With the help of site-directed Gd(III) spin labels, continuous wave electron paramagnetic resonance spectroscopy (CW-EPR) can be used as a powerful probe in determining protein structures under relatively mild conditions. In this research, we aim to utilize this combination to record dynamic structures of live proteins, which will bring us insights into how they work in natural environments. More specifically, by coupling Gd(III) spin labels at strategical positions of proteins, significant dipolar broadenings of CW-EPR line-shape can be observed, from which distance information between the spin labels can be extracted and structures of protein can thus be rebuilt. Analysis of line-shape broadenings is conducted with a modified version of a open-source package named CWdipFit. Profound tests and code analysis of this package enabled us to fully understand and improve its mechanism, where a series of simulated broadened line-shapes with varying distance distributions are compared and then optimized with respect to the experimental data obtained. An optimal fit of experimental data is thus achieved, providing us with reliable dynamic distance measurements up to ~ 3.4 nm even for samples in aquatic environment with temperature above 200 K. We hope that with future improvements, this temperature limit can be further pushed up so that room temperature observation can be performed.
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