Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue.

The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define "fatigability" in vivo of fast vs. slow myosin isoforms, at the molecular level, are not yet fully understood.

Use of pyrene-labelled actin to probe actin-myosin interactions: kinetic and equilibrium studies.

Studying the dynamics of the interaction between actin and myosin and how this is modulated by ATP and other nucleotides is fundamental to any understanding of myosin motor protein activity. The fluorescent label pyrene, covalently attached to actin (at Cys 374), has been one of the most useful optical probes to report myosin binding to actin. The unique spectral features of pyrene make it sensitive to changes in the microenvironment of the probe and allow to monitor processes such as conformational changes and protein-protein interactions.

Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP, contractility, and relaxation.

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca(2+)-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca(2+) fluorescence were monitored by video microscopy.

Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V.

The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements.

Myo1c mutations associated with hearing loss cause defects in the interaction with nucleotide and actin.

Three heterozygous missense mutations in the motor domain of myosin 1c (Myo1c), which mediates adaptation in the inner ear, are associated with bilateral sensorineural hearing loss in humans. With transient kinetic analyses, steady-state ATPase and motility assays, and homology modeling, we studied the interaction of these mutants with nucleotide and actin using a truncated construct, Myo1c(1IQ-SAH), which includes an artificial lever arm. Results indicate that mutation R156W, near switch 1, affects the nucleotide-binding pocket and the calcium binding by disrupting switch 1 movement.

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.

Direct kinetic evidence that lysine 215 is involved in the phospho-transfer step of human 3-phosphoglycerate kinase.

3-Phosphoglycerate kinase (PGK) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. PGK is a key enzyme in glycolysis; it catalyzes the reversible reaction 1,3-bisphosphoglycerate + ADP <--> 3-phosphoglycerate + ATP. Here we explored the catalytic role in human PGK of the highly conserved Lys 215 that has been proposed to be essential for PGK function by a transient and equilibrium kinetic study with the active site mutant K215A.

Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear.

The class I myosin Myo1c is a mediator of adaptation of mechanoelectrical transduction in the stereocilia of the inner ear. Adaptation, which is strongly affected by Ca(2+), permits hair cells under prolonged stimuli to remain sensitive to new stimuli. Using a Myo1c fragment (motor domain and one IQ domain with associated calmodulin), with biochemical and kinetic properties similar to those of the native molecule, we have performed a thorough analysis of the biochemical cross-bridge cycle.

The slow skeletal muscle isoform of myosin shows kinetic features common to smooth and non-muscle myosins.

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform.

What limits the velocity of fast-skeletal muscle contraction in mammals?

In rat skeletal muscle the unloaded shortening velocity (Vo) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit Vo. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.

Kinetic mechanism of myosin IXB and the contributions of two class IX-specific regions.

Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted.