Site Directed Spin Labelling (SDSL) reactions coupled to Electron Paramagnetic Resonance Spectroscopy (EPR) are powerful biophysical methods for the characterization of proteins. Indeed, the incorporation of paramagnetic tags (spin labels) onto these biological systems permits to explore their structural dynamics and their conformational changes at local level using both continuous wave and pulse EPR experiments. In the last decade, SDSL-EPR spectroscopy is emerged also as an optimal approach to probe these aspects directly inside cells (in-cell EPR). In this way, proteins can be investigated in their physiological environments without perturbing their native states. Here, I will present the application of nitroxide-based spin labels to perform structural investigations on cytosolic proteins in various cellular milieu through in-cell EPR spectroscopy. In parallel, I will show some optimal methodologies for protein internalization in both prokaryotic and eukaryotic cells in order to carry out in-cell EPR measurements with various nitroxide-labelled proteins. These results highlighted the potentialities of EPR experiments to provide information at structural level for proteins in real cellular conditions, enhancing at the same time the state of the art of this magnetic resonance technique in the field of structural biology.

Conformational substates of proteins play a key role in protein folding and molecular recognition that ultimately results in conformational exchange under equilibrium and nonequilibrium conditions. In the context of large interdomain rearrangements associated with ligand binding, the free energy landscape describing the transition from the apo form to the bound form, just as in protein folding, is likely to be rugged with numerous local minima along the pathway. Here, we provide simple tools to help you build complex fitting models for non-linear least-squares problems and apply these models to DEER data. This section gives an overview of the concepts and describes how to set up and perform simple fits. Some basic knowledge of Python and modeling data are assumed – this is a tutorial on why or how to perform a minimization or fit data, and is aimed at explaining how to use global analysis to answer biomolecular questions by DEER spectroscopy.

Electron paramagnetic resonance (EPR) is the method of choice to investigate and quantify paramagnetic species in many applications in materials science, biology, and chemistry. In these fields, typical sample states include thin films and solutions. Of particular interest are dynamic processes in solution. Their investigation, however, is limited by the form factor of the utilized microwave (MW) resonators as the entire process needs to be confined to the resonator. The EPR-on-a-Chip (EPRoC) dipstick device circumvents these limitations by integrating the entire EPR spectrometer into a single microchip, covered with a protective coating that enables the operation of the EPRoC submerged directly in the sample solution, thereby expanding the accessible sample environments for EPR measurements. In this approach, instead of a MW resonator, the coil of a voltage-controlled oscillator (VCO) with a size of a few hundred micrometers is simultaneously used as MW source and detector. As a test for the EPRoC dipstick and its protective coating, differently charged electrolyte solutions of a Vanadium redox flow battery with pH < 1 were investigated with the EPRoC and compared to conventional EPR results. The same linear relationship of EPRoC and EPR signal intensities with respect to the state of charge (SOC) was found, so that these experiments serve as proof-of-principle for a quantitative EPRoC dipstick device operating in a harsh sample environment. Dipstick EPRoC with its inherent fast rapid scan (RS) capability using frequency sweeps allows the investigation of processes, in which time resolution is important. It has been shown recently that the SNR per allocated acquisition time can significantly be enhanced, if measurement time for obtaining a full spectrum is the limiting factor, such as in oximetry. In combination with a permanent magnet, of which a first prototype will be presented, the EPRoC dipstick may find its way beyond the laboratory as a quantification tool for paramagnetic species in solution.

In this tutorial I will use some of our recent applications to discuss how we test structural hypotheses by site directed spin-labelling and pulse dipolar EPR spectroscopy (PDS). The tutorial is aimed at broader audience of EPR spectroscopists who are interested in spin-labelling and PDS as a methodology to test structural models, but do not (yet) have extensive experience with this. I will cover a general strategy including advantages and disadvantages of different spin labels for PDS and experimental aspects, both in the wet lab and at the spectrometer. Finally, I will share our take on data analysis and its robustness.

Maintaining a balance, or homeostasis, of Reactive Oxygen / Nitrogen Species (RONS) with antioxidants is crucial for keeping healthy cells in our body. When a high level of RONS is observed in our body, it triggers oxidative stress associated with multiple conditions, such as cancer and cardiovascular diseases. Although it is critical to know the RONS levels in vivo, there is no reliable way to measure and image RONS in a live animal today. For instance, fluorescent probes suffer from light absorption and scattering within the tissues. Hydroxylamines or nitrones can detect ROS using EPR; nevertheless, the nitroxide radicals formed upon reaction with ROS are unstable in vivo, which does not allow enough in vivo accumulation of the nitroxide radical to be detected by EPR. To overcome the issues, we demonstrate the synthesis and characterization of a phosphine derivative of triarylmethyl radicals (TAM-PPh2) spin probe for detecting RONS using EPR. My talk will focus on the progress in the TAM-PPh2 probe development. 

Pulsed dipole EPR spectroscopy is widely used to investigate structure and functions of biological molecules such as proteins and nucleic acids. In my talk I’ll review our recent papers concerning the application of spin tags based on triarylmethyl and stereo-substituted nitroxyl radicals to study the complexes of human ribosomes and RNAs modeling translation and to study the penetration of the unstructured RL2 proteins into lung cancer cells.

Are the structural and dynamic properties of proteins affected by their native cellular environment, suggesting the presence of a quinary structure? To explore the implication of potential quinary structure for globular proteins, we studied the dynamics and conformations of Escherichia coli (E. coli) peptidyl-prolyl cis/transisomerase B (PpiB) in E. coli cells. We applied electron paramagnetic resonance (EPR) techniques, utilizing both Gadolinium (Gd(III)) and nitroxide spin labels. In addition to using standard spin labeling approaches with genetically engineered cysteines, we incorporated an unnatural amino acid to achieve Gd(III)-nitroxide orthogonal labeling. We show that E. coli PpiB, a main cytosolic chaperone, experiences a significant reduction in residue-specific dynamics and an increase in the number of PpiB conformations in E. coli cells compared to solution owing, in part, to interactions with cell components.