Surface-bound casein modulates the adsorption and activity of kinesin on SiO2 surfaces.
Conventional kinesin is routinely adsorbed to hydrophilic surfaces such as SiO(2). Pretreatment of surfaces with casein has become the standard protocol for achieving optimal kinesin activity, but the mechanism by which casein enhances kinesin surface adsorption and function is poorly understood. We used quartz crystal microbalance measurements and microtubule gliding assays to uncover the role that casein plays in enhancing the activity of surface-adsorbed kinesin. On SiO(2) surfaces, casein adsorbs as both a tightly bound monolayer and a reversibly bound second layer that has a dissociation constant of 500 nM and can be desorbed by washing with casein-free buffer. Experiments using truncated kinesins demonstrate that in the presence of soluble casein, kinesin tails bind well to the surface, whereas kinesin head binding is blocked. Removing soluble casein reverses these binding profiles. Surprisingly, reversibly bound casein plays only a moderate role during kinesin adsorption, but it significantly enhances kinesin activity when surface-adsorbed motors are interacting with microtubules. These results point to a model in which a dynamic casein bilayer prevents reversible association of the heads with the surface and enhances association of the kinesin tail with the surface. Understanding protein-surface interactions in this model system should provide a framework for engineering surfaces for functional adsorption of other motor proteins and surface-active enzymes.
Ozeki
Tomomitsu
T
ULVAC Inc., Kanagawa 253-8543, Japan.
Verma
Vivek
V
Uppalapati
Maruti
M
Suzuki
Yukiko
Y
Nakamura
Mikihiko
M
Catchmark
Jeffrey M
JM
Hancock
William O
WO
D000042:Absorption; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D008027:Light; D008870:Microtubules / Q000378:metabolism; D011485:Protein Binding; D012542:Scattering, Radiation; D012822:Silicon Dioxide / Q000737:chemistry*
D002364:Caseins; D012822:Silicon Dioxide; D016547:Kinesins