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Christine R. Cremo - One of the best experts on this subject based on the ideXlab platform.
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Both heads of tissue-derived smooth muscle Heavy Meromyosin bind to actin in the presence of ADP.
Journal of Biological Chemistry, 2002Co-Authors: Patricia Ellison, Zachary S. Depew, Christine R. CremoAbstract:The effect of ADP and phosphorylation upon the actin binding properties of Heavy Meromyosin was investigated using three fluorescence methods that monitor the number of Heavy Meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-Heavy Meromyosin complex, and (iii) amplitudes of the association of Heavy Meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated Heavy Meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% Heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of Heavy Meromyosin with uncleaved Heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240-23245), which showed that one head of the unphosphorylated Heavy Meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated Heavy Meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-Heavy Meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states.
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Structural model of the regulatory domain of smooth muscle Heavy Meromyosin.
Journal of Biological Chemistry, 2002Co-Authors: Jan L. Wahlstrom, M. Allen Randall, J. David Lawson, Derek E. Lyons, William F. Siems, Greg J. Crouch, Regina Barr, Kevin C. Facemyer, Christine R. CremoAbstract:The goal of this study was to provide structural information about the regulatory domains of double-headed smooth muscle Heavy Meromyosin, including the N terminus of the regulatory light chain, in both the phosphorylated and unphosphorylated states. We extended our previous photo-cross-linking studies (Wu, X., Clack, B. A., Zhi, G., Stull, J. T., and Cremo, C. R. (1999) J. Biol. Chem. 274, 20328-20335) to determine regions of the regulatory light chain that are cross-linked by a cross-linker attached to Cys(108) on the partner regulatory light chain. For this purpose, we have synthesized two new biotinylated sulfhydryl reactive photo-cross-linking reagents, benzophenone, 4-(N-iodoacetamido)-4'-(N-biotinylamido) and benzophenone, 4-(N-maleimido)-4'-(N-biotinylamido). Cross-linked peptides were purified by avidin affinity chromatography and characterized by Edman sequencing and mass spectrometry. Labeled Cys(108) from one regulatory light chain cross-linked to (71)GMMSEAPGPIN(81), a loop in the N-terminal half of the regulatory light chain, and to (4)RAKAKTTKKRPQR(16), a region for which there is no atomic resolution data. Both cross-links were to the partner regulatory light chain and occurred in unphosphorylated but not phosphorylated Heavy Meromyosin. Using these data, data from our previous study, and atomic coordinates from various myosin isoforms, we have constructed a structural model of the regulatory domain in an unphosphorylated double-headed molecule that predicts the general location of the N terminus. The implications for the structural basis of the phosphorylation-mediated regulatory mechanism are discussed.
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Kinetics of Smooth Muscle Heavy Meromyosin with One Thiophosphorylated Head
Journal of Biological Chemistry, 2000Co-Authors: Patricia A. Ellison, James R. Sellers, Christine R. CremoAbstract:Actin-activated MgATPase of smooth muscle Heavy Meromyosin is activated by thiophosphorylation of two regulatory light chains, one on each head domain. To understand cooperativity between heads, we examined the kinetics of Heavy Meromyosin (HMM) with one thiophosphorylated head. Proteolytic gizzard Heavy Meromyosin regulatory light chains were partially exchanged with recombinant thiophosphorylated His-tagged light chains, and HMM with one thiophosphorylated head was isolated by nickel-affinity chromatography. In vitro motility was observed. By steady-state kinetic analysis, one-head thiophosphorylated Heavy Meromyosin had a similar K(m) value for actin but a V(max) value of approximately 50% of the fully thiophosphorylated molecule. However, single turnover analysis, which is not sensitive to small amounts of active heads, showed that one-head thiophosphorylated Heavy Meromyosin was 46-120 times more active than unphosphorylated HMM but only 7-19% as active as the fully thiophosphorylated molecule. Discrepancy between the single turnover and steady-state values could be explained by a small fraction of rigor heads. These rigor heads would have a large effect on the steady-state kinetics of one-head thiophosphorylated HMM. In summary, thiophosphorylation of one head leads to a molecule with unique intermediate kinetics suggesting that thiophosphorylation of one head cooperatively alters the kinetics of the partner head and vice versa.
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Phosphorylation-dependent structural changes in the regulatory light chain domain of smooth muscle Heavy Meromyosin.
Journal of Biological Chemistry, 1999Co-Authors: Beatrice A. Clack, Gang Zhi, James T. Stull, Christine R. CremoAbstract:Abstract Smooth muscle Heavy Meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation-dependent structural changes near the head-tail junction, we prepared five well regulated Heavy Meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross-linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated Heavy Meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 A) the regulatory light chain of the partner head and that thiophosphorylation abolishes proximity. This behavior was also observed for the Q15C mutant on a truncated Heavy Meromyosin lacking both catalytic domains. For the actin-Heavy Meromyosin complex, cross-links were formed in both de- and thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated Heavy Meromyosin, suggesting positions outside the region of interaction of the regulatory light chains.
James R. Sellers - One of the best experts on this subject based on the ideXlab platform.
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two headed binding of the unphosphorylated nonmuscle Heavy Meromyosin adp complex to actin
Biochemistry, 2004Co-Authors: Mihaly Kovacs, Judit Toth, Laszlo Nyitray, James R. SellersAbstract:The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA Heavy Meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated Heavy Meromyosin bind very strongly to actin (Kd < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (Kd around 1 μM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myo...
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Kinetics of Smooth Muscle Heavy Meromyosin with One Thiophosphorylated Head
Journal of Biological Chemistry, 2000Co-Authors: Patricia A. Ellison, James R. Sellers, Christine R. CremoAbstract:Actin-activated MgATPase of smooth muscle Heavy Meromyosin is activated by thiophosphorylation of two regulatory light chains, one on each head domain. To understand cooperativity between heads, we examined the kinetics of Heavy Meromyosin (HMM) with one thiophosphorylated head. Proteolytic gizzard Heavy Meromyosin regulatory light chains were partially exchanged with recombinant thiophosphorylated His-tagged light chains, and HMM with one thiophosphorylated head was isolated by nickel-affinity chromatography. In vitro motility was observed. By steady-state kinetic analysis, one-head thiophosphorylated Heavy Meromyosin had a similar K(m) value for actin but a V(max) value of approximately 50% of the fully thiophosphorylated molecule. However, single turnover analysis, which is not sensitive to small amounts of active heads, showed that one-head thiophosphorylated Heavy Meromyosin was 46-120 times more active than unphosphorylated HMM but only 7-19% as active as the fully thiophosphorylated molecule. Discrepancy between the single turnover and steady-state values could be explained by a small fraction of rigor heads. These rigor heads would have a large effect on the steady-state kinetics of one-head thiophosphorylated HMM. In summary, thiophosphorylation of one head leads to a molecule with unique intermediate kinetics suggesting that thiophosphorylation of one head cooperatively alters the kinetics of the partner head and vice versa.
Jerry H. Brown - One of the best experts on this subject based on the ideXlab platform.
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X-Ray Solution Scattering of Squid Heavy Meromyosin: Strengthening the Evidence for an Ancient Compact off State
2016Co-Authors: Richard E. Gillilan, V. S. Senthil Kumar, Carolyn Cohen, Jerry H. BrownAbstract:The overall conformations of regulated myosins or Heavy Meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the ‘‘off’ ’ state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the ‘‘on’ ’ state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on Heavy Meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca2+ the radius of gyration increases. Differences in the squid ‘‘on’ ’ and ‘‘off’ ’ states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca2+-free squid Heavy Meromyosin that is compact, but which becomes extended when Ca2+ is bound. Further, the scattering profile derived from the current model of tarantula Heavy Meromyosin in the ‘‘off’ ’ state is in excellent agreement with the measured ‘‘off’ ’ state scattering profile for squid Heavy Meromyosin. The previous and current studies together provide significant evidence that regulated myosin’s compact off-state conformation is an ancient trait, inherited from
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X-ray solution scattering of squid Heavy Meromyosin: strengthening the evidence for an ancient compact off state.
PLOS ONE, 2013Co-Authors: Richard E. Gillilan, V. S. Senthil Kumar, Elizabeth O'neall-hennessey, Carolyn Cohen, Jerry H. BrownAbstract:The overall conformations of regulated myosins or Heavy Meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the “off” state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the “on” state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on Heavy Meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca2+ the radius of gyration increases. Differences in the squid “on” and “off” states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca2+-free squid Heavy Meromyosin that is compact, but which becomes extended when Ca2+ is bound. Further, the scattering profile derived from the current model of tarantula Heavy Meromyosin in the “off” state is in excellent agreement with the measured “off” state scattering profile for squid Heavy Meromyosin. The previous and current studies together provide significant evidence that regulated myosin's compact off-state conformation is an ancient trait, inherited from a common ancestor during divergent evolution.
Richard E. Gillilan - One of the best experts on this subject based on the ideXlab platform.
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X-Ray Solution Scattering of Squid Heavy Meromyosin: Strengthening the Evidence for an Ancient Compact off State
2016Co-Authors: Richard E. Gillilan, V. S. Senthil Kumar, Carolyn Cohen, Jerry H. BrownAbstract:The overall conformations of regulated myosins or Heavy Meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the ‘‘off’ ’ state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the ‘‘on’ ’ state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on Heavy Meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca2+ the radius of gyration increases. Differences in the squid ‘‘on’ ’ and ‘‘off’ ’ states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca2+-free squid Heavy Meromyosin that is compact, but which becomes extended when Ca2+ is bound. Further, the scattering profile derived from the current model of tarantula Heavy Meromyosin in the ‘‘off’ ’ state is in excellent agreement with the measured ‘‘off’ ’ state scattering profile for squid Heavy Meromyosin. The previous and current studies together provide significant evidence that regulated myosin’s compact off-state conformation is an ancient trait, inherited from
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X-ray solution scattering of squid Heavy Meromyosin: strengthening the evidence for an ancient compact off state.
PLOS ONE, 2013Co-Authors: Richard E. Gillilan, V. S. Senthil Kumar, Elizabeth O'neall-hennessey, Carolyn Cohen, Jerry H. BrownAbstract:The overall conformations of regulated myosins or Heavy Meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the “off” state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the “on” state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on Heavy Meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca2+ the radius of gyration increases. Differences in the squid “on” and “off” states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca2+-free squid Heavy Meromyosin that is compact, but which becomes extended when Ca2+ is bound. Further, the scattering profile derived from the current model of tarantula Heavy Meromyosin in the “off” state is in excellent agreement with the measured “off” state scattering profile for squid Heavy Meromyosin. The previous and current studies together provide significant evidence that regulated myosin's compact off-state conformation is an ancient trait, inherited from a common ancestor during divergent evolution.
Mihaly Kovacs - One of the best experts on this subject based on the ideXlab platform.
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two headed binding of the unphosphorylated nonmuscle Heavy Meromyosin adp complex to actin
Biochemistry, 2004Co-Authors: Mihaly Kovacs, Judit Toth, Laszlo Nyitray, James R. SellersAbstract:The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA Heavy Meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated Heavy Meromyosin bind very strongly to actin (Kd < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (Kd around 1 μM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myo...