Photocrosslinking and Click Chemistry Enable the Specific Detection of Proteins Interacting with Phospholipids at the Membrane Interface
Photocrosslinking and Click Chemistry Enable the Specific Detection of Proteins Interacting with Phospholipids at the Membrane Interface
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DOI：
10.1016/j.chembiol.2008.11.009
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发表时间：
2009-01-30
影响因子：
--
通讯作者：
de Kroon, Anton I. P. M.
中科院分区：
文献类型：
--
作者：
Gubbens, Jacob;Ruijter, Eelco;de Kroon, Anton I. P. M.
新的脂质类似物模仿丰富的膜！磷脂磷脂酰胆碱被开发用于光交联在膜界面处与磷脂头基相互作用的蛋白质。除了连接到头基的苯基叠氮化物或二苯甲酮光活化部分之外，脂质类似物含有作为诱饵连接到酰基链的叠氮化物。在生物膜中原位光交联后，这些诱饵用于使用点击化学附着荧光四甲基罗丹明炔缀合物或生物素-炔缀合物，分别允许交联产物的选择性检测和纯化。检测与来自酿酒酵母的线粒体内膜中的脂质类似物交联的蛋白质，并随后通过质谱法鉴定。已建立的相互作用伙伴的磷脂酰胆碱被发现，以及已知的整体和外周内膜蛋白，和蛋白质，以前不被认为是线粒体内膜蛋白。
New lipid analogs mimicking the abundant membrane! phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively, Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins.