AR_TM1-3) in the C41(DE3) strain of Escherichia coli using an AT-rich gene tag previously reported to enhance cellfree expression yields. The resulting protein, with 6 additional N-terminal residues due to the expression tag, was purified using high performance liquid chromatography (HPLC). Farultraviolet circular dichroism spectropolarimetry demonstrates that AR_TM1-3 has the predicted ~40% α-helical character in membrane-mimetic environments. 1 H-15 N HSQC NMR experiments imply amenability to high-resolution NMR structural characterization and stability in solution for weeks. Notably, this small expression tag approach may also be generally applicable to other membrane proteins that are difficult to express in E. coli.

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Section: Solution-state Nmr Spectroscopysupporting

confidence: 58%

Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor

Pandey

Sarker

Liu

et al. 2014

Biochem. Cell Biol.

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“…In addition, NMR spectroscopy can provide insight into time-dependent chemical phenomena, including reaction kinetics and dynamics in solution and the solid state at atomic resolution [33]. Although the cumulative molecular mass of a protein-detergent complex, the oligomerization state of the protein and a limited dispersion of an NMR spectrum may pose formidable hurdles for the structural study of membrane proteins by solution-state NMR, significant advances in sample preparation and experimental NMR methods have established NMR spectroscopy as a powerful method for studying membrane proteins both in the solution and in the solid state [34,35].…”

Section: Nmr Spectroscopy Of Membrane Proteinsmentioning

confidence: 99%

Structure of the mammalian TSPO/PBR protein

Jaremko

Jaipuria

et al. 2015

Biochemical Society Transactions

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The 3D structure of the 18-kDa transmembrane (TM) protein TSPO (translocator protein)/PBR (peripheral benzodiazepine receptor), which contains a binding site for benzodiazepines, is important to better understand its function and regulation by endogenous and synthetic ligands. We have recently determined the structure of mammalian TSPO/PBR in complex with the diagnostic ligand PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; Science 343, 1363-1366], providing for the first time atomic-level insight into the conformation of this protein, which is up-regulated in various pathological conditions including Alzheimer's disease and Parkinson's disease. Here, we review the studies which have probed the structural properties of mammalian TSPO/PBR as well as the homologues bacterial tryptophan-rich sensory proteins (TspOs) over the years and provide detailed insight into the 3D structure of mouse TSPO (mTSPO)/PBR in complex with PK11195.

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“…Structure determination by NMR requires the sequential assignment of backbone resonances, a process that is particularly laborious for α-helical MPs (discussed in ref. 15). An alternative method of backbone resonance assignment utilizing dual selective 15 N and 13 C labeling and heteronuclear 13 C-15 N spin coupling was originally proposed by Kainosho and Tsuji (22) and improved later by the high selectivity of isotope labeling in CF synthesis and the efficiency of modern NMR heteronuclear techniques (23).…”

Section: Sequence-optimized Isotope Labeling Scheme For Rapid Resonancementioning

confidence: 99%

Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis

Maslennikov

Klammt

Hwang

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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115

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NMR structural studies of membrane proteins (MP) are hampered by complications in MP expression, technical difficulties associated with the slow process of NMR spectral peak assignment, and limited distance information obtainable for transmembrane (TM) helices. To overcome the inherent challenges in the determination of MP structures, we have developed a rapid and cost-efficient strategy that combines cell-free (CF) protein synthesis, optimized combinatorial dual-isotope labeling for nearly instant resonance assignment, and fast acquisition of long-distance information using paramagnetic probes. Here we report three backbone structures for the TM domains of the three classes of Escherichia coli histidine kinase receptors (HKRs). The ArcB and QseC TM domains are both two-helical motifs, whereas the KdpD TM domain comprises a four-helical bundle with shorter second and third helices. The interhelical distances (up to 12 Å) reveal weak interactions within the TM domains of all three receptors. Determined consecutively within 8 months, these structures offer insight into the abundant and underrepresented in the Protein Data Bank class of 2-4 TM crossers and demonstrate the efficiency of our CF combinatorial dual-labeling strategy, which can be applied to solve MP structures in high numbers and at a high speed. Our results greatly expand the current knowledge of HKR structure, opening the doors to studies on their widespread and pharmaceutically important bacterial signaling mechanism.backbone NMR structure | cell-free synthesis | combinatorial selective labeling

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Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins

Cited by 142 publications

References 420 publications

Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor

Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor

Structure of the mammalian TSPO/PBR protein

Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis

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