Isotope labeling of mammalian GPCRs in HEK293 cells and characterization of the C-terminus of bovine rhodopsin by high resolution liquid NMR spectroscopy

Werner, Karla; Richter, Christian; Klein‐Seetharaman, Judith; Schwalbe, Harald

doi:10.1007/s10858-007-9205-3

Cited by 66 publications

(50 citation statements)

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“…Recent advances in the expression and purification of membrane proteins have been described for various expression hosts, for example: Escherichia coli (Drew et al 2003(Drew et al , 2005Grisshammer et al 2005), yeast (Wedekind et al 2006;Lee et al 2007), insect cells (Massotte 2003), mammalian cells (Yin et al 2005;Werner et al 2008) and cell-free systems (Klammt et al 2007). However, from approximately 1000 known GPCRs, only five high-resolution 3-D structures of two distinct receptor types have been reported: bovine rhodopsin (Palczewski et al 2000) and opsin (Park et al 2008), squid rhodopsin (Murakami and Kouyama 2008), the human b2-adrenergic receptor Rosenbaum et al 2007) and the turkey b1-adrenergic receptor (Warne et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

2008

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The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His) 6 tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.

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Section: Introductionmentioning

confidence: 99%

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

2008

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“…[220] Distance constraints determined by SSNMR spectroscopy facilitated the determination of the high-resolution structure of the TTR(105-115) monomer, [71] and constrained the interstrand and intersheet arrangements. TTR (105)(106)(107)(108)(109)(110)(111)(112)(113)(114)(115) peptides were arranged into parallel, in-register b-sheets stacked antiparallel to one another (Figure 8). …”

Section: Accessibilitymentioning

confidence: 99%

“…Although these systems offer higher flexibility for proper folding and PTMs, the high cost of production and low degree of isotopic labeling are common problems. [115][116][117] …”

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confidence: 99%

Recent Advances in Magic‐Angle Spinning Solid‐State NMR of Proteins

Ladizhansky¹

2014

Israel Journal of Chemistry

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Magic‐angle spinning (MAS) solid‐state NMR (SSNMR) spectroscopy is emerging as an important technique for the determination of three‐dimensional structures of biological molecules and for the characterization of their dynamics. While there is an established suite of MAS SSNMR experiments for protein structure determination in small‐ and medium‐sized proteins, these methods face many challenges in large systems. In this review, recent progress in MAS NMR spectroscopy is discussed, specifically focusing on the emerging developments aimed at improving the sensitivity and resolution of SSNMR that are likely to determine its future applications. These developments include sample preparation and isotopic labeling strategies, fast MAS, proton detection, and paramagnetic NMR spectroscopy.

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“…Their structures have been studied using crystallography [3][4][5][6] , NMR [7,8] , and computational methodologies [9,10] , in an effort to discern new ligands using structure-based virtual screening [11] . When an agonist binds to a GPCR, a signal is transduced across the membrane through heterotrimeric G proteins leading to activation of second messenger systems.…”

Section: Introductionmentioning

confidence: 99%