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Photoswitchable phospholipid FRET acceptor: Detergent free intermembrane transfer assay of fluorescent lipid analogs.

ABSTRACT: We have developed and characterized a novel photoswitchable phospholipid analog termed N-nitroBIPS-DPPG. The fluorescence can be switched on and off repeatedly with minimal photobleaching by UV or visible light exposure, respectively. The rather large photochromic head group is inserted deeply into the interfacial membrane region conferring a conical overall lipid shape, preference for a positive curvature and only minimal intermembrane transfer. Utilizing the switchable NBD fluorescence quenching ability of N-nitroBIPS-DPPG, a detergent free intermembrane transfer assay system for NBD modified lipids was demonstrated and validated. As NBD quenching can be turned off, total NBD associated sample fluorescence can be determined without the need of detergents. This not only reduces detergent associated systematic errors, but also simplifies assay handling and allows assay extension to detergent insoluble lipid species.

SUBMITTER: Sumi M

PROVIDER: S-EPMC5460167 | biostudies-other | 2017 Jun

REPOSITORIES: biostudies-other

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Photoswitchable phospholipid FRET acceptor: Detergent free intermembrane transfer assay of fluorescent lipid analogs.

Sumi Mariko M Makino Asami A Inaba Takehiko T Sako Yasushi Y Fujimori Fumihiro F Greimel Peter P Kobayashi Toshihide T

Scientific reports 20170606 1

We have developed and characterized a novel photoswitchable phospholipid analog termed N-nitroBIPS-DPPG. The fluorescence can be switched on and off repeatedly with minimal photobleaching by UV or visible light exposure, respectively. The rather large photochromic head group is inserted deeply into the interfacial membrane region conferring a conical overall lipid shape, preference for a positive curvature and only minimal intermembrane transfer. Utilizing the switchable NBD fluorescence quenchi ...[more]

PMID: 28588242

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Project description:The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

Dataset Information

Photoswitchable phospholipid FRET acceptor: Detergent free intermembrane transfer assay of fluorescent lipid analogs.

Publications

Photoswitchable phospholipid FRET acceptor: Detergent free intermembrane transfer assay of fluorescent lipid analogs.

Similar Datasets

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