Solid-state NMR methods for oriented membrane proteins

Hansen, Sara K.; Bertelsen, Kresten; Paaske, Berit; Nielsen, Niels Chr.; Vosegaard, Thomas

doi:10.1016/j.pnmrs.2015.05.001

Cited by 33 publications

(27 citation statements)

References 209 publications

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“…Although there have been several advancements in the field of OS-ssNMR, 22 there are still limitations for larger membrane proteins that present significant overlap of resonances in the SLF experiments. WE experiments under MAS conditions have been successfully used for studying G protein-coupled receptor, potassium channel, and M2 channel membrane proteins, 14 but the spectral simplification via the WE experiments presented in this article could be a turning point for new methodological developments to determine the 3D structure of larger, oriented membrane proteins.…”

Section: Discussionmentioning

confidence: 99%

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Probing Residue-Specific Water–Protein Interactions in Oriented Lipid Membranes via Solid-State NMR Spectroscopy

et al. 2016

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Water plays a central role in membrane protein folding and function. It not only catalyzes lipid membrane self-assembly but also affects the structural integrity and conformational dynamics of membrane proteins. Magic angle spinning (MAS) solid-state NMR (ssNMR) is the technique of choice for measuring water accessibility of membrane proteins, providing a measure for membrane protein topology and insertion within lipid bilayers. However, the sensitivity and resolution of membrane protein samples for MAS experiments are often dictated by hydration levels, which affect the structural dynamics of membrane proteins. Oriented-sample ssNMR (OS-ssNMR) is a complementary technique to determine both structure and topology of membrane proteins in liquid crystalline bilayers. Recent advancements in OS-ssNMR involve the use of oriented bicellar phases that have improved both sensitivity and resolution. Importantly, for bicelle formation and orientation, lipid bilayers must be well organized and hydrated, resulting in the protein's topology being similar to that found in native membranes. Under these conditions, the NMR resonances become relatively narrow, enabling a better separation of 1H–15N dipolar couplings and anisotropic 15N chemical shifts with separated local field (SLF) experiments. Here, we report a residue-specific water accessibility experiment for a small membrane protein, sarcolipin (SLN), embedded in oriented lipid bicelles as probed by new water-edited SLF (WE-SLF) experiments. We show that SLN's residues belonging to the juxtamembrane region are more exposed to the water–lipid interface than the corresponding membrane-embedded residues. The information that can be obtained from the WE-SLF experiments can be interpreted using a simple theoretical model based on spin-diffusion theory and offers a complete characterization of membrane proteins in realistic membrane bilayer systems.

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Section: Discussionmentioning

confidence: 99%

“…20–22 In fact, the formation of lipid phases, either mechanically or magnetically oriented, requires high hydration levels to reach liquid crystalline lipid phases. 20 These systems are suitable for characterizing water–protein interactions under native-like conditions.…”

Section: Introductionmentioning

confidence: 99%

Probing Residue-Specific Water–Protein Interactions in Oriented Lipid Membranes via Solid-State NMR Spectroscopy

et al. 2016

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“…Solid-state NMR spectroscopy enables studies of the structure and dynamics of membrane proteins in near-native phospholipid bilayer environments (37)(38)(39)(40). There are several examples where solid-state NMR methods have been used to characterize the structures of ligands bound to GPCRs (41)(42)(43)(44) as well as GPCRs themselves.…”

Section: Introductionmentioning

confidence: 99%

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

Park

Berkamp

Radoicic

et al. 2017

Biophysical Journal

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The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G proteincoupled receptor, located in the cell membrane of neutrophils. Here, we describe 1 H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn 2þ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

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“…Solid-state NMR is an emerging method to investigate the structure of membrane proteins in a native-like lipid environment (for recent contributions see for example (McDermott 2009;Renault et al 2010;Kaur et al 2015;Hansen et al 2015;Shahid et al 2015;Mehler et al 2015;Mandal et al 2015;Fu et al 2015)). Still, as for other techniques in structure biology, sample preparation of membrane proteins remains challenging, as it is often difficult to express the proteins in a well-folded form, with the isotopic labeling and in the milligram amounts needed for NMR spectroscopy.…”

Section: Introductionmentioning

confidence: 99%

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

et al. 2016

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Solid-state NMR methods for oriented membrane proteins

Cited by 33 publications

References 209 publications

Probing Residue-Specific Water–Protein Interactions in Oriented Lipid Membranes via Solid-State NMR Spectroscopy

Probing Residue-Specific Water–Protein Interactions in Oriented Lipid Membranes via Solid-State NMR Spectroscopy

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

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