Dresden 2009 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 12: Single Molecules

BP 12.2: Hauptvortrag

Mittwoch, 25. März 2009, 10:00–10:30, HÜL 186

Illuminating the way Kinesin-1 walks using FRET between the motor domains — •Erwin Peterman — VU University, Amsterdam, the Netherlands

Kinesin-1 is a motor protein that walks processively along microtubules in a hand-over-hand manner driving intracellular transport of vesicles and organelles. Each step of 8 nm requires the hydrolysis of one ATP and takes about 10 ms at cellular ATP concentrations. Key aspects of kinesin*s walking mechanism are not fully understood. One important question concerns the configuration of the two motor domains during processive motion.

Here, we use a novel assay based on single-molecule confocal fluorescence microscopy to characterize Kinesin-1*s stepping mechanism in vitro. A key advantage of our approach over conventional wide-field methods is that our time resolution is far better, less than 0.1 ms. We apply this approach to kinesin constructs that are labeled with a donor fluorophore on the one motor domain and an acceptor on the other. We follow the distance between the motor domains during stepping with Förster Resonance Energy Transfer. We use four different homodimeric kinesin constructs with dye molecules attached to different sites of the motor domain. With this approach, we can identify an intermediate state in the stepping process that lasts 2-3 ms at saturating ATP concentration. In this intermediate state one motor domain is bound to the microtubule and the other is rotated and substantially less than 8 nm away.