Abstract:In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tappingmode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, whichsdepending on the concentration of the constituentssseparates into liquid-disordered (ld), liquid-ordered (lo), and solid-ordered (so) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquiddisordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is ld > lo . so. Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the ld/lo phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of the demixed phases. Such an interfacial adsorption effect may serve as an alternative vehicle for association processes of signaling proteins in membranes.
Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid-phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N-terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.
The total synthesis of palmarumycin CP1 (4) and CP2 (5) and racemic CJ‐12.371 methyl ether (17) is described using the Diels–Alder reaction of benzoquinone 1,8‐dihydroxynaphthalene acetal (10) with 1‐methoxy‐1,3‐butadiene under neat reaction conditions. The stereochemistry of adduct 15 was confirmed by single‐crystal X‐ray analysis. The transformation of 15 into target products 4, 5, and 17 involved dehydrogenation, methyl ether cleavage, and reduction and oxidation steps.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.