Kinesin crouches to sprint but resists pushing

130

135

Kinesin-1 is a motor protein that carries cellular cargo such as membrane-bounded organelles along microtubules (MTs). The homodimeric motor molecule contains two N-terminal motor domains (the motor ''heads''), a long coiled-coil domain (the ''rod'' or ''stalk''), and two small globular ''tail'' domains. Much has been learned about how kinesin's heads step along a MT and how the tail is involved in cargo binding and autoinhibition. However, little is known about the role of the rod. Here, we investigate the extension of the rod during active transport by measuring the height at which MTs glide over a kinesin-coated surface in the presence of ATP. To perform height measurements with nanometer precision, we used fluorescence interference contrast microscopy, which is based on the self-interference of fluorescent light from objects near a reflecting surface. Using an in situ calibrating method, we determined that kinesin-1 molecules elevate gliding MTs 17 ؎ 2 nm (mean ؎ SEM) above the surface. When varying the composition of the surrounding nucleotides or removing the negatively charged -COOH termini of the MTs by subtilisin digestion, we found no significant changes in the measured distance. Even though this distance is significantly shorter than the contour length of the motor molecule (Ϸ60 nm), it may be sufficient to prevent proteins bound to the MTs or prevent the organelles from interfering with transport. molecular structure ͉ motor conformation ͉ motility assay

Section: Discussionmentioning

confidence: 99%

The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy

Kerssemakers

Howard

Hess

et al. 2006

130

135

“…As our model shows, electrostatic effects combined with a modulation of the barriers for proton transfer are sufficient to achieve the gating required for highly efficient proton pumping. The mechanisms identified here for energy transduction by using redox chemistry may also be useful in studies of other molecular machines (24) and may aid in the design of novel bio-inspired fuel cells.…”

Section: Effects Of D Channel Mutationsmentioning

confidence: 99%

“…Only the key redox and proton sites are retained, with the effects of the other sites absorbed into effective free energies and rate coefficients in a master equation that governs the dynamics. Master equations provide a powerful framework in which to study nonequilibrium processes in enzymes and molecular motors (21)(22)(23)(24), and have been used by us to show how redoxcoupled proton pumping could be accomplished in principle (25). The model developed here is explicitly designed to reproduce known thermodynamic and kinetic properties of CcO.…”

mentioning

confidence: 99%

Kinetic gating of the proton pump in cytochrome c oxidase

Kim

Wikström

Hummer

2009

Self Cite

Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, reduces oxygen to water and uses the released energy to pump protons across a membrane. Here, we use kinetic master equations to explore the energetic and kinetic control of proton pumping in CcO. We construct models consistent with thermodynamic principles, the structure of CcO, experimentally known proton affinities, and equilibrium constants of intermediate reactions. The resulting models are found to capture key properties of CcO, including the midpoint redox potentials of the metal centers and the electron transfer rates. We find that coarse-grained models with two proton sites and one electron site can pump one proton per electron against membrane potentials exceeding 100 mV. The high pumping efficiency of these models requires strong electrostatic couplings between the proton loading (pump) site and the electron site (heme a), and kinetic gating of the internal proton transfer. Gating is achieved by enhancing the rate of proton transfer from the conserved Glu-242 to the pump site on reduction of heme a, consistent with the predictions of the water-gated model of proton pumping. The model also accounts for the phenotype of D-channel mutations associated with loss of pumping but retained turnover. The fundamental mechanism identified here for the efficient conversion of chemical energy into an electrochemical potential should prove relevant also for other molecular machines and novel fuel-cell designs.bioenergetics ͉ biological machines ͉ kinetic master equation ͉ respiration C ytochrome c oxidase (CcO) captures the energy from the reduction of oxygen to drive the translocation of protons across the inner mitochondrial (or bacterial) membrane (1-8). The resulting electrochemical gradient powers the production of ATP, the energy source of the cell. CcO takes up four electrons from the outside (P side) of the membrane and four protons from the inside (N side) for the reduction of one dioxygen molecule. Four additional protons are translocated across the membrane from the N side to the P side. Crystal structures of the enzyme from various organisms (3, 4, 9, 10) indicate the electron and proton pathways, formed by conserved residues and cofactors. Extensive experimental and theoretical studies have provided valuable information about these pathways, the reaction sequence, and energetic aspects of catalysis (5,7,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). However, some key aspects of the fundamental mechanisms governing the coupling between the chemical reaction and vectorial proton translocation are still unclear.Here, the objective is to explain how vectorial proton translocation is accomplished in CcO, with respect to both the driving forces (i.e., proton and electron affinities expressed as pK a values and midpoint redox potentials, respectively, and Coulombic couplings) and the kinetic control (i.e., the ''gating'' effects that result from a dependence of kinetic barriers on the microscopic states). Specifically, we aim to construct ...

“…Recent singlemolecule (SM) experiments have shown that, with each stepping motion being strongly coupled to the ATP, the kinesin moves toward the (ϩ) ends of MTs by taking discrete 8-nm steps (3-7) in a hand-over-hand fashion (4,7,8). Although the ultimate understanding of kinesin's motility is still far from completion, the SM experiments (3)(4)(5)(6)(7)(8)(9), together with the series of kinetic ensemble measurements (10)(11)(12)(13) and theoretical studies (14)(15)(16)(17)(18)(19), begin providing glimpses to the physical principle of how kinesin walks.…”

Section: F Irst Recognized By Means Of Their Close Relationship Betweenmentioning

confidence: 99%

Mechanical control of the directional stepping dynamics of the kinesin motor

Hyeon

Onuchic²

2007

129

157

Among the multiple steps constituting the kinesin mechanochemical cycle, one of the most interesting events is observed when kinesins move an 8-nm step from one microtubule (MT)-binding site to another. The stepping motion that occurs within a relatively short time scale (Ϸ100 s) is, however, beyond the resolution of current experiments. Therefore, a basic understanding to the real-time dynamics within the 8-nm step is still lacking. For instance, the rate of power stroke (or conformational change) that leads to the undocked-to-docked transition of neck-linker is not known, and the existence of a substep during the 8-nm step still remains a controversial issue in the kinesin community. By using explicit structures of the kinesin dimer and the MT consisting of 13 protofilaments, we study the stepping dynamics with varying rates of power stroke (k p ATPase activity and organelle transport along microtubules (MTs) (1, 2), kinesins have received broad attention as a prototype of molecular motors for the past two decades. Recent singlemolecule (SM) experiments have shown that, with each stepping motion being strongly coupled to the ATP, the kinesin moves toward the (ϩ) ends of MTs by taking discrete 8-nm steps (3-7) in a hand-over-hand fashion (4,7,8). Although the ultimate understanding of kinesin's motility is still far from completion, the SM experiments (3-9), together with the series of kinetic ensemble measurements (10-13) and theoretical studies (14-19), begin providing glimpses to the physical principle of how kinesin walks.Along the kinesin mechanochemical cycle (supporting information (SI) Fig. 7), one of the main observations of the SM experiments is the stepping dynamics that enables the kinesin to move forward. The actual time spent for the stepping motion itself (Շ100 s), compared with the ATP binding and hydrolysis (տ10 ms), is too short, however, to detect the details of the dynamics with the spatial and temporal resolution of current instruments. Thus, it is still difficult to answer many basic questions (20) related to the stepping dynamics. Some of those questions are: (i) During the 8-nm step, how does the swiveling motion of the tethered head occur? Does any detectable substep exist that reflects a transient intermediate (5,21,22)? (ii) What fraction of the time and length scales is contributed from the power stroke and the diffusional search? (iii) Does the kinesin walk parallel to the single protofilament (PF) or walk astride by using two parallel PFs (23, 24)? To shed light on these questions, we propose to take advantage of the native topology of kinesins and MTs. For instance, our earlier study clarified the regulation mechanism between the two heads (6, 25) by using the native topology-based, two-head bound model of kinesin on the MT (19). Following the same line of thought, we show that even the dynamical pathways of kinesins reflect ''the topological constraints emanating from the molecular architecture.'' In the present work, we have adapted our previous two-head bound model (19) to stud...