UC San Diego

Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy is a structural biology technique capable of characterizing complex and heterogenous samples. Here, we develop novel ways of performing MAS NMR to characterize two complex systems - the phase separated environment formed by heterochromatin protein 1α (HP1α), and the cellular interior of mammalian cells. HP1α is a protein fundamental to the organization and gene regulation of heterochromatic genome territories. HP1α can undergo a process called liquid-liquid phase separation, generating concentrated hubs of heterochromatin components. We applied MAS NMR to track the structural dynamics of phase separated HP1α through its development into a gel which resembles the matured state of heterochromatin in cells. Beyond the capabilities of other structural techniques, our methodology was able to identify the residues in HP1α that participated in the crosslinking interactions in the gel state. The gelation process slowed in the presence of chromatin, leading us to further probe the interactions between HP1 and nucleosomes. We investigated the interactions of HP1 with the nucleosome, and studied how HP1 phase separation might influence other proteins that act on the nucleosome, such as transcription factors and chromatin remodelers. We have also developed structural biology methodology for proteins in mammalian cells. Our efforts center around dynamic nuclear polarization (DNP), a tool to enhance the sensitivity of NMR, aiding the detection of biomolecules directly in their native cellular environment. To conduct DNP, samples must be doped with a biradical polarization agent (PA) that transfers polarization to nearby nuclei. We first designed and synthesized a novel bio-orthogonal PA, TTz, that employs a tetrazine-based reaction to target proteins in the cellular milieu. Our strategy was generalized to several proteins and was able to selectively enhance the NMR signal of our protein of interest over the background. We then developed the PA, POPAPOL, to address the susceptibility of PAs to radical reduction in the cellular environment. POPAPOL exhibited longer radical lifetimes than other popular PAs, promising a new design route for future PAs for applications in cells.