Identification of lipid-specific proteins with high-density lipid-immobilized beads.

In biological membranes, lipids often interact with membrane proteins (MPs), regulating the localization and activity of MPs in cells. Although elucidating lipid-MP interactions is critical to comprehend the physiological roles of lipids, a systematic and comprehensive identification of lipid-binding proteins has not been adequately established. Therefore, we report the development of lipid-immobilized beads where lipid molecules were covalently immobilized. Owing to the detergent tolerance, these beads enable screening of water-soluble proteins and MPs, the latter of which typically necessitate surfactants for solubilization. Herein, two sphingolipid species-ceramide and sphingomyelin-which are major constituents of lipid rafts, were immobilized on the beads. We first showed that the density of immobilized lipid molecules on the beads was as high as that of biological lipid membranes. Subsequently, we confirmed that these beads enabled the selective pulldown of known sphingomyelin- or ceramide-binding proteins (lysenin, p24, and CERT) from protein mixtures, including cell lysates. In contrast, commercial sphingomyelin beads, on which lipid molecules are sparsely immobilized through biotin-streptavidin linkage, failed to capture lysenin, a well-known protein that recognizes clustered sphingomyelin molecules. This clearly demonstrates the applicability of our beads for obtaining proteins that recognize not only a single lipid molecule but also lipid clusters or lipid membranes. Finally, we demonstrated the screening of lipid-binding proteins from Neuro2a cell lysates using these beads. This method is expected to significantly contribute to the understanding of interactions between lipids and proteins and to unravel the complexities of lipid diversity.

Truncated derivatives of amphidinol 3 reveal the functional role of polyol chain in sterol-recognition and pore formation.

Here we examined the membrane binding and pore formation of amphidinol 3 (AM3) and its truncated synthetic derivatives. Importantly, both of the membrane affinity and pore formation activity were well correlated with the reported antifungal activity. Our data clearly demonstrated that the C1-C30 moiety of AM3 plays essential roles both in sterol recognition and stable pore formation. Based on the current findings, we updated the interacting model between AM3 and sterol, in which the moiety encompassing from C21 to C67 accommodates a sterol molecule with forming hydrogen bonds with the sterol hydroxy group and van der Waals contact between AM3 polyol and sterol skeleton. Although the conformation of the C1-C20 moiety of AM3 is hard to specify due to its flexibility, the region likely contributes to stabilization of pore structure.

Cardiolipin binding enhances KcsA channel gating via both its specific and dianion-monoanion interchangeable sites.

KcsA is a potassium channel with a plethora of structural and functional information, but its activity in the KcsA-producing actinomycete membranes remains elusive. To determine lipid species involved in channel-modulation, a surface plasmon resonance (SPR)-based methodology, characterized by immobilization of membrane proteins under a membrane environment, was applied. Dianionic cardiolipin (CL) showed extremely higher affinity for KcsA than monoanionic lipids. The SPR experiments further demonstrated that CL bound not only to the N-terminal M0 helix, a lipid-sensor domain, but to the M0 helix-deleted mutant. In contrast, monoanionic lipids interacted primarily with the M0 helix. This indicates the presence of an alternative CL-binding site, plausibly in the transmembrane domain. Single-channel recordings demonstrated that CL enhanced channel opening in an M0-independent manner. Taken together, the action of monoanionic lipids is exclusively mediated by the M0 helix, while CL binds both the M0 helix and its specific site, further enhancing the channel activity.