A Homobifunctional Rhodamine for Labeling Proteins with Defined Orientations of a Fluorophore

Corrie, John E. T.; Craik, J. S.; Munasinghe, V. Ranjit N.

doi:10.1021/bc970174e

Cited by 65 publications

(47 citation statements)

References 20 publications

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“…Inhomogenities were minimized by focusing the exciting laser light on a single half-sarcomere. The dye was immobilized during critical transient states of the contraction cycle, in spite of the fact that it was attached to LC1 at one point only, rather than two points, which is more stable (9). By using the mesoscopic approach, we were able to measure the average kinetics of ϳ20 XBs.…”

Section: Discussionmentioning

confidence: 99%

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions

Duggal

Nagwekar

Rich

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

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J. Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions. Am J Physiol Heart Circ Physiol 308: H1248 -H1257, 2015. First published March 13, 2015; doi:10.1152/ajpheart.00834.2014 is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ϳ20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contractionrelaxation cycle may contribute to altered contractility and the development of FHC. left ventricle; mutation of regulatory light chain; polarization of fluorescence INTERACTIONS OF MYOSIN WITH actin and associated proteins (sarcomeric proteins) are responsible for muscle contraction (19,24,48). These interactions include the mode of attachment of actin to myosin during different physiological states and the kinetics of conformational changes of myosin when interacting with actin. Familial hypertrophic cardiomyopathy (FHC) has been recognized to be caused by mutations in all major sarcomeric proteins of the heart, including the myosin regulatory light chain (RLC) (1). Compared with the myosin heavy chain, mutations in the RLC are rare, but often associated with malignant outcomes (18,27,41). It is therefore important to find out whether these FHC-linked mutations result in alterations of the mode of attachment of myosin to actin, and/or whether they lead to alterations of kinetics of acto-myosin interactions. These changes may ultimately contribute to the development of FHC. In this report we have focused on the K104E (lysine to glutamic acid) substitution in the myosin RLC expressed in the heart of transgenic (Tg) mice (25). The mutation was identified in a Danish population by Andersen et al. (2) and was associated with left ventricu...

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Section: Discussionmentioning

confidence: 99%

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions

Duggal

Nagwekar

Rich

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

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“…Forty percent of the native TnC in muscle fibers was replaced by one of the mutant TnCs labelled with BR (43). Polarized fluorescence intensities were recorded using a new setup (Fig.…”

Section: Methodsmentioning

confidence: 99%

Structural dynamics of troponin during activation of skeletal muscle

Fusi

Brunello

Sevrieva

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s −1 ) matches the rate of Ca 2+ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s −1 ) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s −1 ) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions. muscle regulation | excitation-contraction coupling | muscle signaling C ontraction of skeletal and cardiac muscle is initiated by a transient increase in the concentration of intracellular Ca 2+ ions, which bind to troponin in the thin filaments of the muscle sarcomere. This leads to azimuthal movement of tropomyosin around the thin filament, which uncovers the myosin binding sites on actin and allows the head domain of myosin from the thick filaments to bind to actin and generate force (1, 2). In vitro studies using isolated protein components showed that myosin head binding can produce a further motion of tropomyosin, at least in low [ATP] or rigor-like conditions (2-4), but the functional significance of this effect in physiological conditions and intact sarcomeres is not clear.To elucidate the molecular structural basis of muscle regulation and the role of myosin binding in situ, we introduced bifunctional fluorescent probes into the calcium-binding subunit of troponin, troponin C (TnC) (Fig. 1, yellow), in demembranated fibers from skeletal muscle (5-7). One probe cross-linked a pair of cysteines introduced into the C helix of TnC (Fig. 1, green), close to the regulatory Ca 2+ binding sites (Fig. 1, black spheres) in its N-terminal lobe, and reports the rotation and opening of this lobe on binding Ca 2+ (5). The N-lobe opening is associated with binding of the switch peptide of troponin I (TnI) (Fig. 1, blue) to a hydrophobic pocket on its surface, and this is a key step in the signaling pathway of calcium regulation (8, 9).A second probe was attached to the E helix of TnC (Fig. 1, magenta) in its C-terminal lobe, which contains a pair of divalent cation binding sites (Fig. 1, gray spheres) that can bind Mg 2+ as well as Ca 2+ . The C lobe of TnC is clasped between two long helices of TnI, one of which forms a coiled coil with part of the tropomyosin-binding component of troponin, troponin T (TnT) (Fig. 1, orange). The C lobe of TnC and these long TnI and TnT helices form a well-defined structural domain called the "IT arm" (9, 10). Although the C-lobe E helix of TnC is continuous with the N-lob...

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“…Fluorogenic Probe in LGX test (1) Rhodamine 110 (2) represents one of the most active fluorophores known, with a high extinction coefficient and a fluorescence quantum yield which is near unity. Rhodamine has been used as a marker in a wide variety of enyzmic activity studies (serine protease 6 , esterase 7 , caspase 8 , DT diaphorase 9 ). Essentially the NH 2 groups of rhodamine 110 are conjugated to a polypeptide, which mimics the natural substrate of the enzyme in question.…”

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

Synthetic scale-up of a novel fluorescent probe and its biological evaluation for surface detection of Staphylococcus aureus

Bywaters

Mulcahy-Ryan

Fielder

et al. 2017

Molecular and Cellular Probes

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A Homobifunctional Rhodamine for Labeling Proteins with Defined Orientations of a Fluorophore

Cited by 65 publications

References 20 publications

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions

Structural dynamics of troponin during activation of skeletal muscle

Synthetic scale-up of a novel fluorescent probe and its biological evaluation for surface detection of Staphylococcus aureus

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