Cysteine Mapping of Cytoplasmic Dynein Motor Domain

Abstract

Cytoplasmic dynein is a large cytoskeletal protein complex comprised of a heterodimer of heavy chains, intermediate chains, light intermediate chains and light chains. Cytoplasmic dynein is responsible for transporting cargo, other proteins, vesicles and organelles, throughout the cell by movement along microtubules in a retrograde fashion. This activity is mediated by a series of conformational changes to the motor domain induced by ATP binding, hydrolysis and the release of ADP to give a power-stroke motion. There are, however, many unknowns regarding the conformational changes and structure of the motor domain. The extremely large size of the motor domain (380 kDa) makes structural characterization a challenging task. As an initial step towards this goal, cysteine mapping of the motor domain was performed. Preliminary results from fluorescence spectroscopy indicate that 6 out of the 47 cysteines react with ThioGlo(r)1 (methyl 10-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-benzo[f]chromene-2-carboxylate) in the motor domain's native state. Under denaturing conditions, an additional 15 cysteines are revealed. The non-reactivity of the remaining 26 cysteines suggests the presence of 13 disulfide bonds in the motor domain. These results are being analyzed with mass spectrometry to confirm and identify the accessible, buried and oxidized cysteines. This information will be instrumental in mapping the location of residues within cytoplasmic dynein's motor domain. In addition to characterizing the structure of the motor domain, gold particle-bearing labels reactive with surface-accessible cysteines are being explored to provide cryoelectron microscopy data on the motor domain.