Protein structural dynamics in living cells by EPR spectroscopy – into_the_cell

Exploring biomolecules in the context of their intracellular environment has been recognized as being the gold standard to approach the complexity of the cellular milieu. Therefore, obtaining a holistic understanding of protein function requires to probe structural dynamics of proteins and protein interactions at the molecular level in their native environment, that is, in cell.
As the cellular environment is hardly reproducible in vitro, investigation of biomolecules directly inside cells have attracted a growing interest in the past decade.
Developing methodologies that merge advantages of high sensitivity and nearly no size limit of the biomolecule with the ability to perform in-cell studies of structural transitions and interactions represent a major and challenging goal in the biostructural field for the future. Site-Directed Spin Labeling (SDSL) coupled to Electron Paramagnetic Resonance (EPR) spectroscopy is part of the toolbox available and exhibits competitive advantages but this is still impeded by a series of limitations.
The aim of this project is to develop improved probes and methods to make Site-Directed Spin Labeling (SDSL) combined with EPR an innovative approach for the investigation of proteins structural dynamics, protein-protein interaction and the associated structural changes at the molecular level inside cells.
Site-Directed Spin Labeling coupled to EPR spectroscopy (SDSL-EPR) has demonstrated to be an accurate and powerful approach to study structural and conformational changes in soluble but also membrane proteins. The SDSL-EPR approach, combined with mutagenesis, relies on the selective grafting of paramagnetic labels (usually a nitroxide radical, S=1/2, but also Gd(III)) mostly on cysteine or tyrosine residues and on the study of EPR spectra whose parameters provide detailed information on the local mobility of the grafted label. The dynamics of the spin label is related to the structural properties of the protein under investigation and can be used to follow protein’s structural changes and to detect interaction sites in complexes.
Although several studies demonstrated the feasibility of in-cell EPR experiments, performing EPR in living cells is still challenging and strongly impeded by i) nitroxides instable in the cellular context; ii) the lack of appropriate methods to delivery labeled proteins inside cells and iii) the lack of methods enabling the structural characterization of protein complexes in a natural cellular environment.
In order to bring SDSL-EPR beyond its actual limits and towards the possibility to perform EPR analysis at the molecular level inside cells, we propose: i) the design and synthesis of improved nitroxide labels (ex situ labeling) and non-natural amino acids carrying a nitroxide spin label (in cell labeling), stable in the cellular environment and characterized by enhanced EPR properties for protein studies directly inside cells; ii) the development of an efficient intracellular delivery methods of the labeled proteins inside bacterial cells; iii) a robust and accurate method to explore protein dynamics and protein-protein interaction by EPR (CW and advanced experiments) inside cells.
In the frame of the proposed project, the development of an efficient method to deliver efficiently labeled protein inside cells and new spin labels for in cell studies, will enable high resolution EPR studies of biomolecules in their native environment and will help in addressing the questions of how the intracellular medium modulate protein structural dynamics and protein-protein interactions. The new developed tools will be used to study two bacterial chaperones, NarJ and UreG inside E. coli and B. pasteurii cells, respectively.