High sensitivity and versatility of the DEER experiment on nitroxide radical pairs at Q-band frequencies

Polyhach, Yevhen; Bordignon, Enrica; Tschaggelar, René; Gandra, Sandhya; Godt, Adelheid; Jeschke, Gunnar

doi:10.1039/c2cp41520h

Cited by 184 publications

(200 citation statements)

References 41 publications

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“…All experiments were performed on a customized Bruker Elexsys E580 Q-band spectrometer [35] with a previously described extension by a 12 GS/s AWG (Agilent M8190A) [30]. The temperature was 10 K and the repetition rate of the pulse sequences was 342 µs in the case of Gd-ruler 1 3 and 716 µs for all the other Gd-rulers.…”

Section: Epr Experimentsmentioning

confidence: 99%

Gd(III)–Gd(III) distance measurements with chirp pump pulses

Doll

Wili

et al. 2015

Journal of Magnetic Resonance

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110

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The broad EPR spectrum of Gd(III) spin labels restricts the dipolar modulation depth in distance measurements between Gd(III) pairs to a few percent. To overcome this limitation, frequency-swept chirp pulses are utilized as pump pulses in the DEER experiment. Using a model system with 3.4 nm Gd-Gd distance, application of one single chirp pump pulse at Q-band frequencies leads to modulation depths beyond 10%. However, the larger modulation depth is counteracted by a reduction of the absolute echo intensity due to the pump pulse. As supported by spin dynamics simulations, this effect is primarily driven by signal loss to double-quantum coherence and specific to the Gd(III) high spin state of S = 7/2. In order to balance modulation depth and echo intensity for optimum sensitivity, a simple experimental procedure is proposed. An additional improvement by 25% in DEER sensitivity is achieved with two consecutive chirp pump pulses. These pulses pump the Gd(III) spectrum symmetrically around the observation position, therefore mutually compensating for dynamical Bloch-Siegert phase shifts at the observer spins. The improved sensitivity of the DEER data with modulation depths on the order of 20% is due to mitigation of the echo reduction effects by the consecutive pump pulses. In particular, the second pump pulse does not lead to additional signal loss if perfect inversion is assumed. Moreover, the compensation of the dynamical Bloch-Siegert phase prevents signal loss due to spatial dependence of the dynamical phase, which is caused by inhomogeneities in the driving field. The new methodology is combined with pre-polarization techniques to measure long distances up to 8.6 nm, where signal intensity and modulation depth become attenuated by long dipolar evolution windows. In addition, the influence of the zero-field splitting parameters on the echo intensity is studied with simulations. Herein, larger sensitivity is anticipated for Gd(III) complexes with zero-field splitting that is smaller than for the employed Gd-PyMTA complex.

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Section: Epr Experimentsmentioning

confidence: 99%

Gd(III)–Gd(III) distance measurements with chirp pump pulses

Doll

Wili

et al. 2015

Journal of Magnetic Resonance

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110

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“…For DEER measurements in detergent solution, double cys-mutants of TM287/288 were spin-labeled with MTSL [(1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate] and incubated in the presence or absence of 2.5 mM AMP-PNP and 2.5 mM MgCl 2 at 25°C prior to flash freezing in cold liquid pentane. DEER traces were recorded at Q band with all pulses set to 12 ns and frequency separation of 100 MHz (37). Data analysis and simulation of the MTSL rotamers on the X-ray structures were performed with the softwares DeerAnalysis2013 (38) and MMM2013.2 (19).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter

Höhl

Hürlimann

Böhm

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.membrane transport | X-ray crystallography | allosteric communication A BC exporters are found in every organism (1, 2). They minimally consist of four domains and exist as homodimers or heterodimers. Two transmembrane domains (TMDs) span the membrane with a total of 12 transmembrane helices and form the substrate permeation pathway by alternating between inward-and outward-oriented states (Fig. S1A). A pair of nucleotide binding domains (NBDs) is connected to the TMDs via coupling helices and drive conformational cycling of the transporter by binding and hydrolysis of ATP, a process which is linked to NBD dimerization and dissociation (3).In their closed state, the NBDs sandwich two ATP molecules at the dimer interface by composite ATP binding sites involving conserved sequence motifs contributed by both subunits (4, 5). The A-loop and Walker A motif of one NBD and the ABC signature motif of the opposite NBD are involved in nucleotide binding. The Walker B glutamate and the switch-loop histidine constitute a catalytic dyad required for ATP hydrolysis (6, 7). In heterodimeric ABC exporters with asymmetric ATP binding sites, these catalytic residues are noncanonical at the degenerate site and ATP is therefore primarily, if not exclusively, hydrolyzed at the consensus site (8). The Q-and D...

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“…X-band measurements were per-formed on a Bruker ElexSys 580 spectrometer equipped with a Bruker split-ring or dielectric resonator (ER 4118X -MS3 or ER 4118 MD5). Q-band measurements were performed on a home-built high power spectrometer [48,49] equipped with a custom resonator for samples with 3 mm outer diameter [50]. The S-band measurements were performed on a home built spectrometer [51].…”

Section: Epr Measurementsmentioning

confidence: 99%

Distance determination from dysprosium induced relaxation enhancement: a case study on membrane-inserted WALP23 polypeptides

et al. 2013

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Abstract. Membrane incorporated synthetic α-helical polypeptides labeled with Dy(III) chelate complexes and nitroxide radicals were studied by the inversion recovery (IR) technique and Dy(III)-nitroxide distances were obtained. A comparison of obtained distances with the previously reported Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) calibration data was performed and revealed reliability of the IR-based technique for the distance determination in membrane-incorporated biomacromolecules. The presented distance determination technique is 'spectroscopically orthogonal' to DEER-based distance measurements and can be potentially combined with DEER to study multiply spin-labeled biomacromolecules. The key steps of the data processing, the types of obtained distance information and the areas of possible application of the technique are discussed.

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High sensitivity and versatility of the DEER experiment on nitroxide radical pairs at Q-band frequencies

Cited by 184 publications

References 41 publications

Gd(III)–Gd(III) distance measurements with chirp pump pulses

Gd(III)–Gd(III) distance measurements with chirp pump pulses

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter

Distance determination from dysprosium induced relaxation enhancement: a case study on membrane-inserted WALP23 polypeptides

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