13C- and 1H-detection under fast MAS for the study of poorly available proteins: application to sub-milligram quantities of a 7 trans-membrane protein

Dannatt, Hugh R. W.; Taylor, Garrick F.; Varga, Krisztina; Higman, Victoria Ann; Pfeil, Marc Philipp; Asilmovska, Lubica; Judge, Peter J.; Watts, Anthony

doi:10.1007/s10858-015-9911-1

Cited by 16 publications

(7 citation statements)

References 46 publications

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“…1). The 15 N-1 H correlation and the observed 13 Cα and 13 Cβ chemical shifts agree well with previous NMR studies of purple membranes 30,31 . However, none of these assigned backbone resonances correspond to residues involved in proton pumping; we therefore applied the following strategy to study sites that contribute to proton displacements during BR molecular action.…”

Section: Resultssupporting

confidence: 90%

Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

et al. 2020

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Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. Here, we demonstrate that reversible proton displacement in the active site of bacteriorhodopsin already takes place in its equilibrated dark-state, providing new information on the underlying hydrogen exchange processes. In particular, MAS NMR reveals proton exchange at D85 and the retinal Schiff base, suggesting a tautomeric equilibrium and thus partial ionization of D85. We provide evidence for a proton cage and detect a preformed proton path between D85 and the proton shuttle R82. The protons at D96 and D85 exchange with water, in line with ab initio molecular dynamics simulations. We propose that retinal isomerization makes the observed proton exchange processes irreversible and delivers a proton towards the extracellular release site.

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Section: Resultssupporting

confidence: 90%

Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

et al. 2020

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“…However, dramatic improvements in sensitivity have been obtained by the implementation of 1 H-detected magic angle spinning (MAS) solid-state NMR methods (46,47), especially when fast MAS is combined with perdeuteration of the samples (46,(48)(49)(50)(51). This approach is being applied to an increasing number of membrane proteins (52)(53)(54)(55).…”

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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“…With the advent of fast magic angle spinning (≥ 40 kHz), proton-detection even on fully protonated proteins, first demonstrated by Rienstra and co-workers (Zhou et al, 2007a) has become feasible with improvements in resolution and sensitivity scaling with the MAS rate (Agarwal et al, 2014; Lewandowski et al, 2011a; Marchetti et al, 2012). Further, the sensitivity gains of 1 H detection enable the use of very small sample amounts (Agarwal et al, 2014; Dannatt et al, 2015). Recent works of note in the application of 1 H proton detection include studies of RNA-protein interfaces by Asami et al (Asami et al, 2013), structure determination of superoxide dismutase (SOD) by Knight et al (Knight et al, 2012), and measurements of heteronuclear dipolar couplings in Pf1 bacteriophage by Mueller and co-workers (Park et al, 2013).…”

Section: Current Methodology For Structural and Dynamics Analysis mentioning

confidence: 99%

Structural biology of supramolecular assemblies by magic-angle spinning NMR spectroscopy

Quinn

Polenova

2017

Quart. Rev. Biophys.

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In recent years, exciting developments in instrument technology and experimental methodology have advanced the field of magic angle spinning (MAS) NMR to new heights. Contemporary MAS NMR yields atomic-level insights into structure and dynamics of an astounding range of biological systems, many of which cannot be studied by other methods. With the advent of fast magic angle spinning, proton detection, and novel pulse sequences, large supramolecular assemblies, such as cytoskeletal proteins and intact viruses, are now accessible for detailed analysis. In this review, we will discuss the current MAS NMR methodologies that enable characterization of complex biomolecular systems and will present examples of applications to several classes of assemblies comprising bacterial and mammalian cytoskeleton as well as HIV-1 and bacteriophage viruses. The body of work reviewed herein is representative of the recent advancements in the field, with respect to the complexity of the systems studied, the quality of the data, and the significance to the biology.

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13C- and 1H-detection under fast MAS for the study of poorly available proteins: application to sub-milligram quantities of a 7 trans-membrane protein

Cited by 16 publications

References 46 publications

Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

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

Structural biology of supramolecular assemblies by magic-angle spinning NMR spectroscopy

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