The ability to sense molecular tension is crucial for a wide array of cellular processes, including the detection auditory stimuli, control cell shape, and internalization transport membranes. We show that myosin I, motor protein has been implicated in powering key steps these dramatically alters its motile properties response tension. measured displacement generated by single I molecules, we determined actin-attachment kinetics with varying tensions using an optical trap. rate detachment...

Cell movement and cytokinesis are facilitated by contractile forces generated the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups N-terminal motor domains on opposite sides. The motors then bind pull actin filaments toward NMII-F, thus driving contraction. Inside crawling cells, NMIIA-Fs large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we...

To understand the mechanism underlying ability of individual AdiC molecules to transport arginine and agmatine, we used a recently developed high-resolution single-molecule fluorescence-polarization microscopy method investigate conformation-specific changes in emission polarization bifunctional fluorophore attached an molecule. With this capability, resolved AdiC’s four conformations characterized by distinct spatial orientations absence or presence two substrates, furthermore, each...

Tertiapin-Q (TPNQ) is a derivative of honey bee toxin tertiapin (TPN) whose methionine residue replaced with glutamine residue. TPNQ inhibits the ROMK1 and GIRK1/4 inward-rectifier K+ channels affinities very similar to TPN. However, unlike native TPN, nonoxidizable by air. The stability allows us investigate how it interacts targeted channels. We found that interaction between channel bimolecular reaction, i.e., one molecule binds channel. surface in primarily formed its α helix rather than...

Myosin-Is are molecular motors that link cellular membranes to the actin cytoskeleton, where they play roles in mechano-signal transduction and membrane trafficking. Some myosin-Is proposed act as force sensors, dynamically modulating their motile properties response changes tension. In this study, we examined sensing by widely expressed myosin-I isoform, myo1b, which is alternatively spliced its light chain binding domain (LCBD), yielding proteins with lever arms of different lengths. We...

Myo1c is an unconventional myosin involved in cell signaling and membrane dynamics. Calcium binding to the regulatory-domain-associated calmodulin affects myo1c motor properties, but kinetic details of this regulation are not fully understood. We performed actin gliding assays, ATPase measurements, fluorescence spectroscopy, stopped-flow kinetics determine biochemical parameters that define calmodulin-regulatory-domain interaction. found calcium moderately increases actin-activated activity...

Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes thought to play roles in membrane trafficking dynamics. It one of the best characterized isoforms appears have unique biochemical properties tuned for tension sensing or maintenance. We determined key rate constants define actomyo1b ATPase cycle at 37 °C measured temperature dependence ATP binding, ADP release, transition from nucleotide-inaccessible state nucleotide-accessible (kα). The...

Myosin V (myoV) is a two-headed myosin capable of taking many successive steps along actin per diffusional encounter, enabling it to transport vesicular and ribonucleoprotein cargos in the dense complex environment within cells. To better understand how myoV navigates actin, we used polarized total internal reflection fluorescence microscopy examine angular changes bifunctional rhodamine probes on lever arms single molecules vitro. With newly developed analysis technique, rotational motions...

Lq2 is a unique scorpion toxin. Acting from the extracellular side, blocks ion conduction pore in not only voltage- and Ca2+-activated channels, but also inward-rectifier K+ channels. This finding argues that three-dimensional structures of pores these channels are similar. However, amino acid sequences form external part minimally conserved among various classes Because can bind to all three we use as structural probe examine how non-conserved pore-forming arranged space similar structures....

Most membrane protein molecules undergo conformational changes as they transition from one functional state to another one. An understanding of the mechanism underlying these requires ability resolve individual states, whose often occur on millisecond and angstrom scales. Tracking such acquiring a sufficiently large amount data remain challenging. Here, we use amino-acid transporter AdiC an example demonstrate application high-resolution fluorescence-polarization-microscopy method in...