2P169 Single-molecule imaging analysis of cellobiohydrolase hydrolyzing crystalline cellulose(11. Molecular motor,Poster)

Abstract

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...