Orthogonal Spin Labeling and Gd(III)–Nitroxide Distance Measurements on Bacteriophage T4-Lysozyme

Garbuio, Luca; Bordignon, Enrica; Brooks, Evan K.; Hubbell, Wayne L.; Jeschke, Gunnar; Yulikov, Maxim

doi:10.1021/jp401806g

Cited by 97 publications

(101 citation statements)

References 41 publications

(88 reference statements)

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“…In this regime, the pump pulse cannot be considered as an adiabatic pulse and therefore does not provide a perfect population inversion. Note that for distance measurements between a Gd(III) center and a nitroxide, a related result was obtained for the optimum flip angle of a monochromatic pump pulse [15]. The found correlations may be used to set up DEER experiments with close to optimum sensitivity.…”

Section: Deer With a Single Chirp Pump Pulsementioning

confidence: 65%

“…According to the procedure reported in [15], a stock solution of Gd(III)-DOTA complex (DOTA = 1,4,7,10-…”

Section: Gd(iii)-dota Complexmentioning

confidence: 99%

“…5c, the reduction of the echo intensity V 0 /V ref is more pronounced for Gd-DOTA (D < 1 GHz) than for Gd-PyMTA (mostly D > 1 GHz). In the context of distance measurements between Gd(III) centers and nitroxides with monochromatic pulses, comparison of V 0 /V ref between Gd-DOTA and Gd-DTPA (D > 1 GHz) also revealed a stronger relative reduction effect for Gd-DOTA than for Gd-DTPA [15]. Since the echo V ref in absence of the pump pulse itself depends strongly on D, the absolute echo intensity V 0 in the presence of the pump pulse is required to calculate the DEER sensitivity η = V 0 · λ.…”

Section: Dependence Of Echo Intensity On Zfs Splittingmentioning

confidence: 99%

“…Modifications of this approach involve the use of non-identical spin labels, in particular a Gd(III) center and a nitroxide [3,4], as well as different experimental schemes, such as relaxation-induced dipolar modulation (RIDME) [5] or continuous-wave EPR [6]. Distance measurements involving Gd spin labels have been successfully demonstrated on a number of systems, including model compounds, peptides, nanoparticles, proteins and DNAs [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Moreover, a promising biochemical property of Gd spin labels is the stability under reducing conditions, which recently enabled Gd-Gd distance measurements of peptides and proteins embedded in cellular environments [22,23].…”

Section: Introductionmentioning

confidence: 99%

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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: Deer With a Single Chirp Pump Pulsementioning

confidence: 65%

“…According to the procedure reported in [15], a stock solution of Gd(III)-DOTA complex (DOTA = 1,4,7,10-…”

Section: Gd(iii)-dota Complexmentioning

confidence: 99%

Section: Dependence Of Echo Intensity On Zfs Splittingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…-Gd 3? PELDOR distance measurements, simultaneous two-site attachment is required and has been established using chemical tags (Raitsimring et al 2007;Potapov et al 2010;Gordon-Grossman et al 2011;Lueders et al 2011;Song et al 2011;Yagi et al 2011;Garbuio et al 2013;Matalon et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

et al. 2015

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We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1b (IL1b) and measured 1 H, 15 Npseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Dv-tensors associated with each Tm 3? -loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1b-S2R2 and the respective single-loop-LBT constructs IL1b-S2, IL1b-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd 3? as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.

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From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

et al. 2020

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ATP-binding cassette (ABC) exporters have been studied now for more than four decades, and recent structural investigation has produced a large number of protein database entries. Yet, important questions about how ABC exporters function at the molecular level remain debated, such as which are the molecular recognition hotspots and the allosteric couplings dynamically regulating the communication between the catalytic cycle and the export of substrates. This conundrum mainly arises from technical limitations confining all research to in vitro analysis of ABC transporters in detergent solutions or embedded in membrane-mimicking environments. Therefore, a largely unanswered question is how ABC exporters operate in situ, namely in the native membrane context of a metabolically active cell. This review focuses on novel mechanistic insights into type I ABC exporters gained through a unique combination of structure determination, biochemical characterization, generation of conformation-specific nanobodies/sybodies and double electron-electron resonance.

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Orthogonal Spin Labeling and Gd(III)–Nitroxide Distance Measurements on Bacteriophage T4-Lysozyme

Cited by 97 publications

References 41 publications

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

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

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

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