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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 (95 GHz) 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 ∼ 1150 MHz) 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 ∼ 550 MHz) as a 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 sample’s 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. [ABSTRACT FROM AUTHOR]

Double-arm cyclen-based Gd3+ tags are shown to produce accurate nanometer scale Gd3+-Gd3+ distance measurements in double electron-electron resonance (DEER) experiments by confining the space accessible to the metal ion. The results show excellent agreement with predictions both for the maximum and width of the measured distance distributions. For distance measurements in proteins, the tags can be attached to two cysteine residues located in positions i and i+4, or i and i+8, of an α-helix. In the latter case, an additional mutation introducing an aspartic acid at position i+4 achieves particularly narrow distribution widths. The concept is demonstrated with cysteine mutants of T4 lysozyme and maltose binding protein. We report the narrowest Gd3+-Gd3+ distance distributions observed to date for a protein. By limiting the contribution of tag mobility to the distances measured, double-arm Gd3+ tags open new opportunities to study the conformational landscape of proteins in solution with high sensitivity. [ABSTRACT FROM AUTHOR]

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron-electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed GdIII spin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-GdIII is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5-10 μ m. [ABSTRACT FROM AUTHOR]

High‐frequency double electron‐electron resonance (DEER) distance measurements using different Gd3+ tags (Gd‐DOTA and Gd‐C1) were carried out on transmembrane helical peptides (ca. 0.15 nmol; WALP peptides) in a model membrane. The ability to pick up small distance variations, the chemical flexibility of the tags, and the remarkable absolute sensitivity, make this approach attractive for studies of membrane proteins. [ABSTRACT FROM AUTHOR]

Abstandsmessungen an helicalen Transmembranpeptiden (ca. 0.15 nmol; WALP ‐ Peptide) in einer Modellmembran wurden mit der Doppel ‐ Elektron ‐ Elektron ‐ Resonanz (DEER) ‐ Technik bei hoher Frequenz unter Verwendung von Gd3+ ‐ Markern (Gd ‐ DOTA und Gd ‐ C1) durchgeführt. Der Ansatz ist attraktiv für Membranproteinstudien, da kleine Abstandsänderungen detektiert werden können und die Marker chemisch flexibel sind. [ABSTRACT FROM AUTHOR]

Double electron-electron resonance (DEER) distance measurements of a protein complex tagged with two Gd3+ chelates developed for rigid positioning of the metal ion are shown to deliver outstandingly accurate distance measurements in the 6 nm range. The accuracy was assessed by comparison with modeled distance distributions based on the three-dimensional molecular structures of the protein and the tag and further comparison with paramagnetic NMR data. The close agreement between the predicted and experimentally measured distances opens new possibilities for investigating the structure of biomolecular assemblies. As an example, we show that the dimer interface of rat ERp29 in solution is the same as that determined previously for human ERp29 in the single crystal. [ABSTRACT FROM AUTHOR]

The article examines the complexes of the gadolinium as a potential spin labels for high field (HF) pulsed electron paramagnetic resonance (EPR) distance measurements. According to the authors, the study showed that gadolinium complexes have a potential of being used as spin labels for HF in biological systems. They feature high sensitivity and affording high effective values which allowed for very high repetition rates due to short spin called lattice relaxation times.