Enzymatic activity monitoring through dynamic nuclear polarization in Earth magnetic field.

Cost-effective and portable MRI systems operating at Earth-field would be helpful in poorly accessible areas or in developing nations. Furthermore Earth-field MRI can provide new contrasts opening the way to the observation of pathologies at the biochemical level. However low-field MRI suffers from a dramatic lack in detection sensitivity even worsened for molecular imaging purposes where biochemical specificity requires detection of dilute compounds. In a preliminary spectroscopic approach, it is proposed here to detect protease-driven hydrolysis of a nitroxide probe thanks to electron-nucleus Overhauser enhancement in a home-made double resonance system in Earth-field. The combination of the Overhauser effect and the specific enzymatic modification of the probe provides a smart contrast reporting the enzymatic activity. The nitroxide probe is a six-line nitroxide which lines are shifted according to its substrate/product state, which requires quantum mechanical calculations to predict EPR line frequencies and Overhauser enhancements at Earth field. The NMR system is equipped with a 13-mT prepolarization coil, a 153-MHz EPR coil and a 2-kHz NMR coil. Either prepolarized NMR or DNP-NMR without prepolarization provide NMR spectra within 3 min. The frequency dependence of Overhauser enhancement was in agreement with theoretical calculations. Protease-mediated catalysis of the nitroxide probe could only be measured through the Overhauser effect with 5 min time resolution. Future developments shall open the way for the design of new low-field DNP-MRI systems.

Probing the dynamic properties of two sites simultaneously in a protein-protein interaction process: a SDSL-EPR study.

During molecular processes, protein flexibility is a fundamental property allowing protein-protein interaction. Following structural changes during these interactions is then of crucial interest. Site-Directed Spin Labeling (SDSL) combined to EPR spectroscopy is a powerful technique to follow structural modifications within proteins and during protein-protein interactions. Usual nitroxide labels target cysteine residues and afford a 3-line spectrum, whose shape is informative of the structural environment of the label. However, it is not possible to probe two regions of a protein or two partner proteins at the same time because of the overlapping of EPR signatures. Previously, we reported the design and the characterization of a spin label based on a ß-phosphorylated (PP) nitroxide yielding a 6-line spectrum. Here, we report the use of two labels with different EPR signatures, namely maleimido-proxyl (P) and PP, to follow structural changes during a protein-protein interaction process in one single experiment. As a model system, we chose a disordered protein that undergoes an induced α-helical folding upon binding to its partner. We show that the EPR spectrum of a mixture of labeled interacting proteins can be analyzed in terms of structural changes during the interaction. This study represents an important step forward in the extension of the panoply of SDSL-EPR approaches.