In this work, we use a custom interferometric electron spin resonance (ESR) spectrometer to obtain magnetic field-dependent phase-sensitive transient microwave conductivity (TRMC) measurements of device-sized areas of organic semiconducting films. This approach allows for contactless transient microwave conductivity measurements to monitor the optically induced impedance change in device analogous organic semiconductor thin films. The decrease in probed area size allows for more direct comparison to working devices as contrasted with the stacking of films in the standard microwave cavity technique where film to film variation may influence results. 

Dynamic Nuclear Polarization (DNP) has been a rapidly developing field in the past two decades. It has the potential to enhance Nuclear Magnetic Resonance (NMR) signals by orders of magnitude by relying on polarization transfer from electron spins to nuclear spins. The spectrometer combines a 400 GHz amplifier multiplication chain transmitter, a quasi-optical (QO) bridge with induction mode EPR detection, and a phase sensitive super heterodyne receiver. The spectrometer allows for the acquisition of frequency and field swept EPR spectra at 400 GHz. The unique feature of our spectrometer is the position of the QO bridge below the magnet with 400 GHz radiation transmitted through the specially designed windows at the bottom of the cryostat. This allows for a reduced heat load on the cryostat, ample space in the probe for the DNP RF circuitry, and a rapid sample exchange.

EPR distance measurements employed with DNA Cu(II) spin labeling elucidated structural transitions associated with protein-DNA assembly. We show that the deuteration of protein, cryoprotectant, and solvent increases the duration of the measurable signal by over six-fold to measure distances up to 9 nm. Herein we employed our optimized sample preparation for Cu(II) distance measurements to discern conformational changes of the pathogenic Cu(I) metallo-regulator protein Copper Efflux Regulator’s (CueR) specific DNA with respect to varying CueR and Cu(I) equivalents. Our results show that CueR readily bends DNA in the presence of Cu(I) to permit binding of RNA polymerase to initiate transcription even in the absence of Cu(I). As such, a structural perspective of how CueR terminates transcription via Cu(I)-free CueR replacing Cu(I)-bound CueR in the protein-DNA complex is observed.

IDPs are highly regulated proteins, which are subject to numerous post-translational modifications (PTMs) influencing the IDP energy landscapes in the cell. The cellular environment is characterized by molecular crowding and features a huge variety of interaction partners that may modulate the protein structural ensemble and processes of structural reorganization, as well as the oligomerization behavior, resulting fibril conformers and the interaction kinetics with partners like other proteins. Using RS EPR spectroscopy to study αS interactions with negatively charged vesicles in vitro and upon transfection of the protein and lipid vesicles into model cells, we show that protein−vesicle interactions are reflected in RS spectra in vitro and in cells, which enables time-resolved monitoring of protein−membrane interaction upon transfection into cells. Our data suggest binding of a small fraction of αS to endogenous membranes.

Dynamic nuclear polarization (DNP) is a powerful method to enhance NMR sensitivity. Much progress has been achieved recently to optimize DNP performance at high magnetic fields in solid-state samples, mostly by utilizing the solid or the cross effect. In liquids, only the Overhauser mechanism is active, which exhibits a DNP field profile matching the EPR line shape of the radical, distinguishable from other DNP mechanisms. Here, we observe DNP enhancements with a field profile indicative of the solid effect and thermal mixing at ∼320 K and a magnetic field of 9.4 T in the fluid phase of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers doped with the radical BDPA (1,3-bis(diphenylene)- 2-phenylallyl). This interesting observation might open up new perspectives for DNP applications in macromolecular systems at ambient temperatures.

High hydrostatic pressure is a powerful probe of protein conformational flexibility. Pressurization reveals regions of elevated compressibility, and thus flexibility, within individual conformational states, but also shifts conformational equilibria such that “invisible” excited states become accessible for spectroscopic characterization. In this talk, I will describe instrumentation and methods that enable high-pressure site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) experiments on proteins and demonstrate the information content of these experiments by highlighting specific recent applications. Although high-pressure SDSL-EPR is in its infancy, the recent applications presented highlight the considerable potential of the method to populate and characterize functional excited states of proteins undetected at atmospheric pressure.

TRAP transporters are a unique class of ATP-independent bacterial and archaeal membrane transporters that employ a high-affinity periplasmic substrate-binding protein to scavenge e.g. sialic acid and import it into the cytosol. The transport mechanism involves a large number of conformational transitions of the substrate binding protein that are coupled to the elevator mechanism of the transmembrane domains. We used Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) to investigate these transitions. Together with a cryoEM structure of the transporter, our results reveal detailed insights into the transport mechanism. In addition, our data provide an interesting comparison between distance measurements with both, PELDOR/DEER and smFRET spectroscopy, on the same library of double cysteine mutants of substrate-binding proteins.

Pseudo two-dimensional DEER experiments allow one to differentiate distance populations in inter-exchanging biomolecular systems. The reconstruction, however, of 2D distributions of distance populations is challenging since relating the results of successive traces requires additional information, which may not be readily available. To overcome this problem, we introduce the 2D Srivastava-Freed Singular Value Decomposition (2D SF-SVD) method that enables reconstruction of 2D distance distributions in a straightforward fashion, thereby permitting the accurate determination of measurable population changes for each distance. That is, the distance surface one obtains contains both the distance components transformed from the dimension of the dipolar signal and the dimension of traces, revealing changes in each distance population. 

A critical step in CRISPR associated protein DNA target selection is the unwinding of the DNA duplex protospacer segment to form an R-loop, in which the RNA guide hybridizes with one of the DNA strands. The mechanisms of Cas9 and Cas12a DNA target surveillance, discrimination, and interrogation are actively being explored. I will discuss how we leverage site-directed-spin-labeling in conjunction with EPR to investigate mechanisms of CRISPR-Cas mediated target DNA unwinding. Our research approaches utilize R5 nitroxide labeled DNA to probe the unwinding mechanisms of Cas9 and Cas12a. The impact of truncated RNA guides on protospacer adjacent motif-distal (PAM-distal) unwinding by Cas9 will be presented. After that, data showcasing the effect of Cas12a non-target strand trimming on bound DNA conformation will be explored.

A portable biosensor will improve the accessibility of many medical services. Here, we present a prototype of a quantum biosensor enabled by diamond NV centers and SOMAmers (Slow Off-rate Modified Aptamers). The binding interaction between the spin-labeled SOMAmer and its target molecule alters the microenvironment around the spin label, which will change the NV center readout via magnetic dipole-dipole interaction. We demonstrate the viability of this mechanism by solution-state bulk EPR measurements on a spin-labeled model SOMAmer and present a workflow of searching for the optimal labeling site. We hope to extend this workflow to more SOMAmers and continue to engineer the NV center sensing platform.

PDS acquisition schemes conventionally use uniform sampling of the dipolar trace, but non-uniform sampling (NUS) schemes can decrease the total measurement time or increase the accuracy of the resulting distance distributions. NUS requires optimization of the data acquisition scheme, as well as changes in data processing algorithms to accommodate the non-uniformly sampled data. We investigate in silico the applicability of the NUS approach in PDS, considering its effect on random, truncation and sampling noise in the experimental data. NUS schemes seem to be a valid approach for increasing sensitivity and/or throughput in PDS by decreasing and redistributing noise in the distance spectrum so that it has less impact on the distance spectrum.

The catalytic oxidation of para-xylene to terephthalic acid (pTA) is one of the most important industrial-scale processes to produce polyethylene terephthalate (PET), which finds wide applications. The homogeneous catalytic mixture of Co, Mn and Br salts in aqueous acetic acid media is challenging to study for most characterisation techniques due to the harsh operating conditions and vast array of potential organic radical and paramagnetic transition metal intermediates. Herein, we present a more comprehensive investigation into the underlying catalysis using spin trapping to identify radical intermediates, CW linewidth analysis as a probe of changing catalyst coordination sphere, and 3 pulse ESEEM as an empirical method of determining catalyst hydration.

Here, we have recently exploited PDS based modulation depth to demonstrate the surprisingly efficient and robust coordinative copper(II) based spin labelling of double histidine motifs in proteins offering highly precise distance measurements at nanomolar concentrations. Furthermore, parameters of more complex equilibria are accessible from PDS data. In an archaeal single-stranded DNA binding protein binding of short DNA competes with self-dimerisation but leaves the tetramerization to dimers of dimers unaffected. On the other hand, the cooperativity of the templated dimerisation of a model protein can be monitored as a function of conditions. This demonstrates the breadth of applications of PDS available beyond the already extremely valuable extraction of distances.