Neck-Linker-Length Dependence of Processive Kinesin-5 Motility

Abstract

To explore the basic motor activity of the mitotic Kinesin-5, we previously constructed a stable dimeric Kinesin-5 head/Kinesin-1 stalk chimera (Eg5Kin), which contains the motor domain and 14 amino acids of the neck linker of Xenopus leavis Eg5 fused to the neck coiled coil of Drosophila melanogaster Kinesin-1. In contrast to truncated dimeric Eg5-513 (Valentine and Block, 2009, Biophys. J. 97:1671), Eg5Kin is a highly processive motor (Lakämper et al., 2010, J. Mol. Biol. 399:1). We have here investigated the effect of varying neck-linker length on the motile properties of Eg5Kin. As truncated versions of Eg5 contain the native 18 amino acids of the neck linker, we generated six Eg5Kin constructs comprising of 13 to up to the 18 amino acids of the native Eg5 neck linker, possibly providing a physiological context. Using single-molecule fluorescence, we found that all six constructs are active motor molecules capable of processive motility. In a first set of experiments, we found that the neck-linker length influences the run length, but not the velocity of the motor. We thus confirm the findings of Shastry and Hancock (2010, Curr. Biol. 20:939) with a different motor. In addition we used optical-trap assays to investigate the change in the average force the motor constructs generated and found only a small variation. Our data thus suggest that the neck-linker length of Eg5 is at least not the sole determinant for speed and force generation.