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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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Related in: MedlinePlus

DSC scans of single cluster shift mutants with TnT70–170 in the presence and absence of F-actin.Tropomyosin (15 µM) and TnT70–170 (18 µM) were mixed with phalloidin (36 µM) stabilized F-actin (24 µM) in 100 mM NaCl, 10 mM Hepes pH 7.0, 2 mM MgCl2, 1 mM DTT and heated as described in Materials and Methods. The second (with F-actin-solid lines) and third (post-F-actin denaturation-dotted lines, TM- TnT70–170) scans are shown. A. wildtype (black); B. P2Shift (green); C. P3Shift (red); D. Combination of A-C in the presence of F-actin with the color scheme as indicated in A-C. The P2Shift and P3Shift mutants bind F-actin with higher affinity than wildtype. E. Experimental procedure: An excess of unacetylated recombinant tropomyosins and the TnT70–170 fragment was added to phalloidin-stabilized F-actin and the sample was heated a total of three times. An excess was added to ensure maximal binding of tropomyosin. The experiments were done in the presence of TnT70–170 because unacetylated tropomyosin binds poorly to F-actin. First, the sample was heated from 0–70°C (black line) and then cooled. This curve shows the dissociation of tropomyosin from F-actin (main peak) and the unfolding of TM-TnT70–170 . Second, the sample was heated to 90°C (green line) to denature the F-actin and subsequently cooled. In the third scan (magenta line), post-F-actin denaturation, the sample was heated to 70°C and only the TM+TnT70–170 signal is present. The first two scans are similar, and mark the tropomyosin unfolding/dissociation thermal peak, consistent with previously published results [22], [24], [62]. The thermal unfolding of tropomyosin is almost completely reversible, while F-actin denaturation is irreversible.
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pone-0006336-g003: DSC scans of single cluster shift mutants with TnT70–170 in the presence and absence of F-actin.Tropomyosin (15 µM) and TnT70–170 (18 µM) were mixed with phalloidin (36 µM) stabilized F-actin (24 µM) in 100 mM NaCl, 10 mM Hepes pH 7.0, 2 mM MgCl2, 1 mM DTT and heated as described in Materials and Methods. The second (with F-actin-solid lines) and third (post-F-actin denaturation-dotted lines, TM- TnT70–170) scans are shown. A. wildtype (black); B. P2Shift (green); C. P3Shift (red); D. Combination of A-C in the presence of F-actin with the color scheme as indicated in A-C. The P2Shift and P3Shift mutants bind F-actin with higher affinity than wildtype. E. Experimental procedure: An excess of unacetylated recombinant tropomyosins and the TnT70–170 fragment was added to phalloidin-stabilized F-actin and the sample was heated a total of three times. An excess was added to ensure maximal binding of tropomyosin. The experiments were done in the presence of TnT70–170 because unacetylated tropomyosin binds poorly to F-actin. First, the sample was heated from 0–70°C (black line) and then cooled. This curve shows the dissociation of tropomyosin from F-actin (main peak) and the unfolding of TM-TnT70–170 . Second, the sample was heated to 90°C (green line) to denature the F-actin and subsequently cooled. In the third scan (magenta line), post-F-actin denaturation, the sample was heated to 70°C and only the TM+TnT70–170 signal is present. The first two scans are similar, and mark the tropomyosin unfolding/dissociation thermal peak, consistent with previously published results [22], [24], [62]. The thermal unfolding of tropomyosin is almost completely reversible, while F-actin denaturation is irreversible.

Mentions: Introduction of an Ala cluster into P2 or P3 had little effect on the global stability of the molecule, as indicated by the slight decrease in overall TM from 45.0°C to 44.5 and 43.4 °C, respectively (Figure 2A, Table 2). The thermal unfolding of wildtype tropomyosin analyzed using DSC showed the expected two major endotherms (Figure 3A, dotted line, Table 2). In this biphasic unfolding the first endotherm corresponds to the unstable middle region of the molecule including P5 (residues 130–190) and the C-terminal half whereas the second endotherm, 56.0°C in wildtype, is the unfolding of the N-terminal ∼half. In P2Shift both transitions occurred at lower temperatures (Figure 3B, dotted line). The unfolding of P3Shift was more cooperative and lacks the pretransition evident in the melts of the other forms, known to reflect the unfolding of the unstable middle of the molecule (Figure 3C, dotted line). The compensatory A120L mutation in P3Shift may stabilize this region and result in the single, lower temperature transition (49.0°C) with DSC, indicative of increased cooperativity of unfolding. P2P3Shift had similar characteristics and lower stability (Figure 2B, Table 2). The P5→P3 mutant was much less stable than the others; the initial transition reflects the low stability of the P5 region and its effect on the adjacent middle of the molecule (Figure 2C, Table 2). The mutant does not have the A120L mutation that adds stability to the P3Shift mutant. The DSC experiments were carried out in the presence of TnT70–170 (for the experiments with actin, see below), which has little effect on the thermal peaks of tropomyosin, consistent with previous results with full-length TnT (results not shown; [22]).


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

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

DSC scans of single cluster shift mutants with TnT70–170 in the presence and absence of F-actin.Tropomyosin (15 µM) and TnT70–170 (18 µM) were mixed with phalloidin (36 µM) stabilized F-actin (24 µM) in 100 mM NaCl, 10 mM Hepes pH 7.0, 2 mM MgCl2, 1 mM DTT and heated as described in Materials and Methods. The second (with F-actin-solid lines) and third (post-F-actin denaturation-dotted lines, TM- TnT70–170) scans are shown. A. wildtype (black); B. P2Shift (green); C. P3Shift (red); D. Combination of A-C in the presence of F-actin with the color scheme as indicated in A-C. The P2Shift and P3Shift mutants bind F-actin with higher affinity than wildtype. E. Experimental procedure: An excess of unacetylated recombinant tropomyosins and the TnT70–170 fragment was added to phalloidin-stabilized F-actin and the sample was heated a total of three times. An excess was added to ensure maximal binding of tropomyosin. The experiments were done in the presence of TnT70–170 because unacetylated tropomyosin binds poorly to F-actin. First, the sample was heated from 0–70°C (black line) and then cooled. This curve shows the dissociation of tropomyosin from F-actin (main peak) and the unfolding of TM-TnT70–170 . Second, the sample was heated to 90°C (green line) to denature the F-actin and subsequently cooled. In the third scan (magenta line), post-F-actin denaturation, the sample was heated to 70°C and only the TM+TnT70–170 signal is present. The first two scans are similar, and mark the tropomyosin unfolding/dissociation thermal peak, consistent with previously published results [22], [24], [62]. The thermal unfolding of tropomyosin is almost completely reversible, while F-actin denaturation is irreversible.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006336-g003: DSC scans of single cluster shift mutants with TnT70–170 in the presence and absence of F-actin.Tropomyosin (15 µM) and TnT70–170 (18 µM) were mixed with phalloidin (36 µM) stabilized F-actin (24 µM) in 100 mM NaCl, 10 mM Hepes pH 7.0, 2 mM MgCl2, 1 mM DTT and heated as described in Materials and Methods. The second (with F-actin-solid lines) and third (post-F-actin denaturation-dotted lines, TM- TnT70–170) scans are shown. A. wildtype (black); B. P2Shift (green); C. P3Shift (red); D. Combination of A-C in the presence of F-actin with the color scheme as indicated in A-C. The P2Shift and P3Shift mutants bind F-actin with higher affinity than wildtype. E. Experimental procedure: An excess of unacetylated recombinant tropomyosins and the TnT70–170 fragment was added to phalloidin-stabilized F-actin and the sample was heated a total of three times. An excess was added to ensure maximal binding of tropomyosin. The experiments were done in the presence of TnT70–170 because unacetylated tropomyosin binds poorly to F-actin. First, the sample was heated from 0–70°C (black line) and then cooled. This curve shows the dissociation of tropomyosin from F-actin (main peak) and the unfolding of TM-TnT70–170 . Second, the sample was heated to 90°C (green line) to denature the F-actin and subsequently cooled. In the third scan (magenta line), post-F-actin denaturation, the sample was heated to 70°C and only the TM+TnT70–170 signal is present. The first two scans are similar, and mark the tropomyosin unfolding/dissociation thermal peak, consistent with previously published results [22], [24], [62]. The thermal unfolding of tropomyosin is almost completely reversible, while F-actin denaturation is irreversible.
Mentions: Introduction of an Ala cluster into P2 or P3 had little effect on the global stability of the molecule, as indicated by the slight decrease in overall TM from 45.0°C to 44.5 and 43.4 °C, respectively (Figure 2A, Table 2). The thermal unfolding of wildtype tropomyosin analyzed using DSC showed the expected two major endotherms (Figure 3A, dotted line, Table 2). In this biphasic unfolding the first endotherm corresponds to the unstable middle region of the molecule including P5 (residues 130–190) and the C-terminal half whereas the second endotherm, 56.0°C in wildtype, is the unfolding of the N-terminal ∼half. In P2Shift both transitions occurred at lower temperatures (Figure 3B, dotted line). The unfolding of P3Shift was more cooperative and lacks the pretransition evident in the melts of the other forms, known to reflect the unfolding of the unstable middle of the molecule (Figure 3C, dotted line). The compensatory A120L mutation in P3Shift may stabilize this region and result in the single, lower temperature transition (49.0°C) with DSC, indicative of increased cooperativity of unfolding. P2P3Shift had similar characteristics and lower stability (Figure 2B, Table 2). The P5→P3 mutant was much less stable than the others; the initial transition reflects the low stability of the P5 region and its effect on the adjacent middle of the molecule (Figure 2C, Table 2). The mutant does not have the A120L mutation that adds stability to the P3Shift mutant. The DSC experiments were carried out in the presence of TnT70–170 (for the experiments with actin, see below), which has little effect on the thermal peaks of tropomyosin, consistent with previous results with full-length TnT (results not shown; [22]).

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