Rapid Estimation of Membrane Protein Orientation in Liposomes.
Rapid Estimation of Membrane Protein Orientation in Liposomes.
复制标题
脂质体中膜蛋白取向的快速估计。
DOI：
10.1002/cbic.202100543
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发表时间：
2022-01-19
期刊：
影响因子：
3.2
通讯作者：
von Ballmoos, Christoph
中科院分区：
文献类型：
--
作者：
Deutschmann, Sabina;Rimle, Lukas;von Ballmoos, Christoph
关键词：
The topological organization of proteins embedded in biological membranes is crucial for the tight interplay between these enzymes and their accessibility to substrates in order to fulfil enzymatic activity. The orientation of a membrane protein reconstituted in artificial membranes depends on many parameters and is hardly predictable. Here, we present a convenient approach to assess this important property independent of the enzymatic activity of the reconstituted protein. Based on cysteine‐specific chemical modification of a target membrane protein with a cyanine fluorophore and a corresponding membrane‐impermeable fluorescence quencher, the novel strategy allows rapid evaluation of the distribution of the two orientations after reconstitution. The assay has been tested for the respiratory complexes bo3 oxidase and ATP synthase of Escherichia coli and the results agree well with other orientation determination approaches. Given the simple procedure, the proposed method is a powerful tool for optimization of reconstitution conditions or quantitative orientation information prior to functional measurements. A rapid fluorescence‐based method for the estimation of membrane protein (MP) orientation in liposomes was developed. For the determination of MP orientation, the fluorescence of site‐specifically labeled mutants was monitored. Outside‐oriented fluorophores are quenched with membrane‐impermeable corresponding quencher, before solubilization of liposomes by detergent, leading to maximal quench. MP orientation corresponds to the ratio of first to full quench.
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影响因子：
15
作者：
Blackman, Melissa L.;Royzen, Maksim;Fox, Joseph M.
通讯作者：
Fox, Joseph M.
DOI：
10.1111/j.1432-1033.1990.tb19418.x
发表时间：
1990-11-13
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
RICHARD, P;RIGAUD, JL;GRABER, P
通讯作者：
GRABER, P
影响因子：
4.3
作者：
Wiedenmann, Alexander;Dimroth, Peter;von Ballmoos, Christoph
通讯作者：
von Ballmoos, Christoph
影响因子：
4.8
作者：
Skrzypek，Ruth;Iqbal，Shagufta;Callaghan，Richard
通讯作者：
Callaghan，Richard
影响因子：
4.3
作者：
Ishmukhametov, RR;Galkin, MA;Vik, SB
通讯作者：
Vik, SB