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.

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.

Global transformation of carbon dioxide to biomass is almost entirely dependent on the CO 2 -fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Paradoxically this critical catalyst displays a low carboxylation velocity and is prone to confuse CO 2 with oxygen. To mitigate these properties the chemo-and photosynthetic organisms relying on Rubisco activity have evolved elaborate machineries to assist this catalyst. Almost all eukaryotic microalgae utilize CO 2 -concentrating mechanisms (CCMs) that function as carbon dioxide superchargers for Rubisco. The heartpiece of this CCM is a liquid-like Rubisco-containing membraneless organelle known as the pyrenoid. Recently a putative Rubisco linker protein comprised of four highly conserved 60-residue repeats was identified in the green alga Chlamydomonas reinhardtii. We report a reconstituted system using pure components that recapitulates properties of the pyrenoid in vitro. Addition of the linker protein to Rubisco results in a salt sensitive liquid-liquid phase separation. The two proteins rapidly demix from the bulk solution to form dense droplets that fuse and can be harvested by centrifugation. The droplet composition is dynamic and both components exchange rapidly with the bulk solution. Highly homologous Rubisco enzymes from diverse organisms such as higher plants and cyanobacteria show a great variability in their tendency to demix with the algal linker. Linker protein variants containing fewer repeats can phase separate with Rubisco, but require higher protein concentrations. Our data permits first conclusions regarding the interaction of Rubisco and the linker protein to be drawn. The ability to dissect aspects of pyrenoid biochemistry in vitro will permit us to inform and guide synthetic biology ambitions aiming to engineer microalgal CCMs into crop plants.