Because the mitotic kinesin Eg5 is essential for the formation of bipolar spindles during eukaryotic cell division, it has been considered as a potential target for cancer treatment. A number of specific and potent inhibitors of Eg5 are known. S-trityl-L-cysteine is one of the inhibitors of Eg5 whose molecular mechanism of inhibition was well studied. The trityl group of S-trityl-L-cysteine was shown to be a key moiety required for potent inhibition. In this study, we synthesized a novel photochromic S-trityl-L-cysteine analogue, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4'- (N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (ACTAB), composed of a trityl group, azobenzene and N-acetyl-L-cysteine, which exhibits cis-trans photoisomerization in order to photocontrol the function of Eg5. ACTAB exhibited cis-trans photoisomerization upon alternating irradiation at two different wavelengths in the visible range, 400 and 480 nm. ACTAB induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with the cis-trans photoisomerization. Compared with cis-ACTAB, trans-ACTAB reduced ATPase activity and microtubule gliding velocity more significantly. These results suggest that ACTAB could be used as photochromic inhibitor of Eg5 to achieve photocontrol of living cells.
that CENP-E plays an important mitotic role at the kinetochore-associated microtubule tips. To determine the molecular mechanism of CENP-E tip-tracking, we characterized two purified recombinant fragments of CENP-E: one containing the motor and neck domains and the second with the dimeric C-terminal tails. The motor-containing truncated protein walked on the microtubule wall in essentially the same manner as the full length CENP-E, while the C-terminal tail exhibited rapid diffusion. Neither of these fragments showed the tiptracking, however, this activity was recapitulated by artificially joining these two proteins by conjugating to Qdots. A computational model of CENP-E motility successfully described the tip-tracking ability by repeating the cycles of plus-end-directed walking and the tail-mediated diffusion of the microtubule wall-tethered motor. This novel ''tethered motor'' mechanism of tip-tracking does not rely on the specific properties of the assembling or disassembling microtubule tips, explaining why CENP-E can tip-track bi-directionally, i.e. with the growing and shortening microtubule ends. Together, these results establish the requirement for CENP-E in stably linking the kinetochores to dynamic microtubule tips, and provide a detailed molecular mechanism to explain how CENP-E can achieve this function.
Cellobiohydrolase is a new type of linear molecular motor that hydrolyzes crystalline cellulose into water-soluble disaccharide at solid/liquid interface. Here, with single-molecule fluorescence microscopy and highspeed AFM, we revealed the adsorption-desorption dynamics and linear translocation of Trichoderma reesei cellobiohydrolase (TrCel7A) hydrolyzing Type I α crystalline celluloses. TrCel7A showed binding rate constant of 9.7 × 10 6 M -1 μm -2 s -1, including productive and non-productive binding that lasted 8.6 s and 1.2 s respectively. The velocity of movement on cellulose in the productive binding was 5.0 nm/s (5 s -1 in turnover rate). Our results shed light on the enzyme reaction mechanism at solid/liquid interface at the single-molecule level for the first time. Kinesin-1 is a motor protein that walks along microtubules to transport cargoes. The velocity of the movement decreases with increasing load, however it is still unclear which conformational transition is the loaddependent. At the last meeting, Isojima in our lab demonstrated that we could distinguish bound and unbound states of the motor head using darkfield microscopy with 50-μs temporal resolution. In this study, we constrained the kinesin movement by fusing a mutant head that cannot detach from the microtubule to the stalk of wild-type dimer. Then we observed the movement of a wild-type head, which showed discrete 16 nm steps and then stalled after several steps. We will discuss the effect of load on the duration of bound and unbound states of a head. Kinesin-1 walks along microtubules by alternately hydrolyzing ATP and moving two motor domains, although the mechanism of the alternate catalysis remains unknown. Here we focused on the neck linker that connects two motor domains and investigated the effect of the neck linker tension on the motor activity by constraining the neck linker in the forward or backward extended conformation using disulfide-crosslinking. Stopped flow and single molecule measurements showed that the forward-constraint of the neck linker reduced ADP release rate although the backwardconstraint suppresses either ATP hydrolysis or Pi release rate. These results suggest that ATP hydrolysis cycle can be differently regulated depending on the direction of the neck linker tension. We show the nucleotide-dependent displacement of the α-1 helix of kinesin on microtubule by ESR spectroscopy. Kinesin monomer was doubly spinlabeled at α-1 and α-2. The inter-helix distance distribution was determined by spectral broadening and showed that 40% of spins had a peak at 1.4-1.7 nm, which was close to that from crystal structure, but 60% beyond sensitivity (>2.5 nm). The fraction of 1.4-1.7 nm was 20 and 25% in the presence of AMPPNP and ADP, respectively. These nucleotide-induced decreases in the fraction of 1.4-1.7 nm were reversely related to those in the docking fraction of neck-linker on motor core, suggesting that shift of spatial equilibrium of α-1 helix from 1.4-1.7 nm toward >2.5 nm makes its C-terminal end to be exposed and ...
Diffusion coefficients are often measured in small biological systems to characterize their fluctuation. Giant acceleration of diffusion is one of the theories on diffusion in the field of non-equilibrium statistical mechanics. We apply this theory to F 1 -ATPase which is an ATP-driven rotary motor protein. According to the theory, when we apply a constant torque to F 1 by using an electric rotating field, the diffusion coefficient of a rotary probe attached to F 1 as a function of the applied torque exhibits a resonance peak. The resonance peak corresponds to the torque value near the critical tilt of the rotary potential of F 1 . 3P152TF 1 β サブユニットのヌクレオチド結合への Pi の影響 Nucleotide binding is important for the rotation of F 1 -ATPase. Here, we examined nucleotide binding to the isolated β subunit and α 3 β 3 γ subcomplex containing βY341W mutation in relation to the effect of Pi. We found that k on ATP and k on ADP for the isolated β subunit were insensitive to 100 mM Pi. Next we examined the effect of Pi on nucleotide binding to α 3 β 3 γ subcomplex. In order to prevent ATP hydrolysis during experiment, we first used β(E190Q/Y341W) mutant for nucleotide binding to subcomplex. One hundred mM Pi partially released pre-loaded ADP but not bound ATP from α 3 β(E190Q/Y341W) 3 γ subcomplex. Next, we repeated the same experiment using α 3 β(Y341W) 3 γ. In this case, 100mM Pi partially released the bound nucleotide irrespective of ATP or ADP was pre-loaded. Kinesin Eg5 is essential for bipolar spindle formation during eukaryotic cell division. Previously, we prepared mutants of mitotic kinesin Eg5, which have a single cysteine in the functional region and modified with photochromic molecules, iodoacetyl-spiropyran and 4-phenylazomaleinanil. ATPase activities of the modified Eg5 mutants W127C and D130C were photo-reversibly regulated by light irradiations. I this study, we synthesized novel SH group crosslinkable photochromic molecules, iodo-fulgide (IAFG) and iodo-trityl azobenzene (IATAB) and tried to control function of Eg5. IAFI and IATAB were incorporated into the Eg5 mutants, E116C, E118C, Y125C, W127C and D130C. Photocontrol of ATPase and motility activities of the modified Eg5 were investigated. 3P156ヒト細胞質ダイニンのパワーストローク測定
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