Biology subjects

Thomas, D. D.

Publications and source records attributed to Thomas, D. D..

3 recordsLinked to original sources

bioRxiv2018

Age affects myosin relaxation states in skeletal muscle fibers of female but not male mice

The recent discovery that myosin has two distinct states in relaxed muscle - disordered relaxed (DRX) and super-relaxed (SRX) - provides another factor to consider in our fundamental understanding of the aging mechanism in skeletal muscle, since myosin is thought to be a potential contributor to dynapenia. The primary goal of this study was to determine the effects of age on DRX and SRX states and to examine their sex specificity. We have used quantitative fluorescence microscopy of the fluorescent nucleotide analog 2'/3'-O-(N-methylanthraniloyl) ATP (mantATP) to measure single-nucleotide turnover kinetics of myosin in skinned skeletal muscle fibers under relaxing conditions. We examined changes in DRX and SRX in response to the natural aging process by measuring the turnover of mantATP in skinned fibers isolated from psoas muscle of adult young (3-4 months old) and aged (26-28 months old) C57BL/6 female and male mice. Fluorescence decays were fitted to a multi-exponential decay function to determine both the time constants and mole fractions of fast and slow turnover populations, and significance was analyzed by a t-test. We found that in females, both the DRX and SRX lifetimes of myosin ATP turnover at steady state were shorter in aged muscle fibers compared to young muscle fibers (p[≤]0.033). However, there was no significant difference in relaxation lifetime of either DRX (p=0.202) or SRX (p=0.804) between young and aged male mice. No significant effects were measured on the mole fractions (populations) of these states, as a function of sex or age (females, p=0.100; males, p=0.929). The effect of age on the order of myosin heads at rest and their ATPase function is sex specific, affecting only females. These findings provide new insight into the molecular factors and mechanisms that contribute to aging muscle dysfunction in a sex-specific manner.

biochemistry↗

bioRxiv2018

Mavacamten stabilizes the auto-inhibited state of two-headed cardiac myosin

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an auto-inhibited state of two-headed cardiac myosin, not found in the isolated S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single ATP turnover kinetics. A two-headed myosin fragment exhibits distinct auto-inhibited ATP turnover kinetics compared to a single-headed fragment. Mavacamten enhanced this auto-inhibition. It also enhanced auto-inhibition of ADP release. Furthermore, actin changes the structure of the auto-inhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.\n\nSignificance StatementSmall-molecule allosteric effectors designed to target and modulate striated and smooth myosin isoforms for the treatment of disease show promise in preclinical and clinical trials. Beta-cardiac myosin is an especially important target, as heart disease remains a primary cause of death in the U.S. One prevalent type of heart disease is hypertrophic cardiomyopathy (HCM), which is hypothesized to result from dysregulated force generation by cardiac myosin. Mavacamten is a potent cardiac myosin ATPase activity inhibitor that improves cardiac output in HCM animal models. Our results show that mavacamten selectively stabilizes a two-head dependent, auto-inhibited state of cardiac myosin in solution. The kinetics and energetics of this state are consistent with the auto-inhibited super-relaxed state, previously only observed in intact sarcomeres.

biophysics↗

bioRxiv2017

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation

Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100-fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation profoundly alters the structure and dynamics of the DFG-In subpopulation, leading to activation of the kinase. Kinetics experiments tracking structural transitions during nucleotide binding suggest that a substantial DFG-Out subpopulation is an important feature of activated AurA that evolved to optimize the kinetics of substrate binding and product release.

biophysics↗