Configuration Of The Kinesin1 Motor Domains In The ATP-waiting State As Revealed By Fluorescence Polarization Microscopy

Abstract

The molecular mechanism of coordination between kinesin1 heads during processive walking remains unclear, partly due to the lack of structural information on critical intermediates of the kinesin1 mechano-chemical cycle. To address this issue here we used ensemble and single molecule fluorescence polarization microscopy to determine the mobility and orientation of the kinesin motor domains. We first investigated conditions mimicking a state when only one head is bound to the microtubule and the other one is tethered. For this we made heterodimeric constructs with impaired microtubule binding in one head. Our results indicate that the tethered head is very mobile. We then investigated the orientation of the head domains in homodimeric constructs moving processively at saturating or limiting [ATP]. At saturating [ATP] both motor domains are well oriented relative to the microtubule but at limiting [ATP] there is an increase in mobility. This result indicates that before ATP-binding one motor domain is mobile.