We study biological motor proteins in single-molecule experiments with the goal of understanding the physical principles of biological force generation in a multitude of active transport processes. Motor proteins are the ubiquitous nanometer-scale mechanical engines at the basis of many crucial processes of life. Examples are intracellular transport processes, cell division, cell locomotion, and in complex large scale assemblies also macroscopic motion, such as muscle contraction, or flagellar motion. The non-equilibrium dynamics of these specialized enzymes, usually embedded in a complex regulatory and functional environment, are the essence of their function.

	How most motors work: Motor proteins are divided into super-families by sequence similarity. Myosins move on actin filaments, kinesins and dyneins on microtubules. All use ATP as fuel. Many mmembers of these families are multimeric, often homodimers, the motor function sits in the 'heads', while the tails regulate cargo binding.

Observing dynamic events on the scale of single protein molecules is a major experimental challenge. We use light microscopy, combined with optical tweezers and laser interferometry to determine the position of sub-micron probe particles to which motor proteins are attached in a light microscope with nanometer spatial and microsecond temporal resolution. We also use single-molecule fluorescence techniques to extend the range of dynamic parameters we can measure. Several projects are focussed on kinesin-like motor proteins. Kinesin is a motor protein that transduces chemical energy from ATP hydrolysis into movement along the surface of microtubules. Like most proteins, kinesin is a member of a large family of related proteins. The ncd protein, one of the earliest kinesin-like proteins to be identified, is important for normal meiotic spindle formation in female Drosophila and may also have a role in mitosis. Another kinein-like motor protein we are interested in is Eg5. Eg5 is a tetrameric, bipolar motor involved in mitotic spindle formation. It is thought to play a role in the crosslinking of microtubules in the spindle and separation of the two spindle poles.

	We study motor protein dynamics using in vitro motility assays. Most of those assays are variants of two basic schemes: surface sliding assays, where the motor is substrate-bound and the track moves, or single-motor walking assays, where the track is substrate-bound and the motor moves a fluorophore or a microscopic bead.

Current topics of our interest are the mechanism of the kinesin-related ncd and Eg5 motors, the mechanism of processivity in kinesin, the cooperative behavior of multiple motors (both kinesins and myosins), and the function of mitotic kinesins in the mitotic spindle or model systems.

	The most sophisticated configuration is a 'three-bead assay', where the motor is bound to a fixed bead, while the track is suspended over it with the help of two optical traps (see below), holding two beads which are attached to the the track.

Technical issues are the engineering of defined and rigid surface attachment methods, improvements of resolution and stability in the detection methods, and the development of methods that combine optical trapping and single-molecule fluorescence microscopy.

	Typical data from monitoring the motion of a 0.5 micron bead pulled out of the optical trap by a single kinesin motor protein. With increasing resistance from the optical trap the motor slows down until it eventually stalls at a little more than 6 piconewton load.
	A high resolution view of the data demonstrates that the motor protein moves in steps of about 8nm along the microtubule axis.

Collaborators

Günther Woehlke, TU München, Germany
Hernando Sosa, Albert Einstein College of Medicine, Bronx, NY, USA
Jonathan Scholey, UC Davis, Davis, CA, USA