Investigation of protein-protein interactions within flagellar dynein using homobifunctional and zero-length crosslinking reagents.
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.
Benashski
S E
SE
Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06032-3305, USA.
King
S M
SM
D000818:Animals; D002696:Chlamydomonas; D003432:Cross-Linking Reagents; D004398:Dyneins / Q000378:metabolism*; D005407:Flagella / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000378:metabolism*; D011485:Protein Binding
D003432:Cross-Linking Reagents; D020409:Molecular Motor Proteins; D004398:Dyneins