A Comparative Study of Nitroxide-Based Biradicals for Dynamic Nuclear Polarization in Cellular Environments.

Dynamic nuclear polarization (DNP) is a powerful tool to enhance the NMR signals of molecules by transferring polarization from unpaired electron spins to nuclei through microwave irradiation. The resulting signal enhancements can enable the analysis of samples that have previously been intractable by NMR spectroscopy, including proteins, nucleic acids, and metabolites in cells. To carry out DNP, the sample is doped with a polarization agent, a biradical containing two nitroxide moieties. DNP applications in cells, however, present significant challenges as nitroxides are often susceptible to the reducing cellular environment. Here, we introduce a novel polarization agent, POPAPOL, that exhibits increased lifetimes under reducing conditions. We also compare its bioresistance and DNP performance with three popular, commercially available polarization agents. Our work indicates that pyrrolidine-based nitroxides can outperform piperidine-based nitroxides in cellular environments, and that future polarization agent designs must carefully balance DNP performance and stability for cellular applications.

Proteorhodopsin Function Is Primarily Mediated by Oligomerization in Different Micellar Surfactant Solutions.

The diverse functionalities of membrane proteins (MPs) have garnered much interest in leveraging these biomolecules for technological applications. One challenge of studying MPs in artificial micellar surfactant environments is that many factors modulate their structures and functionalities, including the surfactants that interact with the MP or their assembly into oligomers. As oligomerization offers a means by which MPs could selectively interact among the copious environmental factors in biological environments, we hypothesized that MP function is predominantly modified by oligomerization rather than interactions with local surfactants that, by comparison, largely interact with MPs nonspecifically. To test this, we study the light-activated proton pump proteorhodopsin (PR) in micellar surfactant solutions because it is functionally active in monomeric and oligomeric forms, the light-activated functionalities of which can be assessed in detail. The surfactant composition and oligomerization are correlated with PR function, as measured by the protonation behaviors of aspartic acid residue 97, which mediates light-activated proton transport, and the associated photocycle kinetics. The results demonstrate that oligomerization dominantly mediates PR function in different surfactant environments, whereas some surfactants can subtly modulate proton-pumping kinetics. This work underscores the importance of understanding and controlling oligomerization of MPs to study and exploit their function.