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Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that permit lipid exchange on experimentally relevant timescales. Therefore, examining the kinetics and mechanisms of lipid exchange is of great interest. Since the high charge densities of existing anionic polymers can interfere with protein–protein and protein–lipid interactions as well as charge-sensitive analysis techniques, electroneutral nanodisc-forming polymers have been recently introduced. However, it has remained unclear how the electroneutrality of these polymers affects the lipid-exchange behavior of the nanodiscs. Here, we use time-resolved Förster resonance energy transfer to study the kinetics and the mechanisms of lipid exchange among nanodiscs formed by the electroneutral polymer Sulfo-DIBMA. We also examine the role of coulombic repulsion and specific counterion association in lipid exchange. Our results show that Sulfo-DIBMA nanodiscs exchange lipids on a similar timescale as DIBMA nanodiscs. In contrast with nanodiscs made from polyanionic DIBMA, however, the presence of mono- and divalent cations does not influence lipid exchange among Sulfo-DIBMA nanodiscs, as expected from their electroneutrality. The robustness of Sulfo-DIBMA nanodiscs against varying ion concentrations opens new possibilities for investigating charge-sensitive processes involving membrane proteins. [ABSTRACT FROM AUTHOR]

The HDV ribozyme is an RNA enzyme from the human pathogenic hepatitis delta virus (HDV) that has recently also been identified in the human genome. It folds into a compact, nested double-pseudoknot. We examined here the functional relevance of the capping loop L4 and the helical crossover J1/2, which tightly interlace the two helical stacks of the ribozyme. Peripheral structural elements such as these are present in cis-acting, but not trans-acting ribozymes, which may explain the order-of-magnitude decrease in cleavage activity observed in trans-acting ribozymes with promise in gene therapy applications. Comparison of a systematic set of cis- and trans-acting HDV ribozymes shows that the absence of either L4 or J1/2 significantly and independently impacts catalytic activity. Using terbium(III) footprinting and affinity studies, as well as distance measurements based on time-resolved fluorescence resonance energy transfer, we find that J1/2 is most important for conferring structural properties similar to those of the cis-acting ribozyme. Our results are consistent with a model in which removal of either a helical crossover or surprisingly a capping loop induces greater dynamics and expansion of the catalytic core at long range, impacting local and global folding, as well as catalytic function. [ABSTRACT FROM AUTHOR]