Modification of Loop 1 Affects the Nucleotide Binding Properties of Myo1c, the Adaptation Motor in the Inner Ear

Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotide- and actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, we expressed in insect cells mutants of a truncated form of Myo1c, Myo1c1IQ, as well as chimeras of Myo1c1IQ with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties, or Ca2+ sensitivity of Myo1c1IQ. Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c1IQ-tonic) or replacement with a single glycine (Myo1c1IQ-G) accelerated the release of ADP from A.M 2?3-fold in Ca2+, whereas substitution with loop 1 from phasic muscle myosin II (Myo1c1IQ-phasic) accelerated the release of ADP 35-fold. Motility assays with chimeras containing a single ?-helix, or SAH, domain showed that Myo1cSAH-tonic translocated actin in vitro twice as fast as Myo1cSAH-WT and 3-fold faster than Myo1cSAH-G. The studies show that changes induced in Myo1c via modification of loop 1 showed no resemblance to the behavior of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras

A micro-volume adaptation of a stopped-flow system; use with μg quantities of muscle proteins

Stopped-flow spectroscopy is a powerful method for measuring very fast biological and chemical reactions. The technique however is often limited by the volumes of reactants needed to load the system. Here we present a simple adaptation of commercial stopped-flow system that reduces the volume needed by a factor of 4 to ≈120 μl. After evaluation the volume requirements of the system we show that many standard myosin based assays can be performed using <100 μg of myosin. This adaptation both reduces the volume and therefore mass of protein required and also produces data of similar quality to that produced using the standard set up. The 100 μg of myosin required for these assays is less than that which can be isolated from 100 mg of muscle tissue. With this reduced quantity of myosin, assays using biopsy samples become possible. This will allow assays to be used to assist diagnoses, to examine the effects of post translational modifications on muscle proteins and to test potential therapeutic drugs using patient derived samples

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch, slow-twitch, and cardiac muscle

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ~0.05 s-1) and super-relaxed states (SRX, 0.005 s-1) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J. 100, 1969–1976). Studies have normally used purified proteins, myosin filaments or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording time scales from 0.1 – 1000 sec provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labelled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C – 30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared to myofibrils from fast twitch muscle, slow-twitch and cardiac muscles myofibrils require a 10-fold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human derived iPSCs

Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into “ghost muscle fibers”. The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1

The Mechanism of Thin Filament Regulation: Models in Conflict?

In a recent article in this journal, Heeley and colleagues (Heeley, White, and Taylor 2019 J Gen Physiol 151, 628-634) reopened the debate about 2 vs 3 state models of thin filament regulation. The authors review their work, which measures the rate constant of Pi release from myosin.ADP.Pi activated by actin or thin filaments under a variety of conditions. They conclude that their data can be described by a 2-state model and raise doubts about the generally accepted 3-state model as originally formulated by McKillop and Geeves (Biophysical Journal 65: 693–701, 1993). However, in the following article, we follow Plato’s dictum that “twice and thrice over, as they say, good it is to repeat and review what is good”. We have therefore reviewed the evidence for the 3- and 2-state models and present our view that the evidence is overwhelmingly in favor of three structural states of the thin filament, which regulate access of myosin to its binding sites on actin and, hence, muscle contractility

Functional and biophysical analyses of the class XIV Toxoplasma gondii Myosin D

Summary: The obligate intracellular parasite Toxoplasma gondii uses gliding motility to migrate across the biological barriers of the host and to invade cells. This unique form of locomotion requires an intact actin cytoskeleton and involves at least one motor protein (TgMyoA) that belongs to the class XIV of the myosin superfamily. TgMyoA is anchored in the inner membrane complex and is essential for the gliding motion, host cell invasion and egress of T. gondii tachyzoites. TgMyoD is the smallest T. gondii myosin and is structurally very closely related to TgMyoA. We show here that TgMyoD exhibits similar transient kinetic properties as the fast single-headed TgMyoA. To determine if TgMyoD also contributes to parasite gliding motility, the TgMyoD gene was disrupted by double homologous recombination. In contrast to TgMyoA, TgMyoD gene is dispensable for tachyzoite propagation and motility. Parasites lacking TgMyoD glide normally and their virulence is not compromised in mice. The fact that TgMyoD is predominantly expressed in bradyzoites explains the absence of a phenotype observed with myodko in tachyzoites and does not exclude a role of this motor in gliding that would be restricted to the cyst forming but nevertheless motile stage of the parasit