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A peek into tropomyosin binding and unfolding on the actin filament.

Singh A, Hitchcock-Degregori SE - PLoS ONE (2009)

Bottom Line: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding.The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample.We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America. Abhishek.Singh@ucsf.edu

ABSTRACT

Background: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin.

Principal findings: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism).

Conclusions: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

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The effect the mutations on the actin affinity measured by cosedimentation with F-actin.Binding to filamentous actin. Tropomyosin (0.1–10 µM, depending on the tropomyosin) and 0.12–12 µM troponin T70–170 [44] were combined with 5 µM actin and sedimented at 20°C in 250 mM NaCl, 10 mM TrisHCl, pH 7.5, 2 mM MgCl2, and 0.5 mM DTT. Stoichiometric binding of 1 tropomyosin: 7 actins is represented by the 1.0 fraction of maximal binding. The apparent Kapps are reported in Table 2. A. Symbols: •, wildtype; ○, P3Shift, ▾, P2Shift. B. Symbols: •, wildtype; ○, P2P3Shift. C. Symbols: •, wildtype; ○, P5→P3. The mutations affect the affinity and cooperativity of binding.
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pone-0006336-g006: The effect the mutations on the actin affinity measured by cosedimentation with F-actin.Binding to filamentous actin. Tropomyosin (0.1–10 µM, depending on the tropomyosin) and 0.12–12 µM troponin T70–170 [44] were combined with 5 µM actin and sedimented at 20°C in 250 mM NaCl, 10 mM TrisHCl, pH 7.5, 2 mM MgCl2, and 0.5 mM DTT. Stoichiometric binding of 1 tropomyosin: 7 actins is represented by the 1.0 fraction of maximal binding. The apparent Kapps are reported in Table 2. A. Symbols: •, wildtype; ○, P3Shift, ▾, P2Shift. B. Symbols: •, wildtype; ○, P2P3Shift. C. Symbols: •, wildtype; ○, P5→P3. The mutations affect the affinity and cooperativity of binding.

Mentions: The values for Kapp, shown with standard errors. The data were fit to the Hill equation, and the Kapp and αH, Hill coefficient, are those reported by SigmaPlot (Figure 6).


A peek into tropomyosin binding and unfolding on the actin filament.

Singh A, Hitchcock-Degregori SE - PLoS ONE (2009)

The effect the mutations on the actin affinity measured by cosedimentation with F-actin.Binding to filamentous actin. Tropomyosin (0.1–10 µM, depending on the tropomyosin) and 0.12–12 µM troponin T70–170 [44] were combined with 5 µM actin and sedimented at 20°C in 250 mM NaCl, 10 mM TrisHCl, pH 7.5, 2 mM MgCl2, and 0.5 mM DTT. Stoichiometric binding of 1 tropomyosin: 7 actins is represented by the 1.0 fraction of maximal binding. The apparent Kapps are reported in Table 2. A. Symbols: •, wildtype; ○, P3Shift, ▾, P2Shift. B. Symbols: •, wildtype; ○, P2P3Shift. C. Symbols: •, wildtype; ○, P5→P3. The mutations affect the affinity and cooperativity of binding.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2710508&req=5

pone-0006336-g006: The effect the mutations on the actin affinity measured by cosedimentation with F-actin.Binding to filamentous actin. Tropomyosin (0.1–10 µM, depending on the tropomyosin) and 0.12–12 µM troponin T70–170 [44] were combined with 5 µM actin and sedimented at 20°C in 250 mM NaCl, 10 mM TrisHCl, pH 7.5, 2 mM MgCl2, and 0.5 mM DTT. Stoichiometric binding of 1 tropomyosin: 7 actins is represented by the 1.0 fraction of maximal binding. The apparent Kapps are reported in Table 2. A. Symbols: •, wildtype; ○, P3Shift, ▾, P2Shift. B. Symbols: •, wildtype; ○, P2P3Shift. C. Symbols: •, wildtype; ○, P5→P3. The mutations affect the affinity and cooperativity of binding.
Mentions: The values for Kapp, shown with standard errors. The data were fit to the Hill equation, and the Kapp and αH, Hill coefficient, are those reported by SigmaPlot (Figure 6).

Bottom Line: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding.The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample.We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America. Abhishek.Singh@ucsf.edu

ABSTRACT

Background: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin.

Principal findings: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism).

Conclusions: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

Show MeSH
Related in: MedlinePlus