G‐protein‐coupled receptor structures were not built in a day

Blois, Tracy M.; Bowie, James U.

doi:10.1002/pro.165

Cited by 28 publications

(17 citation statements)

References 69 publications

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“…To expand the applications to larger and more biologically complex systems demands improvement in both experimental sensitivity and resolution. For instance, G-protein coupled receptors, which are carriers of important cellular signal transduction events and prime drug targets, often have 30–70 kDa molecular weights (Filmore 2004; Blois and Bowie 2009). Furthermore, mammalian membrane proteins are notoriously difficult to express in milligram quantities when minimal growth media are used for isotope enrichment.…”

Section: Introductionmentioning

confidence: 99%

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

et al. 2012

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Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.

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Section: Introductionmentioning

confidence: 99%

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

et al. 2012

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“…32 The topological comparison of these crystal structures and their implications for GPCR activation has been reviewed extensively 33-37 . All GPCR conformations observed so far have corresponded to the inactive state of the respective receptor, except for bovine opsin 28 and nanobody stabilized human β2AR bound to an agonist 16 .…”

Section: Introductionmentioning

confidence: 99%

Bihelix: Towards de novo structure prediction of an ensemble of G‐protein coupled receptor conformations

2011

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G-Protein Coupled Receptors (GPCRs) play a critical role in cellular signal transduction pathways and are prominent therapeutic targets. Recently there has been major progress in obtaining experimental structures for a few GPCRs. Each GPCR, however, exhibits multiple conformations that play a role in their function and we have been developing methods aimed at predicting structures for all these conformations. Analysis of available structures shows that these conformations differ in relative helix tilts and rotations. The essential issue is, determining how to orient each of the 7 helices about its axis since this determines how it interacts with the other 6 helices. Considering all possible helix rotations to ensure that no important packings are overlooked, and using rotation angle increments of 30° about the helical axis would still lead to 127 or 35 million possible conformations each with optimal residue positions. We show in this paper how to accomplish this. The fundamental idea is to optimize the interactions between each pair of contacting helices while ignoring the other 5 and then to estimate the energies of all 35 million combinations using these pair-wise interactions. This BiHelix approach dramatically reduces the effort to examine the complete set of conformations and correctly identifies the crystal packing for the experimental structures plus other near-native packings we believe may play an important role in activation. This approach also enables a detailed structural analysis of functionally distinct conformations using helix-helix interaction energy landscapes and should be useful for other helical transmembrane proteins as well.

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“…The observance of functionally relevant differences in TM and loop conformation demonstrates the need for further structural characterization, especially for non‐Class A receptors. A number of elegant reviews have appeared during the last ten years, which analyze, compare, and infer changes in conformation based on the structures published to date . Therefore this review will not cover the crystallographic analyses of intact GPCRs.…”

Section: Introductionmentioning

confidence: 99%

Invited review GPCR structural characterization: Using fragments as building blocks to determine a complete structure

et al. 2014

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The structural characterization of G protein-coupled receptors has surged since the development of methodologies to facilitate the crystallization of these highly helical, seven transmembrane, integral membrane receptors. In the past seven years, eighteen GPCR structures were determined by X-ray crystallography. The crystal structures represent a static picture of these conformationally flexible signal transducers. Analyses that probe their dynamics and conformational changes require other techniques, in particular solution state nuclear magnetic resonance studies. Such investigations are challenged by the size of GPCRs, their α-helical structure, which limits resonance dispersion, their tendencies to aggregate in micellar preparations and their conformational heterogeneity. For many years, groups have been studying GPCR fragments as a means to overcome some of these difficulties. The results of these fragment analyses are presented here. Review of the literature reveals that much of the original work depended on circular dichroism, infra-red spectroscopy and fluorescence approaches. High resolution structures obtained by NMR are compared, where applicable, to the available crystal structures. In most cases, the work done on fragments by biophysical analysis is validated by these comparisons. Our perspective on the field of GPCR fragment analysis is presented together with the future goals that must be considered if work with fragments is continued.

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G‐protein‐coupled receptor structures were not built in a day

Cited by 28 publications

References 69 publications

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

Bihelix: Towards de novo structure prediction of an ensemble of G‐protein coupled receptor conformations

Invited review GPCR structural characterization: Using fragments as building blocks to determine a complete structure

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