Analysis of membrane protein clusters using fluorescence lifetime FRET

为了利用荧光寿命FRET监测膜蛋白的聚类，在细胞中表达gfp或rfp标记的编码酵母色氨酸渗透酶Tat2的Tat2。然而，荧光信号太低，无法进行有效的光子计数。修改策略，执行本机PAGE来阐明etat2集群。在用非离子洗涤剂适度处理后，大多数Tat2蛋白以单体形式迁移。采用高压时间分辨荧光各向异性测量系统，对深海亲压生物的质膜流动性进行了分析。这种膜显示出是刚性的，这与深缝微生物的膜是高度流动的一般概念相反。

To monitor clustering of the membrane protein using fluorescence lifetime FRET, GFP-or RFP-tagged Tat2, which encodes yeast tryptophan permease Tat2, was expressed in cells. However, the fluorescence signal was too lowto obtain operative photon counting. Revisingthe strategy, native PAGE was performed toelucidateTat2 clusters. The majority of Tat2 proteins appeared to migrate as monomers after the membrane fraction was moderately treated with a non-ionic detergent. By means of high-pressure time-resolved fluorescence anisotropy measurement system, fluidity of the plasma membrane was analyzed in a deep-sea piezophile. The membrane revealed to be rigid as opposed to a general concept that the membrane of deep-seamicrobes is highly fluid.