title: Nucleotide-free kinesin motor domains reversibly convert to an inactive conformation with characteristics of a molten globule.
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.
authors: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA, USA. Electronic address: ddh@andrew.cmu.edu.
authors: David D
authors: DD
authors: Hackney
authors: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA, USA.
authors: Marshall S
authors: MS
authors: McGoff
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002942:Circular Dichroism; D004331:Drosophila melanogaster; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016547:Kinesins / Q000737:chemistry*; D007700:Kinetics; D011485:Protein Binding; D000072417:Protein Domains; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D000817:Anilino Naphthalenesulfonates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins; D005990:Glycerol
keywords: Conformational selection; Induced fit; Kinesin; Molten globule; Osmolyte effect