Mian Qi

Gd(III)-PyMTA Label Is Suitable for In-Cell EPR

Distance measurement in the nanometer range by electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling is a very powerful tool to monitor the structure and dynamics of biomacromolecules in their natural environment. However, in-cell application is hampered by the short lifetime of the commonly used nitroxide spin labels in the reducing milieu inside a cell. Here, we demonstrate that the Gd(III) based spin label Gd-PyMTA is suitable for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was proven to be inert in in-cell extracts of Xenopus laevis oocytes at 18 °C for more than 24 h. The proline rich peptide H-AP10CP10CP10-NH2 was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2, as well as the model compound Gd-spacer-Gd, which consists of a spacer of well-known stiffness, were microinjected into Xenopus laevis oocytes, and the Gd(III)-Gd(III) distances were determined by double electron-electron resonance (DEER) spectroscopy. To analyze the intracellular peptide conformation, a rotamer library was set up to take the conformational flexibility of the tether between the Gd(III) ion and the Cα of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2 is inserted into cell membranes, coinciding with a conformational change of the oligoproline from a PPII into a PPI helix.

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

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.

RIDME Spectroscopy with Gd(III) Centers

The relaxation induced dipolar modulation enhancement (RIDME) technique is applied at W-band microwave frequencies around 94 GHz to a pair of Gd(III) complexes that are connected by a rodlike spacer, and the extraction of the interspin distance distribution is discussed. A dipolar pattern derived from RIDME experimental data is a superposition of Pake-like dipolar patterns corresponding to the fundamental dipolar interaction and higher harmonics thereof. Intriguingly, the relative weights of the stretched patterns do not depend significantly on mixing time. As much larger modulation depths can be achieved than in double electron-electron resonance distance measurements at the same frequency, Gd(III)-Gd(III) RIDME may become attractive for structural characterization of biomacromolecules and biomolecular complexes.

Spacers for Geometrically Well-Defined Water-Soluble Molecular Rulers and Their Application

The synthesis and application of monodisperse oligo(para-phenyleneethynylene)s (oligoPPEs) with side chains that are adjustable to specific needs, such as water solubility, on a very late stage of the multistep synthesis are described. The adjustable side chains allow for circumventing problems associated with the isolation of highly hydrophilic compounds during early stages of a synthesis. Furthermore, these oligoPPEs can be stocked as adaptable advanced building blocks for rapid assembly of tailor-made spacers and rulers. A rapid growth synthesis provides oligoPPEs with alkyne termini protected with the orthogonal groups trimethylsilyl and 1-hydroxy-1-methylethyl (HOP) and with propargyloxy side chains protected with the triisopropylsilyl group. The three protecting groups allow independent modification of the two termini and the side chains. The HOP group not only acts as a protecting group but also as a polar tag for simple product isolation. We demonstrate one application of these oligoPPEs as spacers for the water-soluble Gd rulers of the type Gd-PyMTA-spacer-Gd-PyMTA with Gd-Gd distances of 2.1-10.9 nm. For this purpose, the terminal alkyne units were used for backbone assembly and attachment of the ligand PyMTA, and the porpargyloxy side chains were used to attach water solubilizing poly(ethylene glycol) chains through a click reaction after spacer assembly.

EPR characterization of Mn(II) complexes for distance determination with pulsed dipolar spectroscopy

The four Mn(ii) complexes Mn-DOTA, Mn-TAHA, Mn-PyMTA, and Mn-NO3Py were characterized by electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and relaxation measurements, to predict their relative performance in the EPR pulse dipolar spectroscopy (PDS) experiments. High spin density localization on the metal ions was proven by ENDOR on 1H, D, 14N, and 55Mn nuclei. The transverse relaxation of the Mn(ii) complexes appears to be slow enough for PDS-based spin-spin distance determination. Rather advantageous ratios of T1/Tm were measured allowing for good relaxation induced dipolar modulation enhancement (RIDME) performance and, in general, fast shot repetitions in any PDS experiment. Relaxation properties of the Mn(ii) complexes correlate with the strengths of their zero field splitting (ZFS). Further, a comparison of Mn(ii)-DOTA and Gd(iii)-DOTA based spin labels is presented. The RIDME technique to measure nanometer-range Mn(ii)-Mn(ii) distances in biomolecules is discussed as an alternative to the well-known DEER technique that often appears challenging in cases of metal-metal distance measurements. The use of a modified kernel function that includes dipolar harmonic overtones allows model-free computation of the Mn(ii)-Mn(ii) distance distributions. Mn(ii)-Mn(ii) distances are computed from RIDME data of Mn-rulers consisting of two Mn-PyMTA complexes connected by a rodlike spacer of defined length. Level crossing effects seem to have only a weak influence on the distance distributions computed from this set of Mn(ii)-Mn(ii) RIDME data.

Averaging of nuclear modulation artefacts in RIDME experiments

The presence of artefacts due to Electron Spin Echo Envelope Modulation (ESEEM) complicates the analysis of dipolar evolution data in Relaxation Induced Dipolar Modulation Enhancement (RIDME) experiments. Here we demonstrate that averaging over the two delay times in the refocused RIDME experiment allows for nearly quantitative removal of the ESEEM artefacts, resulting in potentially much better performance than the so far used methods. The analytical equations are presented and analyzed for the case of electron and nuclear spins S=1/2,I=1/2. The presented analysis is also relevant for Double Electron Electron Resonance (DEER) and Chirp-Induced Dipolar Modulation Enhancement (CIDME) techniques. The applicability of the ESEEM averaging approach is demonstrated on a Gd(III)-Gd(III) rigid ruler compound in deuterated frozen solution at Q band (35GHz).

Gd(III) complexes as paramagnetic tags: Evaluation of the spin delocalization over the nuclei of the ligand

Complexes of the Gd(III) ion are currently being established as spin labels for distance determination in biomolecules by pulse dipolar spectroscopy. Because Gd(III) is an f ion, one expects electron spin density to be localized on the Gd(III) ion - an important feature for the mentioned application. Most of the complex ligands have nitrogens as Gd(III) coordinating atoms. Therefore, measurement of the (14)N hyperfine coupling gives access to information on the localization of the electron spin on the Gd(III) ion. We carried out W-band, 1D and 2D (14)N and (1)H ENDOR measurements on the Gd(III) complexes Gd-DOTA, Gd-538, Gd-595, and Gd-PyMTA that serve as spin labels for Gd-Gd distance measurements. The obtained (14)N spectra are particularly well resolved, revealing both the hyperfine and nuclear quadrupole splittings, which were assigned using 2D Mims ENDOR experiments. Additionally, the spectral contributions of the two different types of nitrogen atoms of Gd-PyMTA, the aliphatic N atom and the pyridine N atom, were distinguishable. The (14)N hyperfine interaction was found to have a very small isotropic hyperfine component of -0.25 to -0.37MHz. Furthermore, the anisotropic hyperfine interactions with the (14)N nuclei and with the non-exchangeable protons of the ligands are well described by the point-dipole approximation using distances derived from the crystal structures. We therefore conclude that the spin density is fully localized on the Gd(III) ion and that the spin density distribution over the nuclei of the ligands is rightfully ignored when analyzing distance measurements.

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

Chirp and shaped pulses have been recently shown to be highly advantageous for improving sensitivity in DEER (double electron-electron resonance, also called PELDOR) measurements due to their large excitation bandwidth. The implementation of such pulses for pulse EPR has become feasible due to the availability of arbitrary waveform generators (AWG) with high sampling rates to support pulse shaping for pulses with tens of nanoseconds duration. Here we present a setup for obtaining chirp pulses on our home-built W-band (95GHz) spectrometer and demonstrate its performance on Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements. We carried out an extensive optimization procedure on two model systems, Gd(III)-PyMTA-spacer-Gd(III)-PyMTA (Gd-PyMTA ruler; zero-field splitting parameter (ZFS) D∼1150MHz) as well as nitroxide-spacer-nitroxide (nitroxide ruler) to evaluate the applicability of shaped pulses to Gd(III) complexes and nitroxides, which are two important classes of spin labels used in modern DEER/EPR experiments. We applied our findings to ubiquitin, doubly labeled with Gd-DOTA-monoamide (D∼550MHz) asa model for a system with a small ZFS. Our experiments were focused on the questions (i) what are the best conditions for positioning of the detection frequency, (ii) which pump pulse parameters (bandwidth, positioning in the spectrum, length) yield the best signal-to-noise ratio (SNR) improvements when compared to classical DEER, and (iii) how do the samples spectral parameters influence the experiment. For the nitroxide ruler, we report an improvement of up to 1.9 in total SNR, while for the Gd-PyMTA ruler the improvement was 3.1-3.4 and for Gd-DOTA-monoamide labeled ubiquitin it was a factor of 1.8. Whereas for the Gd-PyMTA ruler the two setups pump on maximum and observe on maximum gave about the same improvement, for Gd-DOTA-monoamide a significant difference was found. In general the choice of the best set of parameters depends on the D parameter of the Gd(III) complex.

Gd3+-Gd3+ distances exceeding 3 nm determined by very high frequency continuous wave electron paramagnetic resonance

Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling is a very powerful tool for elucidating the structure and organization of biomolecules. Gd3+ complexes have recently emerged as a new class of spin labels for distance determination by pulsed EPR spectroscopy at Q- and W-band. We present CW EPR measurements at 240 GHz (8.6 Tesla) on a series of Gd-rulers of the type Gd-PyMTA-spacer-Gd-PyMTA, with Gd-Gd distances ranging from 1.2 nm to 4.3 nm. CW EPR measurements of these Gd-rulers show that significant dipolar broadening of the central |-1/2〉 → |1/2〉 transition occurs at 30 K for Gd-Gd distances up to ∼3.4 nm with Gd-PyMTA as the spin label. This represents a significant extension for distances accessible by CW EPR, as nitroxide-based spin labels at X-band frequencies can typically only access distances up to ∼2 nm. We show that this broadening persists at biologically relevant temperatures above 200 K, and that this method is further extendable up to room temperature by immobilizing the sample in glassy trehalose. We show that the peak-to-peak broadening of the central transition follows the expected 1/r3 dependence for the electron-electron dipolar interaction, from cryogenic temperatures up to room temperature. A simple procedure for simulating the dependence of the lineshape on interspin distance is presented, in which the broadening of the central transition is modeled as an S = 1/2 spin whose CW EPR lineshape is broadened through electron-electron dipolar interactions with a neighboring S = 7/2 spin.

Bis-Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization

High-spin complexes act as polarizing agents (PAs) for dynamic nuclear polarization (DNP) in solid-state NMR spectroscopy and feature promising aspects towards biomolecular DNP. We present a study on bis(Gd-chelate)s which enable cross effect (CE) DNP owing to spatial confinement of two dipolar-coupled electron spins. Their well-defined Gd⋅⋅⋅Gd distances in the range of 1.2-3.4 nm allowed us to elucidate the Gd⋅⋅⋅Gd distance dependence of the DNP mechanism and NMR signal enhancement. We found that Gd⋅⋅⋅Gd distances above 2.1 nm result in solid effect DNP while distances between 1.2 and 2.1 nm enable CE for 1 H, 13 C, and 15 N nuclear spins. We compare 263 GHz electron paramagnetic resonance (EPR) spectra with the obtained DNP field profiles and discuss possible CE matching conditions within the high-spin system and the influence of dipolar broadening of the EPR signal. Our findings foster the understanding of the CE mechanism and the design of high-spin PAs for specific applications of DNP.