RESUMEN
Membrane proteins (MPs) are affected by binding of specific lipids. We previously developed a methodology for systematically analyzing MP-lipid interactions leveraging surface plasmon resonance (SPR). In this method, the gold sensor chip surface was modified with a self-assembled monolayer (SAM), which allowed for a larger amount of MP-immobilization. However, the laborious lipid purification step remained a bottleneck. To address this issue, a new strategy has been developed utilizing gold nanoparticles (AuNPs) instead of the gold sensor chip. AuNPs were coated with SAM, on which MP was covalently anchored. The MP-immobilized AuNPs were mixed with a lipid mixture, and the recovered lipids were quantified by LC-MS. Bacteriorhodopsin (bR) was used as an MP to demonstrate this concept. We optimized immobilization conditions and confirmed the efficient immobilization of bR by dynamic light scattering and electron micrographs. Washing conditions for pulldown experiments were optimized to efficiently remove non-specific lipids. A new binding index was introduced to qualitatively reproduce the known affinity of lipids for bR. Consequently, the low-abundant and least-studied lipid S-TeGD was identified as a candidate for bR-specific lipids. This technique can skip the laborious lipid purification process, accelerating the screening of MP-specific lipids from complex lipid mixtures.
Asunto(s)
Lípidos de la Membrana , Nanopartículas del Metal , Oro , Proteínas de la Membrana , Resonancia por Plasmón de Superficie/métodosRESUMEN
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.
Asunto(s)
Esfingomielinas , Esfingomielinas/química , Animales , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Ceramidas/química , Toxinas BiológicasRESUMEN
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.
Asunto(s)
Anfidinoles , Esteroles , Esteroles/farmacología , Esteroles/química , Alquenos/química , Piranos/químicaRESUMEN
Ginsenoside Rh2 (Rh2) is a ginseng saponin comprising a triterpene core and one unit of glucose and has attracted much attention due to its diverse biological activities. In the present study, we used small-angle X-ray diffraction, solid-state NMR, fluorescence microscopy, and MD simulations to investigate the molecular interaction of Rh2 with membrane lipids in the liquid-disordered (Ld) phase mainly composed of palmitoyloleoylphosphatidylcholine compared with those in liquid-ordered (Lo) phase mainly composed of sphingomyelin and cholesterol. The electron density profiles determined by X-ray diffraction patterns indicated that Rh2 tends to be present in the shallow interior of the bilayer in the Ld phase, while Rh2 accumulation was significantly smaller in the Lo phase. Order parameters at intermediate depths in the bilayer leaflet obtained from 2H NMR spectra and MD simulations indicated that Rh2 reduces the order of the acyl chains of lipids in the Ld phase. The dihydroxy group and glucose moiety at both ends of the hydrophobic triterpene core of Rh2 cause tilting of the molecular axis relative to the membrane normal, which may enhance membrane permeability by loosening the packing of lipid acyl chains. These features of Rh2 are distinct from steroidal saponins such as digitonin and dioscin, which exert strong membrane-disrupting activity.
RESUMEN
BACKGROUND: N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required. RESULTS: In this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates. SIGNIFICANCE AND NOVELTY: In conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.