Spins in cells and afar : exploring the limits of pulse dipolar EPR spectroscopy

Obtaining structural information on biomolecules in their native cellular environment and over extended nanometre distance ranges remain major challenges in integrative structural biology. Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) is uniquely positioned to address both. A workflow for introducing spin-labelled proteins into mammalian cells was established based on literature known procedures using electroporation (EP). The studies demonstrate that spin label, cell type, transfection efficiency, and post-EP recovery determine the achievable signal-to-noise ratio (SNR) and the analysability of the resulting measurements. It could be shown that in different cell types measurements with tetrathiatriarylmethyl (trityl or TAM) and gadolinium (Gdᴵᴵᴵ) based spin labels are dependent on the recovery time within these cells. These findings highlight benefits and drawbacks of certain label, cell, and protein combinations in the cellular environment. Complementary to protein delivery into cells, an endogenous labelling strategy based on Cuᴵᴵ-NTA coordination in E. coli was adapted from recent literature to enable in-cell measurements in the absence of protein transfection. The results confirm that genetically encoded metal-binding motifs can be efficiently labelled within living bacteria and yield measurable dipolar signals using PDS. It was shown that the applicability of this method critically depends on the utilised binding motifs persistence. In addition to gaining further insights into in-cell methodologies, the accessible distance range of PDS was systematically investigated. Using different PDS experiments (3,4,5p experiments), optimised experimental conditions (temperature and concentration), and protein deuteration, the achievable upper distance limits were significantly extended. The results demonstrate that substantially longer distances can be measured than previously established and might pave the way for the study of large biomolecular assemblies. The combination of improved in-cell methodologies and expanded long-range distance measurements brings PDS closer to bridging the gap between in vitro structural studies and the complex environments in cell.