Modeling thin filament cooperativity.
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Actins↗
Biomedical subjects
S S Lehrer
Publications and source records attributed to S S Lehrer.
Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence.
The regulatory properties of naturally occurring tropomyosins (Tms) of differing lengths have been examined. These Tms span from 4 to 7 actin subunits. Native proteins have been used to study the common 7 actin-spanning skeletal and smooth muscle variants and expressed recombinant proteins to study the shorter fibroblast 5a, 5b, yeast Tm1 and yeast Tm2 Tms (6, 6, 5, and 4 actin-spanning variants, respectively). The yTm2 has been overexpressed in Escherichia coli with N-terminal constructs equivalent to those previously used for yTm1 [Maytum, R., et al. (2000) Biochemistry 39, 11913]. The regulation of myosin subfragment 1 (S1) binding to actin by Tm has been assessed using a sensitive S1 binding titration. The equilibrium between closed and open (C to M states, KT = 0.1-0.14) was similar for all vertebrate Tms. Apart from skTm where the apparent cooperative unit size (n) is the same as the structural size (n = 7 actin sites), the other vertebrate Tms that were studied exhibited large n values (n = 12-14). The yeast Tms also exhibited large values of n (6-9) in comparison to their structural sizes (4-5). The determined value of KT depended on the N-terminal sequence (KT = 0.15-1). These results are compared with the effect of S1 upon Tm's affinity for actin. The yeast Tms have regulatory parameters similar to those of skTm, but unlike skTm, S1 has little effect upon their actin affinity. This shows that an actin state with a high affinity for S1 and Tm is not necessary for regulation, and the higher affinity of S1 for actin in the presence of vertebrate Tms is probably the result of a direct interaction of S1 with Tm.
Actins↗
Local heterogeneity in the pressure denaturation of the coiled-coil tropomyosin because of subdomain folding units.
Coiled-coil domains mediate the oligomerization of many proteins. The assembly of long coiled coils, such as tropomyosin, presupposes the existence of intermediates. These intermediates are not well-known for tropomyosin. Hydrostatic pressure affects the equilibrium between denatured and native forms in the direction of the form that occupies a smaller volume. The hydrophobic core is the region more sensitive to pressure, which leads in most cases to the population of intermediates. Here, we used N-(1-pyrenyl)iodoacetamide covalently bound to cysteine residues of tropomyosin (PIATm) and high hydrostatic pressure to assess the chain interaction and the inherent instability of the coiled-coil molecule. The native and denatured states of tropomyosin were determined from the pyrene excimer fluorescence. The combination of low temperature and high pressure permitted the attainment of the full denaturation of tropomyosin without the separation of the subunits. High-temperature denaturation of Tm leads to a great exchange between labeled and unlabeled Tm subunits, indicating subunit dissociation linked to unfolding. In contrast, under high pressure, unlabeled and labeled tropomyosin molecules do not exchange, demonstrating that the denatured species are dimeric. The decrease of the concentration dependence of PIATm corroborates the idea that pressure produces subdomain denaturation and that the polypeptide chains do not separate. Substantial unfolding of tropomyosin was also verified by measurements of tyrosine fluorescence and bis-ANS binding. Our results indicate the presence of independent folding subdomains with different susceptibilities to pressure along the length of the coiled-coil structure of tropomyosin.
Animals↗
Inhibition of actin-myosin subfragment 1 ATPase activity by troponin I and IC: relationship to the thin filament states of muscle.
Troponin I (TnI) is the component of the troponin complex that inhibits actomyosin ATPase activity, and Ca(2+) binding to the troponin C (TnC) component reverses the inhibition. Effects of the binding of TnI and the TnI-TnC (TnIC) complex to actin-tropomyosin (actinTm) on ATPase and on the binding kinetics of myosin subfragment 1 (S1) were studied to clarify the mechanism of the inhibition. TnI and TnIC in the absence of Ca(2+) bind to actinTm and inhibit ATPase to similar levels with a stoichiometry of one TnI or one TnIC per one Tm and seven actin subunits. TnI also binds to actinTmTn in the presence of Ca(2+) with a stoichiometry and inhibition constant similar to those for the binding to actinTm of TnI and Tn in the absence of Ca(2+). Thus, in the presence of Ca(2+), the intrinsic TnI which is released from its binding site on actinTm does not interfere with the binding of an extra molecule of TnI to actinTmTn. The rate of S1 binding to actinTmTnI and to actinTmTnTnI in the presence of Ca(2+) was inhibited to the same extent as upon removal of Ca(2+) from actinTmTn. These studies show that TnI inhibits ATPase by the same mechanism as Tn in the absence of Ca(2+), by shifting the thin filament equilibria from the open state to the closed and blocked states.
Actin Cytoskeleton↗
Binding of troponin I and the troponin I-troponin C complex to actin-tropomyosin. Dissociation by myosin subfragment 1.
Troponin I (TnI) is the component of the troponin complex, TnI, TnC, TnT, that is responsible for inhibition of actomyosin ATPase activity. Using the fluorescence of pyrene-labeled tropomyosin (Tm), we probed the interaction of TnI and TnIC with Tm on the reconstituted muscle thin filament. The results indicate that TnI and TnIC(-Ca(2+)) bind specifically and strongly to actin-Tm with a stoichiometry of 1 TnI or 1 TnIC/1 Tm/7 actin, in agreement with previous results. The binding of myosin heads (S1) to actin-Tm at low levels of saturation caused TnI and TnIC to dissociate from actin-Tm. These results are interpreted in terms of the S1-binding state allosteric-cooperative model of the actin-Tm thin filament, closed/open. Thus, TnI and TnIC(-Ca(2+)) bind to the closed state of actin-Tm and their binding is greatly weakened in the S1-induced open state, indicating that they act as allosteric inhibitors. The fluorescence change and the stoichiometry indicate that the TnI-binding site is composed of regions from both actin and Tm probably in the vicinity of Cys 190.
Actins↗
Smooth muscle alpha-tropomyosin crosslinks to caldesmon, to actin and to myosin subfragment 1 on the muscle thin filament.
To obtain proximity information between tropomyosin (Tm) and caldesmon (CaD) on the muscle thin filament, we cloned gizzard alphaTm and created two single Cys mutants S56C/C190S (56Tm) and D100C/C190S (100Tm). They were labeled with benzophenone maleimide (BPM) and UV-irradiated on thin filaments. One chain of BPM-56Tm and two chains of BPM-100Tm crosslinked to CaD. Only BPM-100Tm crosslinked to actin in the absence and presence of CaD and binding of low ratios of myosin subfragment 1 (S1) prevented the crosslinking. Tm-S1 crosslinks were produced when actin.Tm was saturated with S1. Thus, CaD on the actin.Tm filament is located <10 A away from Tm amino acids 56 and 100; in the closed state of the actin.Tm filament, Tm residue 100 is located close to the actin surface and is moved further away in the S1-induced open state; in the open state, S1 binds close to Tm.
Actins↗
Cooperativity and switching within the three-state model of muscle regulation.
Thin filament regulation is mediated by the presence of tropomyosin (Tm) and troponin (Tn) on the actin filament. Binding of Tm alone induces two states, closed and open (with the equilibrium between them defined by KT), which differ in their affinity for myosin subfragment 1 (S1). Cooperative switching between the states results in characteristic sigmoidal myosin S1 binding curves. In the presence of Tn and absence of Ca2+, a third state, blocked, has previously been kinetically shown to be present, leading to the three state model of McKillop and Geeves [(1993) Biophys. J. 65, 693-701]. We have measured equilibrium binding of S1 to phalloidin-stabilized pyrene-actin filaments by monitoring the pyrene fluorescence at 50 nM, a concentration 10-fold lower than previously possible. In combination with kinetic studies, we show that the data can be fitted to a modified version of the three-state model with an additional term allowing for a varying apparent cooperative unit size (n). Our results show that the apparent cooperative unit size (n) is dependent upon both the presence of Tn and of Ca2+. Also in the absence of Ca2+, the occupancy of the blocked state (defined by KB) is accompanied by a 2-3-fold reduction in KT. These results are discussed in comparison to the Hill model [(1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190] and a flexible model of thin filament regulation based upon that of Lehrer et al. [(1997) Biochemistry 36, 13449-13455].
Actins↗
The muscle thin filament as a classical cooperative/allosteric regulatory system.
It is generally accepted that the regulation of muscle contraction involves cooperative and allosteric interactions among the protein components, actin, myosin, tropomyosin and troponin. But, as yet, the individual role of each component has not been clearly identified. Here we compare the properties of the components of the muscle regulatory system with the corresponding components of two systems, hemoglobin and aspartate transcarbamylase, that are well described by the classical Monod, Wyman and Changeux (MWC) model. The analogy indicates that actin is the catalytic subunit, tropomyosin is the regulatory subunit and troponin in the absence and presence of Ca2+ is the allosteric inhibitor and activator, respectively. The analogy additionally indicates that the substrate is myosin-ATP (or myosin-ADP-Pi) rather than ATP. Also, in contrast to other MWC systems, the activating ligand for actin-tropomyosin is a myosin-nucleotide intermediate or product that binds tightly to actin, rather than the substrate which binds weakly. This tightly bound intermediate switches the system from the off-state to the on-state (T to R-state in MWC nomenclature) in a concerted transition, affecting n actin subunits, allowing force to be developed.
Actins↗
Unfolding domains and tryptophan accessibility of a 59 kDa coiled-coil light meromyosin.
Light meromyosin (LMM 77), the C-terminal proteolytic peptide from myosin rod, is a 900 A coiled-coil that contains two pairs of tryptophan residues in d-positions of the heptad repeat (abcdefg)n. Previous studies showed that LMM 77 unfolded in two transitions and suggested that both Trp pairs were located in the least stable unfolding domain. Here, the thermal and denaturant unfolding properties of LMM 59, a recombinant N-terminal truncated LMM, containing only one of the Trp pairs, was compared to LMM 77. LMM 59 unfolded in two transitions with similar midpoints to the two transitions of LMM 77. However, only the second transition of LMM 59 affected the Trp fluorescence, indicating that the two pairs of Trp residues in LMM 77 are in different unfolding domains. Disulfide-crosslinked LMM 59 verified this assignment. Solute-quenching studies showed that the accessibility of the Trp in LMM 59 decreased only by 56% on forming filaments. Electron micrographs indicated that all of LMM 59 is located within the core of a bipolar tactoid with the Trp-containing region the most accessible to negative strain, in agreement with the solute-quenching studies. This suggests that part of the core of the myosin thick filament is appreciably exposed to solvent.
Circular Dichroism↗
The stability of tropomyosin at acid pH: effects of anion binding.
The alpha-helical coiled-coil tropomyosin homodimer, alpha alphaTm, unfolds cooperatively with T1/2 = 47 degreesC, at neutral pH, 0.5 M NaCl. At pH 2, where each chain contains 55 positive charges, no cooperative unfolding occurs to at least 80 degreesC. The NaCl and K2SO4 dependence of thermal unfolding of alpha alphaTm and a less stable protein, beta betaTm, were studied with circular dichroism. For alpha alphaTm, 10 mM NaCl, or 0.5 mM K2SO4, was sufficient to increase the unfolding temperature by 80 degreesC. For beta betaTm, similar concentrations of NaCl and K2SO4 as for alpha alphaTm increased the unfolding temperature of the most stable domain. Titrations indicated that two to three anions bind preferentially to the beta betaTm intermediate. Thus anions bind to Tm at acid pH values to greatly stabilize the helix. But even in the absence of added salt, Tm is more stable at pH 2 than pH 7, suggesting destabilization by negatively charged amino acids.
Animals↗
Actin-tropomyosin activation of myosin subfragment 1 ATPase and thin filament cooperativity. The role of tropomyosin flexibility and end-to-end interactions.
Tropomyosin (Tm) bound to actin induces cooperative activation of actomyosin subfragment 1 (actin-S1) ATPase, observed as a sigmoid ATPase vs [S1] dependence. The activation is much steeper for gizzard muscle Tm (GTm) than for rabbit skeletal Tm (RSTm). To investigate if this greater cooperativity is due to increased communication between GTms along the thin filament, we studied effects of S1 binding on the state of actin-Tm using the fluorescence of pyrene-labeled Tm. Kinetic and equilibrium studies provided values for n, the apparent cooperative unit size [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273]. We report comparative studies of Tm-actin-S1 ATPase with values of n using GTm, RSTm, and 5aTm, a 1/7 shorter nonmuscle Tm from rat fibroblast cells [Pittenger, M. F., et al. (1994) Curr. Opin. Cell Biol., 6, 96]. 5aTm and GTm produce similar cooperative activation of actin-S1 ATPase and have similar n values that are 2-fold greater than RSTm, indicating a correlation between ATPase activation and n value. This appears to be due to the similarity of the C-terminal amino acid sequences of 5a and GTm which produce strong end-to-end interactions. The results are discussed in terms of a continuous flexible Tm strand on the actin filament.
Actin Cytoskeleton↗
Effects of two familial hypertrophic cardiomyopathy-causing mutations on alpha-tropomyosin structure and function.
Missense mutations in alpha-tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled tropomyosins differed in their response to myosin S1 binding to the actin-tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.
Actins↗
Intramolecular pyrene excimer fluorescence: a probe of proximity and protein conformational change.
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Animals↗
Ca2+-dependent binding of calcyclin to muscle tropomyosin.
The interaction of calcyclin with tropomyosin and tropomyosin-actin was studied with fluorescence titrations and photo-reactive crosslinking experiments. Titrations of pyreneiodoacetamide-labeled tropomyosin alone or with actin showed binding of calcyclin to tropomyosin with muM dissociation constants when Ca2+ was present. UV irradiation of mixtures of calcyclin and gizzard beta beta-tropomyosin labeled with benzophenone-iodoacetamide at Cys36, with or without actin, produced crosslinks between tropomyosin chains and calcyclin monomers only in the presence of Ca2+. These data provide direct evidence for a Ca+2-dependent tropomyosin-calcyclin interaction at or near Cys36 of tropomyosin and indicate that calcyclin binding to tropomyosin-actin does not cause tropomyosin dissociation.
Actins↗
Separation and characterization of the two functional regions of troponin involved in muscle thin filament regulation.
Mild proteolytic cleavage of the troponin complex yields TnT1, the N-terminal fragment of troponin T, and TnT2IC, a complex of the C-terminal fragment of troponin T (TnT2) with troponin I (TnI) and troponin C (TnC) [Morris, E. P., & Lehrer, S. S. (1984) Biochemistry 23, 2214-2220]. Both TnT1 and TnT2IC bind tightly to the tropomyosin.actin (Tm.actin) thin filament and influence the interaction of myosin subfragment 1 (S1) with Tm.actin. TnT1 does not affect the rate of S1 binding to Tm.actin but does increase the cooperativity with which S1 "turns on" Tm.actin, monitored by the excimer fluorescence of a pyrene label attached to Cys 190 of Tm [Geeves, M.A., & Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. The apparent cooperative unit size of Tm.actin is increased from 6 to 9 by TnT1 and to 12 by whole troponin. In contrast, TnT2IC has no effect on the cooperativity of Tm.actin but does make the apparent S1-binding rate constant, kapp, Ca(2+)-sensitive; i.e., in the absence of Ca2+, kapp is reduced 2-3-fold by both TnT2IC and whole troponin. Thus, the N- and C-terminal regions of TnT appear to act independently in modulating effects of S1 binding to the Tm.actin thin filament that are important in regulation.
Actins↗
Unfolding domains in smooth muscle myosin rod.
Gizzard smooth muscle myosin rod, an alpha-helical coiled coil, exhibits two cooperative thermal or denaturant-induced helix unfolding transitions in solutions containing 0.6 M NaCl at neutral pH, when monitored by circular dichroism at 222 nm. The first smaller transition unfolds part of the subfragment 2 (S2) domain, and the main transition unfolds the remaining helix including the light meromyosin (LMM) domain. These unfolding domains were identified by monitoring the fluorescence of acrylodan, an environmentally sensitive fluorescence probe, and the ESR signal of a maleimide spin-label, sensitive to motion, both specifically attached to Cys 43 in the S2 region of the rod sequence. The identities of the domains were verified by studying the unfolding of the S2 and LMM coiled-coil peptides obtained by proteolytic cleavage of spin-labeled and unlabeled rod. The fluorescence of acrylodan-labeled rod indicated that although the S2 intermediate is unfolded, it is not in a random-coil conformation. The unfolded S2 region stabilized the LMM domain against unfolding, possibly by a direct interaction with the LMM region. Such an interaction may be involved in the salt- and phosphorylation-dependent 6S to 10S shift in configuration of the myosin molecule.
2-Naphthylamine↗
Pyrene excimer fluorescence as a probe of protein conformational change.
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Animals↗
Phalloidin unzips nebulin from thin filaments in skeletal myofibrils.
Fluorescent phallotoxins such as rhodamine-phalloidin take hours to bind uniformly to thin filaments of skeletal myofibrils, after fast initial binding to both ends of thin filaments. Observation of this process in skeletal and cardiac myofibrils and of the resulting re-distribution of nebulin using anti-nebulin antibody showed that: (1) rhodamine-phalloidin binds uniformly to actin in cardiac myofibrils within minutes, in contrast to skeletal myofibrils; (2) overnight pre-incubation of skeletal myofibrils with phalloidin results in uniform initial binding of rhodamine-phalloidin and a changed nebulin localization; (3) pre-incubation of skeletal myofibrils with Ca(2+)-calmodulin results in uniform initial binding of rhodamine-phalloidin; (4) the binding of rhodamine-phalloidin to actin in skeletal myofibrils is unidirectional, i.e. the fluorescence of incorporated rhodamine-phalloidin moves from the pointed ends where it is bound initially toward the barbed end at the Z-band; (5) the unidirectional binding of rhodamine-phalloidin results in redistribution of nebulin, i.e. the initial fluorescent bands associated with the epitopes of bound nebulin antibody change to a single band located close to Z-line. These results indicate that nebulin inhibits rhodamine-phalloidin binding to actin and suggests that the unidirectional rhodamine-phalloidin binding may be due to cooperative competitive binding, i.e. phalloidin 'unzips' nebulin starting from the pointed ends of the thin filaments.
Actins↗