Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels

Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.This work was supported by the German-Israeli Foundation for Scientific Research and Development to E.B. (GIF, Grant Number I-1202-248.9/2012), the Clore Center of Biological Physics at the Weizmann Institute of Science to D.G. and E.B., by a grant from the United States - Israel Binational Science Foundation (BSF), Jerusalem, Israel to H.S.M. and E.B., and by a grant from the Minerva Foundation to D.G. E.H.Y is the recipient of a Professor Rahamimoff Travel Grant from the United States - Israel Binational Science Foundation. T.B. acknowledges financial support from the Minerva Foundation. The authors would like to thank Dr. Akiva Feintuch for his support with Gd(III) AWG-DEER measurements

rDEER: A Modified DEER Sequence for Distance Measurements Using Shaped Pulses

The DEER (double electron-electron resonance, also called PELDOR) experiment, which probes the dipolar interaction between two spins and thus reveals distance information, is an important tool for structural studies. In recent years, shaped pump pulses have become a valuable addition to the DEER experiment. Shaped pulses offer an increased excitation bandwidth and the possibility to precisely adjust pulse parameters, which is beneficial especially for demanding biological samples. We have noticed that on our home built W-band spectrometer, the dead-time free 4-pulse DEER sequence with chirped pump pulses suffers from distortions at the end of the DEER trace. Although minor, these are crucial for Gd(III)-Gd(III) DEER where the modulation depth is on the order of a few percent. Here we present a modified DEER sequence—referred to as reversed DEER (rDEER)—that circumvents the coherence pathway which gives rise to the distortion. We compare the rDEER (with two chirped pump pulses) performance values to regular 4-pulse DEER with one monochromatic as well as two chirped pulses and investigate the source of the distortion. We demonstrate the applicability and effectivity of rDEER on three systems, ubiquitin labeled with Gd(III)-DOTA-maleimide (DOTA, 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid) or with Gd(III)-DO3A (DO3A, 1,4,7,10-Tetraazacyclododecane-1,4,7-triyl) triacetic acid) and the multidrug transporter MdfA, labeled with a Gd(III)-C2 tag, and report an increase in the signal-to-noise ratio in the range of 3 to 7 when comparing the rDEER with two chirped pump pulses to standard 4-pulse DEER

Improved sensitivity for W-band Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements with shaped pulses

Bahrenberg T, Rosenski Y, Carmieli R, et al. Improved sensitivity for W-band Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements with shaped pulses. Journal of Magnetic Resonance. 2017;283:1-13

Gd(III) and Mn(II) complexes for dynamic nuclear polarization : small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins

We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology

Small Gd(III) Tags for Gd(III)-Gd(III) Distance Measurements in Proteins by EPR Spectroscopy

The C7-Gd and C8-Gd tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the C7-Gd (S configuration) and C8-Gd (R configuration) tags loaded with Gd(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band 1H-ENDOR (electron–electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron–electron resonance) measurements were insensitive to the R or S configuration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, the C7-Gd and C8-Gd tags are attractive Gd(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy.D.G. acknowledges the support of the Israel Science Foundation (ISF grant No. 334/ 14). J.C. was supported by NSF grant MCB 1617025. This research was made possible in part by the historic generosity of the Harold Perlman Family (D.G.). D.G. holds the Erich Klieger Professorial Chair in Chemical Physics. T.B. acknowledges financial support from the Minerva Foundation. G.O. thanks the Australian Research Council (ARC) for a Laureate Fellowship, B.G. thanks the ARC for a Future Fellowship, and G.O. and B.G. thank the ARC for financial support

Gd(iii) and Mn(ii) Complexes for Dynamic Nuclear Polarization: Small Molecular Chelate Polarizing Agents and Applications with Site-Directed Spin Labeling of Proteins

We investigate complexes of two paramagnetic metal ions Gd³⁺ and Mn²⁺ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of ¹H, ¹³C, and ¹⁵N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the m[subscript S]= -1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as ¹³C and even ¹⁵N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of > 100 (for ¹H) and on the order of 1000 (for ¹³C) can be achieved in the saturation limit even at 80 K. At larger Gd(iii) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd³⁺ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between ¹³C and ¹⁵N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd³⁺ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology.National Institutes of Health (U.S.) (Grant EB-002026)National Institutes of Health (U.S.) (Grant EB-00280

Small Gd(III) Tags for Gd(III)–Gd(III) Distance Measurements in Proteins by EPR Spectroscopy

The <b>C7-Gd</b> and <b>C8-Gd</b> tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the <b>C7-Gd</b> (<i>S</i> configuration) and <b>C8-Gd</b> (<i>R</i> configuration) tags loaded with Gd­(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd­(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band ¹H-ENDOR (electron–electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron–electron resonance) measurements were insensitive to the <i>R</i> or <i>S</i> configuration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd­(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, the <b>C7-Gd</b> and <b>C8-Gd</b> tags are attractive Gd­(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy