News and Publications

Structure of Molecular Assemblies

R.A. Milligan, J.M. Al-Bassam, C.M. Birdsell, C. Arthur, B.O. Carragher,* A.W. Lin, C.A. Moores, C.S. Potter,* I. Rouiller, B. Sheehan, E.M. Wilson-Kubalek

* Beckman Institute, Urbana, IL

Part of our research focuses on elucidating the structure-function relationships of myosin and kinesin motor proteins. Our goal is to visualize and describe conformational changes in the track-motor complexes that occur during the cycles of interaction. We use electron cryo-microscopy and image analysis to calculate 20-Å-resolution 3-dimensional maps of the complexes in the presence and absence of nucleotide analogs. These maps of the entire complexes and the x-ray structures of the individual components are used to build high-resolution models of the working assemblies. In this way, we obtain a detailed picture of how the motors interact with their tracks at various stages in the chemomechanical cycle.

We showed that members of one class of myosins (class VI) move in the opposite direction to other myosins in the superfamily. Class VI myosins seem to interpret nucleotide-induced motions of the motor core differently so that the lever arm swings in the opposite direction. Encouraged by this surprising result, we are examining members of other myosin classes to determine if they have unusual functional attributes.

Work on microtubule motors belonging to the kinesin family focuses on understanding the molecular basis for directionality and for processivity, the ability of one molecule to move along another molecule for long distances without dissociating. To determine how brain kinesin moves processively toward the microtubule plus end, we attached a gold-cluster label to the putative mechanical element and determined the position of the label relative to the motor in different nucleotide states. The results and other data suggest a model for plus end--directed motion along the microtubule protofilament. The goal of current experiments is to discover how some members of the kinesin superfamily move in the opposite direction. Movies showing the motions of brain kinesin and conventional myosin can be viewed at www.scripps.edu/milligan/projects.html.

We have expanded our research activities to include work on p97, a member of the AAA ATPase family of proteins that is involved in organelle assembly and homotypic membrane fusion. A preliminary examination of the hexameric p97 complex in various nucleotide states revealed that a major conformational change occurs upon ATP binding and not with ATP hydrolysis as had been proposed previously. Our goal is to extend our investigation of the conformational change and to determine how the change affects the disposition of p97's binding partners in the functional complex.

Exciting technical advances in our group include the development of a general approach for helical crystallization of macromolecules on tube-forming lipids and the development and implementation of automatic grid searching, image acquisition, and image analysis protocols for electron microscopic structure determination.

PUBLICATIONS

Carragher, B., Kisseberth, N., Kriegman, D., Milligan, R.A., Potter, C.S., Pulokas, J., Reilein, A. Leginon: An automated system for acquisition of images from vitreous ice specimens. J. Struct. Biol. 132:33, 2000.

Rosenfeld, S.S., Xing, J., Whitaker, M., Cheung, H.C., Brown, F., Wells, A., Milligan, R.A., Sweeney, H.L. Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle. J. Biol. Chem. 275:25418, 2000.

Rouiller, I., Butel, V.M., Latterich, M., Milligan, R.A., Wilson-Kubalek, E.M. A major conformational change in p97 AAA ATPase upon ATP binding. Mol. Cell 6:1485, 2000.

Rouiller, I., Pulokas, J., Butel, V.M., Milligan, R.A., Wilson-Kubalek, E.M., Potter, C.S., Carragher, B.O. Automated image acquisition for single-particle reconstruction using p97 as the biological sample. J. Struct. Biol. 133:102, 2001.

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