A high-field cellular DNP-supported solid-state NMR approach to study proteins with sub-cellular specificity

Studying the structural aspects of proteins within sub-cellular compartments is of growing interest. Dynamic nuclear polarization supported solid-state NMR (DNP-ssNMR) is uniquely suited to provide such information, but critically lacks the desired sensitivity and resolution. Here we utilize SNAPol-1, a novel biradical, to conduct DNP-ssNMR at high-magnetic fields (800 MHz/527 GHz) inside HeLa cells and isolated cell nuclei electroporated with [13C,15N] labeled ubiquitin. We report that SNAPol-1 passively diffuses and homogenously distributes within whole cells and cell nuclei providing ubiquitin spectra of high sensitivity and remarkably improved spectral resolution. For cell nuclei, physical enrichment facilitates a further 4-fold decrease in measurement time and provides an exclusive structural view of the nuclear ubiquitin pool. Taken together, these advancements enable atomic interrogation of protein conformational plasticity at atomic resolution and with sub-cellular specificity. We show that the polarising agent SNAPol-1 enters human cells yielding 800 MHz DNP solid-state NMR data of remarkable sensitivity & resolution thereby revealing structural heterogeneity of ubiquitin at atomic scale and with sub-cellular specificity.

Automated summary

We demonstrate that the polarizing agent SNAPol-1 can enter human cells and enable DNP solid-state NMR measurements at 800 MHz, resulting in remarkable sensitivity and resolution. This allows for the identification of structural heterogeneity of ubiquitin at the atomic scale and with sub-cellular specificity. The use of SNAPol-1 also enables faster measurement times and provides a unique structural view of the nuclear ubiquitin pool.