Gadolinium Tagging for High-Precision Measurements of 6 nm Distances in Protein Assemblies by EPR

Yagi, Hiromasa; Banerjee, Dipanwita; Graham, Bimbil; Huber, Thomas; Goldfarb, Daniella; Otting, Gottfried

doi:10.1021/ja204415w

Cited by 105 publications

(150 citation statements)

References 35 publications

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“…The deviation is up to 1.6% and presumably due to approximations in distance prediction and experimental uncertainty. In comparison to recent experiments at W-band [20], we notice apparent differences for the long Gdruler 1 11 . These are discussed in Section 6.3 of the SI.…”

Section: Application To Long Distancescontrasting

confidence: 79%

“…7a. The black curve represents single pulse data and is identical with the experimental data of Gd-ruler 1 11 in Fig. 5c.…”

Section: Consecutive Chirp Pulsesmentioning

confidence: 68%

“…The experimental data for real (black) and imaginary (gray) components are shown by the solid lines in Figs. 5a and b for Gd-ruler 1 11 that contains Gd-PyMTA and for a solution of Gd-DOTA complex, respectively. The simulated data points are illustrated by the circles in Figs.…”

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

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: Application To Long Distancescontrasting

confidence: 79%

“…7a. The black curve represents single pulse data and is identical with the experimental data of Gd-ruler 1 11 in Fig. 5c.…”

Section: Consecutive Chirp Pulsesmentioning

confidence: 68%

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

110

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“…16 Due to its large D value, the sensitivity of DEER experiments obtained with the Mn 2+ -EDTA tag is less than what has been obtained with the Gd 3+ -DOTA-amide tag. 16 This situation, however, may change completely if tags with low D values become available, in particular, in combination with a bimodal cavity 28 that allows placement of the pump and observe pulses on two neighboring narrow high-intensity hyperfine components (separated by ∼280 MHz).…”

Section: +mentioning

confidence: 92%

Nanometer-Range Distance Measurement in a Protein Using Mn²⁺ Tags

Banerjee

Yagi

Huber

et al. 2012

J. Phys. Chem. Lett.

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Pulse electron paramagnetic resonance measurements of long-range (nm scale) distances between spin labels site-specifically attached to biomacromolecules have proven highly effective in structural studies. The most commonly used spin labels are stable nitroxide radicals, and measurements are usually carried out at X-band frequencies (∼9.5 GHz, 0.35 T). Higher magnetic fields open new possibilities for distance measurements with increased sensitivity using alternative spin labels containing half-integer high-spin metal ions. Here we demonstrate W-band (95 GHz) pulse double electron−electron resonance (DEER) distance measurements in a protein labeled with two Mn 2+ -EDTA tags. The distance distribution obtained is in excellent agreement with model calculations based on the known solution NMR structure. Thus, sitespecific labeling with Mn 2+ tags opens a highly promising approach to nanometer distance measurements in biological macromolecules. SECTION: Biophysical Chemistry L ong-range (nm scale) distance measurements between specific sites in biological macromolecules offer important insight into their structure and interactions. In the past decade, such distance measurements by pulse electron paramagnetic resonance (EPR) techniques, often referred to as pulse dipolar spectroscopy (PDS), have proven highly efficient for nitroxidelabeled proteins and nucleic acids.1−3 Distances in the range of 2−5 nm can be routinely accessed, and distances up to 8 nm can be determined under favorable conditions. 4,5 In proteins, site-directed spin labeling is usually achieved by covalent attachment of nitroxide compounds to cysteine residues that are either native or, more commonly, have been introduced at strategically chosen positions by site-directed mutagenesis. where r is the electron−electron distance and θ is the angle between the interelectron vector r and the external magnetic field. The measurements are carried out at low temperatures on frozen solutions. The most popular PDS experiment is the four-pulse double electron−electron resonance (DEER) sequence, 7 often also referred to as PELDOR (pulse electron double resonance). DEER carried out at standard X-band frequencies (∼9.5 GHz, 0.35 T) has become well-established. An order of magnitude increase in sensitivity can be gained by high-frequency/highfield DEER, provided that the microwave (mw) power is sufficient to generate short enough microwave pulses, as recently demonstrated at the W-band (95 GHz, ∼3.5 T). 8,9 In addition, high-field measurements open new opportunities for the use of metal ions as spin labels. The EPR spectra of highspin transition-metal ions with half-filled valence orbitals, such as Gd 3+ (S = 7/2) and Mn 2+ (S = 5/2), become much simpler at high fields, displaying an intense and relatively narrow central |−1/2⟩ → |1/2⟩ transition. 10,11 The width of this transition is proportional to D 2 /ν 0 , where D is the zero-field splitting (ZFS) parameter and ν 0 is the spectrometer frequency, leading to increased sensitivity with increased freque...

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