The use of the Rx spin label in orientation measurement on proteins, by EPR

Stevens, Michael A.; McKay, Johannes E.; Robinson, J. L. S.; Mkami, Hassane El; Smith, Graham; Norman, D.

doi:10.1039/c5cp04753f

Cited by 18 publications

(22 citation statements)

References 39 publications

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“…These experiments were based on the docking protocol ( 18 ) used to solve the structure of the Vps75 tetramer from distance restraints ( 18 ). Briefly, R1 spin label ensembles were simulated in MTSSLwizard ( 35 ) and Rx2 spin labels in XPLOR-NIH ( 36 ). Model distance measurements between spin label ensembles and cross-linking restraints between lysine side chains (Nζ–Nζ) were input as NOE distance restraints.…”

Section: Methodsmentioning

confidence: 99%

The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3–H4 tetramers and Asf1–H3–H4 complexes

et al. 2016

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Vps75 is a histone chaperone that has been historically characterized as homodimer by X-ray crystallography. In this study, we present a crystal structure containing two related tetrameric forms of Vps75 within the crystal lattice. We show Vps75 associates with histones in multiple oligomers. In the presence of equimolar H3–H4 and Vps75, the major species is a reconfigured Vps75 tetramer bound to a histone H3–H4 tetramer. However, in the presence of excess histones, a Vps75 dimer bound to a histone H3–H4 tetramer predominates. We show the Vps75–H3–H4 interaction is compatible with the histone chaperone Asf1 and deduce a structural model of the Vps75–Asf1-H3–H4 (VAH) co-chaperone complex using the Pulsed Electron-electron Double Resonance (PELDOR) technique and cross-linking MS/MS distance restraints. The model provides a molecular basis for the involvement of both Vps75 and Asf1 in Rtt109 catalysed histone H3 K9 acetylation. In the absence of Asf1 this model can be used to generate a complex consisting of a reconfigured Vps75 tetramer bound to a H3–H4 tetramer. This provides a structural explanation for many of the complexes detected biochemically and illustrates the ability of Vps75 to interact with dimeric or tetrameric H3–H4 using the same interaction surface.

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Section: Methodsmentioning

confidence: 99%

The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3–H4 tetramers and Asf1–H3–H4 complexes

et al. 2016

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“…At present, such studies are performed predominantly using the four-pulse DEER experiment at W-band frequencies recorded for a set of pump and observation positions [49][50][51][52] . Multiple DEER experiments are therefore required, which results in a longer acquisition time than for 2D SIFTER.…”

Section: Epr-correlated Siftermentioning

confidence: 99%

EPR-correlated dipolar spectroscopy by Q-band chirp SIFTER

Doll

Jeschke

2016

Phys. Chem. Chem. Phys.

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While two-dimensional correlation spectra contain more information as compared to one-dimensional spectra, typical spectral widths encountered in electron paramagnetic resonance (EPR) spectroscopy largely restrict the applicability of correlation techniques. In essence, the monochromatic excitation pulses established in pulsed EPR often cannot uniformly excite the entire spectrum. Here, this restriction is alleviated for nitroxide spin labels at Q-band microwave frequencies around 35 GHz. This is achieved by substitution of monochromatic pulses by frequency-swept chirp pulses tailored for uniform excitation. Unwanted interference effects brought by this substitution are analyzed for a pair of electron spins with secular dipolar coupling. Experimentally, the dipole-dipole interaction can be separated from other interactions by a constant-time Zeeman-compensated solid echo sequence called SIFTER. Such SIFTER experiments usually yield a one-dimensional dipolar spectrum. EPR-correlated dipolar spectra can be obtained when the four pulses are replaced by chirp pulses. These two-dimensional spectra encode additional information on the geometrical arrangement of the two spin labels. With the excitation parameters achieved by a home-built Q-band spectrometer capable of frequency-swept excitation, unwanted interference effects can be largely neglected for the examined model compound with a spin-spin distance of 4 nm. The experimentally obtained correlation pattern conforms to the expectation based on the inter-spin geometry of the investigated rigid model compound.

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“…3). RX delivers welldefined distance distributions and is distinctive particularly in context of orientation studies (Norman et al 2015), but it requires two cysteine residues in close proximity to each other, which limits the range of its applications. In TOAC, the nitroxide group is incorporated in a sixmembered ring attached to the backbone α-carbon, which also allows for distance measurements with higher accuracy (Karim et al 2004).…”

Section: Investigations Of Tm Peptides With Topp Labels Topp: a Novelmentioning

confidence: 99%

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels

2021

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Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5–10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes. Graphical Abstract

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The use of the Rx spin label in orientation measurement on proteins, by EPR

Abstract: Alternative labeling sites using the ‘rigid’ Rx spin label on protein secondary structures are explored and high field orientation measurements are made.

Cited by 18 publications

References 39 publications

The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3–H4 tetramers and Asf1–H3–H4 complexes

The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3–H4 tetramers and Asf1–H3–H4 complexes

EPR-correlated dipolar spectroscopy by Q-band chirp SIFTER

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels

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