Identification of the walking mechanism of conventional kinesin by single molecule fluorescence resonance energy transfer (smFRET) measurement on heterodimeric kinesins constructs

• Antragsteller: Professor Dr. Arne Gennerich
• Fachliche Zuordnung: Biophysik
• Förderung: Förderung von 2004 bis 2007
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 5427581

The objective of the project is to identify the walking mechanism of conventional kinesin and to find substantial evidence for the neck-linker structural change that has been proposed to enable the processive movement of the motor enzymes. The issues of the project will be addressed by the generation of both a kinesin heterodimer in which one of the heads is a mutant motor with impaired neck-linker mobility and two dimeric kinesin constructs with specific dye-labeled residues for single molecule fluorescence resonance energy transfer (smFRET) measurements. The kinesin constructs will be used in single and multiple molecule processivity assays. Quantitative information on motor movement and structural changes within the dye-labeled dimeric kinesins will be obtained using single particle tracking (SPT) and smFRET techniques together with total internal reflection fluorescence (TIRF) microscopy. By simultaneously measuring, under various biochemical conditions, the spatial movement and specific distances between carefully chosen FRET donor- and acceptor-labeled positions in the catalytic cores and the neck-linkers of processive kinesin enzymes, these studies will offer the detailed conformational information needed to unequivocally identify the correct walking mechanism of conventional kinesin.

• DFG-Verfahren: Forschungsstipendien
• Internationaler Bezug: USA
• Kooperationspartner: Professor Dr. Ronald D. Vale