Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II.

We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeast Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro.

Structure and Regulation of the Movement of Human Myosin VIIA.

Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail).

Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR.

In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening.

Biochemical characterization of the novel rice kinesin K23 and its kinetic study using fluorescence resonance energy transfer between an intrinsic tryptophan residue and a fluorescent ATP analogue.

We previously demonstrated that the rice kinesin K16, which belongs to the kinesin-7 subfamily, has unique enzymatic properties and atomic structure within key functional regions. In this study, we focused on a novel rice plant kinesin, K23, which also belongs to the kinesin-7 subfamily. The biochemical characterization of the K23 motor domain (K23MD) was studied and compared with the rice kinesin K16 and other related kinesins.

Photocontrol of calmodulin interaction with target peptides using azobenzene derivative.

Calmodulin (CaM), a physiologically important Ca(2+)-binding protein, participates in numerous cellular regulatory processes. It is dumbbell shaped and contains two globular domains connected by a short alpha-helix. Each of the globular domains has two Ca(2+)-binding sites, the EF hands.

Conformational dynamics of loops L11 and L12 of kinesin as revealed by spin-labeling EPR.

The EPR spectra of the spin labels attached to loops L11 and L12 of kinesin were resolved into slow (rotational correlation time, tau=10-45 ns) and fast (tau=2 ns) components. The fraction of the slow component increased considerably when kinesin was complexed with a microtubule (MT). On MT binding and in the presence of nucleotides ADP and AMPPNP, the spin labels on L11, particularly at A252C and L249C, significantly decreased the fraction of the slow component.

Photocontrol of kinesin ATPase activity using an azobenzene derivative.

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin to reversibly regulate the ATPase activity of kinesin by photoirradiation. We prepared five kinesin motor domain mutants, A247C, L249C, A252C, G272C and S275C, which contained a single reactive cysteine residue in loops L11 and L12.