Direct observation of intermediate states during the stepping motion of kinesin-1.

The dimeric motor protein kinesin-1 walks along microtubules by alternatingly hydrolyzing ATP and moving two motor domains ('heads'). Nanometer-precision single-molecule studies demonstrated that kinesin takes regular 8-nm steps upon hydrolysis of each ATP; however, the intermediate states between steps have not been directly visualized. Here, we employed high-temporal resolution dark-field microscopy to directly visualize the binding and unbinding of kinesin heads to or from microtubules during processive movement. Our observations revealed that upon unbinding from microtubules, the labeled heads were displaced rightward and underwent tethered diffusive movement. Structural and kinetic analyses of wild-type and mutant kinesins with altered neck linker lengths provided evidence that rebinding of the unbound head to the rear-binding site is prohibited by a tension increase in the neck linker and that ATP hydrolysis by the leading head is suppressed when both heads are bound to the microtubule, thereby explaining how the two heads coordinate to move in a hand-over-hand manner.

High-speed angle-resolved imaging of a single gold nanorod with microsecond temporal resolution and one-degree angle precision.

We developed two types of high-speed angle-resolved imaging methods for single gold nanorods (SAuNRs) using objective-type vertical illumination dark-field microscopy and a high-speed CMOS camera to achieve microsecond temporal and one-degree angle resolution. These methods are based on: (i) an intensity analysis of focused images of SAuNR split into two orthogonally polarized components and (ii) the analysis of defocused SAuNR images. We determined the angle precision (statistical error) and accuracy (systematic error) of the resultant SAuNR (80 nm × 40 nm) images projected onto a substrate surface (azimuthal angle) in both methods. Although both methods showed a similar precision of ∼1° for the azimuthal angle at a 10 µs temporal resolution, the defocused image analysis showed a superior angle accuracy of ∼5°. In addition, the polar angle was also determined from the defocused SAuNR images with a precision of ∼1°, by fitting with simulated images. By taking advantage of the defocused image method's full revolution measurement range in the azimuthal angle, the rotation of the rotary molecular motor, F1-ATPase, was measured with 3.3 µs temporal resolution. The time constants of the pauses waiting for the elementary steps of the ATP hydrolysis reaction and the torque generated in the mechanical steps have been successfully estimated. The high-speed angle-resolved SAuNR imaging methods will be applicable to the monitoring of the fast conformational changes of many biological molecular machines.

CK2 activates kinesin via induction of a conformational change.

Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain. Furthermore, we show that treatment of kinesin with CK2 prevents and reverses this repositioning. Finally, we demonstrate that CK2 treatment facilitates ADP dissociation from the motor, resulting in a nucleotide-free state that promotes microtubule binding. Thus, we propose that kinesin inactivation results from neck-linker repositioning and that CK2-mediated reactivation results from CK2's dual ability to reverse this repositioning and to promote ADP release.

Photoclickable dendritic molecular glue: noncovalent-to-covalent photochemical transformation of protein hybrids.

A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu(+))/benzophenone (BP) pairs at its periphery (Glue(BP)-FITC) serves as a "photoclickable molecular glue". By multivalent salt-bridge formation between Gu(+) ions and oxyanions, Glue(BP)-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of "optical tweezers".