Protein engineering approaches to study the dynein mechanism using a Dictyostelium expression system.
Dyneins are microtubule-based motor complexes that power a wide variety of motile processes within eukaryotic cells, including the beating of cilia and flagella and intracellular trafficking along microtubules. Mechanistic studies on dynein have been hampered by their enormous size (molecular masses of 0.5-3MDa) and molecular complexity. However, the recent establishment of recombinant expression systems for cytoplasmic dynein, together with structural and functional analyses, has advanced our understanding of the molecular mechanisms of dynein motility. Here, we describe several protocols for protein engineering approaches to the dynein mechanism using a Dictyostelium discoideum expression system. We first describe the design and preparation of recombinant dynein suitable for mechanistic studies. We then discuss two distinct functional assays that take advantage of the recombinant dynein. One is for detection of dynein's conformational changes during the ATPase cycle. Another is an in vitro motility assay at multiple- and single-molecule levels for examination of the dynamic behavior of dynein moving on a microtubule.
Kon
Takahide
T
Department of Life Sciences, University of Tokyo, Japan.
Shima
Tomohiro
T
Sutoh
Kazuo
K
D000818:Animals; D001681:Biological Assay; D001710:Biotin / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D004398:Dyneins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007734:Kymography; D008870:Microtubules / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D001710:Biotin; D004398:Dyneins