Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, MD simulations, and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events, phosphoryl transfer (>105-fold) and lid-opening (103-fold). In contrast, mutation of an essential active-site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid-opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a pre-organized active site.

Stem cell factor (SCF) gene expression is regulated by FSH in testicular Sertoli cells. Many functions of FSH are mediated through the second messenger cAMP. We show that cAMP activates transcription of the human SCF promoter in a Sertoli cell line. The human SCF promoter was cloned in cosmid vector pWE15, and its DNA sequence was determined for the promoter region extending 2.3 kilobase pairs upstream from the translation start site at +184 bp. The in vivo messenger RNA (mRNA) start site, by primer-extension studies, was located in exon 1 at +109 bp in human testis mRNA, and at +99 bp in mouse SF7 Sertoli cell line or GC1 germ cell line mRNA. To test which regions of the SCF promoter are necessary for regulation by cAMP, a series of 5'-end deletions of this region were cloned onto the luciferase reporter gene in plasmid pXP1. The SCF promoter region was fused to luciferase downstream (at +120) from its +109 mRNA start site, extending upstream a variable distance to BstXI (-162), BamHI (-313), Bgl2 (-853), or XbaI (-2185). The shortest of these fragments extending only to -162 bp, contains possible SP1 and AP-2 elements. When mouse Sertoli SF7 or human JEG.3 cell lines were transfected with these plasmids, all of the mutants were regulated by 8Br-cAMP or forskolin, as expected for the SCF gene, whereas FSH and TPA had no effect. In the shortest promoter deletion -162, luciferase expression from SF7 cells in serum-free media was at a moderate basal level, but it was induced in six h about 2-fold by 8Br-cAMP, and over 7-fold by forskolin (an adenylate cyclase activator) to high levels, similar to the SV40 positive control promoter. In SCF-luc plasmids extending to -853 or -2185, luciferase expression was still inducible by 8Br-cAMP and forskolin to high levels, but basal promoter activity was repressed to levels over 15-fold lower, in both the absence or presence of testosterone in the media for SF7 cells. The distal portion of the human SCF promoter (between -313 and -853, and also -853 and -2185) inhibits the basal level of transcription, while the proximal region (5' of -162) can mediate activation by cAMP.

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 (age-associated loss of muscle strength independent of atrophy). 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.

NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.

The recent discovery that myosin has two distinct states in relaxed muscledisordered 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.

Actin’s interactions with myosin and other actin-binding proteins is essential for cellular viability in numerous cell types, including muscle. In a previous high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) screen, we identified a class of compounds that bind to actin and affect actomyosin structure and function. For clinical utility, it is highly desirable to identify compounds that affect skeletal and cardiac muscle differently. Because actin is more highly conserved than myosin and most other muscle proteins, most such efforts have not targeted actin. Nevertheless, in the current study, we tested the specificity of the previously discovered actin-binding compounds for effects on skeletal and cardiac α-actins as well as on skeletal and cardiac myofibrils. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that several of these effects were different for skeletal and cardiac actin isoforms. We also found that several of these compounds affected ATPase activity differently in skeletal and cardiac myofibrils. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

The myosin super-relaxed state (SRX) in skeletal muscle is hypothesized to play an important role in regulating muscle contractility and thermogenesis in humans, but has only been examined in model organisms. Here we report the first human skeletal muscle SRX measurements, using quantitative epifluorescence microscopy of fluorescent 2'/3'-O-(N-methylanthraniloyl) ATP (mantATP) single-nucleotide turnover. Myosin heavy chain (MHC) isoform expression was determined using gel electrophoresis for each permeabilized vastus lateralis fiber, to allow for novel comparisons of SRX between fiber-types. We find that the fraction of myosin in SRX is less in MHC IIA fibers than in MHC I and IIAX fibers (p = 0.008). ATP turnover of SRX is faster in MHC IIAX fibers compared to MHC I and IIA fibers (p = 0.001). We conclude that SRX biochemistry is measurable in human skeletal muscle, and our data indicate that SRX depends on fiber type as classified by MHC isoform. Extension from this preliminary work would provide further understanding regarding the role of SRX in human muscle physiology.

We have used spectroscopic and functional assays to evaluate the effects of a group of actin-binding compounds on striated muscle protein structure and function. Actin is present in every human cell, and its interaction with multiple myosin isoforms and multiple actin-binding proteins is essential for cellular viability. A previous highthroughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay from our group identified a class of compounds that bind to actin and affect actomyosin structure and function. In the current study, we tested their effects on the two isoforms of striated muscle α-actins, skeletal and cardiac. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that these effects were different for the two actin isoforms, suggesting a different mode of action. To determine the effects of these compounds on sarcomeric function, we further tested their activity on skeletal and cardiac myofibrils. We found that several compounds affected ATPase activity of skeletal and cardiac myofibrils differently, suggesting different mechanisms of action of these compounds for the two muscle types. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.