Investigation of protein-protein interactions within flagellar dynein using homobifunctional and zero-length crosslinking reagents.
Investigation of protein-protein interactions within flagellar dynein using homobifunctional and zero-length crosslinking reagents.
复制标题
使用同双功能和零长度交联试剂研究鞭毛动力蛋白内的蛋白质-蛋白质相互作用。
DOI：
10.1006/meth.2000.1088
复制
发表时间：
2000
期刊：
影响因子：
--
通讯作者：
King,SM
中科院分区：
文献类型：
--
作者：
Benashski,SE;King,SM
动力蛋白分子马达是一种高度复杂的酶，含有多达15种不同的蛋白质组分，并由几个不同的结构域可通过电子显微镜鉴定。目前的挑战之一是了解这种马达的超分子组织，并确定各种组件的位置和功能。最近，我们已经使用共价交联胺选择性试剂和碳二亚胺，这导致在零长度交联，研究蛋白质-蛋白质协会内衣原体鞭毛动力蛋白。这种方法也使我们能够确定以前未描述的动力蛋白武器和鞭毛轴丝的其他组件之间的相互作用。在这份报告中，我们详细介绍了我们已经开发的方法来探测内动力蛋白和轴丝内的相互作用，并讨论了各种因素，需要解决一个成功的交联实验。
The dynein molecular motor is a highly complex enzyme containing up to 15 different protein components and consists of several distinct domains identifiable by electron microscopy. One of the current challenges is to understand the supramolecular organization of this motor and to determine the location and function of the various components. Recently, we have used covalent crosslinking by amine-selective reagents and a carbodiimide, which results in zero-length crosslink, to investigate protein–protein associations within Chlamydomonas flagellar dynein. This approach also has enabled us to identify previously undescribed interactions between the dynein arms and other components of the flagellar axoneme. In this report, we detail methods we have developed to probe intradynein and intraaxonemal interactions and discuss the variety of factors that need be addressed to perform a successful crosslinking experiment.