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Abstract

Nucleotide-free kinesin motor domains from several kinesin families convert reversibly to a refractory conformation that cannot rapidly rebind ADP. In the absence of glycerol, the refractory conformation of Drosophila kinesin motor domains is favored by 50-fold with conversion of the active to the refractory species at ∼0.052 s−1 and reactivating in the presence of ADP at ∼0.001 s−1. This reactivation by ADP is due to conformational selection rather than induced fit because ADP is not bound to the refractory species at concentrations of ADP that are sufficient to saturate the rate of reactivation. Glycerol stabilizes the active conformation by reducing the rate of inactivation, while having little effect on the reactivation rate. Circular dichroism indicates a large conformational change occurs on formation of the refractory species. The refractory conformation binds ANS (8-anilino-1-napthalenesulfonic acid) with a large increase in fluorescence, indicating that it has molten globule character. High ANS binding is also observed with the refractory forms of Eg5 (a kinesin-5) and Ncd (a kinesin-14), indicating that a refractory conformation with molten globule characteristics may be a common feature of nucleotide-free kinesin motor domains.

5. Conclusions and perspectives

Kinesin motor domains in the absence of nucleotide convert reversibly between an active state and an inactive state that has characteristics of a molten globule. This molten globule state is an excellent candidate for more extensive structural studies because it is i) stable; ii) the predominant species under physiological conditions and iii) readily formed without the use of chaotropic agents or extremes of pH. Furthermore, the kinetics and mechanism of the interconversion between native and molten globule states are experimentally accessible because the equilibrium between the native and molten globule states can be shifted by nucleotides and osmolytes such as glycerol.