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Transmission of stability information through the N-domain of tropomyosin is interrupted by a stabilizing mutation (A109L) in the hydrophobic core of the stability control region (residues 97-118).

Kirwan JP, Hodges RS - J. Biol. Chem. (2013)

Bottom Line: The single mutation L110A destabilizes the entire Tm(1-131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction.We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect.We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.

View Article: PubMed Central - PubMed

Affiliation: From the Program in Structural Biology and Biophysics, Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, Colorado 80045.

ABSTRACT
Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97-118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1-131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1-131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.

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Folding and reversibility of Tm(1–131) wild type and mutant proteins using CD spectroscopy.A, CD spectrum scans of Tm(1–131) wild type (black), L110A (red), and A109L (light blue) measured immediately after thermal denaturation to 75 °C and cooling back to 5 °C. These scans indicate helical structure with very little difference in helical content between wild type and mutants. B, overlay of thermal denaturation or unfolding (dark circles) and refolding (open circles) profiles for wild type Tm(1–131) using CD. The profiles are shown overlapping, indicating equilibrium unfolding (reversibility of folding) in the temperature range of 5–75 °C. Tm(1–131) L110A and A109L both exhibited similar overlapping unfolding and refolding profiles. ASTm(1–131) indicates the presence of an N-terminal Ala-Ser dipeptide in these Tm(1–131) sequences. All profiles were measured with a temperature change of 1 °C/min.
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Figure 3: Folding and reversibility of Tm(1–131) wild type and mutant proteins using CD spectroscopy.A, CD spectrum scans of Tm(1–131) wild type (black), L110A (red), and A109L (light blue) measured immediately after thermal denaturation to 75 °C and cooling back to 5 °C. These scans indicate helical structure with very little difference in helical content between wild type and mutants. B, overlay of thermal denaturation or unfolding (dark circles) and refolding (open circles) profiles for wild type Tm(1–131) using CD. The profiles are shown overlapping, indicating equilibrium unfolding (reversibility of folding) in the temperature range of 5–75 °C. Tm(1–131) L110A and A109L both exhibited similar overlapping unfolding and refolding profiles. ASTm(1–131) indicates the presence of an N-terminal Ala-Ser dipeptide in these Tm(1–131) sequences. All profiles were measured with a temperature change of 1 °C/min.

Mentions: Overlaid CD Spectra (195–250 nm) of Tm(1–131) wild type, L110A, and A109L indicated that each protein was fully folded, with [θ]222 (degrees·cm2·dmol−1) values of 39,143 (wild type), 36,412 (L110A), and 34,587 (A109L) in benign conditions (100 mm KCl, 50 mm PO4, pH 7) (Fig. 3A and Table 1). The [θ]222/[θ]208 ratios for these proteins were greater than 1, indicating that all three proteins were folded as coiled-coils (88). However, it was interesting that although A109L was expected to be the most stable mutant, it showed the least helical content of the three proteins (89). Initial thermal unfolding CD experiments (5–75 °C, 1 °C/min) were immediately cooled at the same rate (75–5 °C, 1 °C/min) for comparison of the unfolding and refolding profiles of each protein. Fig. 3B shows that the unfolding and refolding profiles of Tm(1–131) wild type overlay, indicating that the sample was in thermodynamic equilibrium. The unfolding and refolding profiles of Tm(1–131) L110A and A109L overlapped similarly (data not shown) and indicated that the unfolding of all of our samples was reversible. We proceeded to compare the thermal stabilities of the Tm(1–131) proteins evaluated by CD and observed novel results in which the L110A and A109L mutations completely altered the stability of this N-terminal domain in very different ways. Fig. 4 shows their melting profiles and associated non-linear least squares fit according to the Gibbs-Helmholtz equation (Equation 3). Relative to wild type, L110A destabilized the Tm(1–131) protein by more than 6 °C (Fig. 4, A and B, and Table 1). However, A109L simultaneously stabilized 55% of the molecule by more than 11 °C and destabilized 45% of the molecule by more than 12 °C, relative to wild type, based on the fractions of total ellipticity associated with each of two apparent transitions (Fig. 4, A and C, and Table 1). We had expected A109L to increase the stability of the entire protein, but instead Tm(1–131) was divided into two domains of stability, as is clearly shown in Fig. 4D.


Transmission of stability information through the N-domain of tropomyosin is interrupted by a stabilizing mutation (A109L) in the hydrophobic core of the stability control region (residues 97-118).

Kirwan JP, Hodges RS - J. Biol. Chem. (2013)

Folding and reversibility of Tm(1–131) wild type and mutant proteins using CD spectroscopy.A, CD spectrum scans of Tm(1–131) wild type (black), L110A (red), and A109L (light blue) measured immediately after thermal denaturation to 75 °C and cooling back to 5 °C. These scans indicate helical structure with very little difference in helical content between wild type and mutants. B, overlay of thermal denaturation or unfolding (dark circles) and refolding (open circles) profiles for wild type Tm(1–131) using CD. The profiles are shown overlapping, indicating equilibrium unfolding (reversibility of folding) in the temperature range of 5–75 °C. Tm(1–131) L110A and A109L both exhibited similar overlapping unfolding and refolding profiles. ASTm(1–131) indicates the presence of an N-terminal Ala-Ser dipeptide in these Tm(1–131) sequences. All profiles were measured with a temperature change of 1 °C/min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924298&req=5

Figure 3: Folding and reversibility of Tm(1–131) wild type and mutant proteins using CD spectroscopy.A, CD spectrum scans of Tm(1–131) wild type (black), L110A (red), and A109L (light blue) measured immediately after thermal denaturation to 75 °C and cooling back to 5 °C. These scans indicate helical structure with very little difference in helical content between wild type and mutants. B, overlay of thermal denaturation or unfolding (dark circles) and refolding (open circles) profiles for wild type Tm(1–131) using CD. The profiles are shown overlapping, indicating equilibrium unfolding (reversibility of folding) in the temperature range of 5–75 °C. Tm(1–131) L110A and A109L both exhibited similar overlapping unfolding and refolding profiles. ASTm(1–131) indicates the presence of an N-terminal Ala-Ser dipeptide in these Tm(1–131) sequences. All profiles were measured with a temperature change of 1 °C/min.
Mentions: Overlaid CD Spectra (195–250 nm) of Tm(1–131) wild type, L110A, and A109L indicated that each protein was fully folded, with [θ]222 (degrees·cm2·dmol−1) values of 39,143 (wild type), 36,412 (L110A), and 34,587 (A109L) in benign conditions (100 mm KCl, 50 mm PO4, pH 7) (Fig. 3A and Table 1). The [θ]222/[θ]208 ratios for these proteins were greater than 1, indicating that all three proteins were folded as coiled-coils (88). However, it was interesting that although A109L was expected to be the most stable mutant, it showed the least helical content of the three proteins (89). Initial thermal unfolding CD experiments (5–75 °C, 1 °C/min) were immediately cooled at the same rate (75–5 °C, 1 °C/min) for comparison of the unfolding and refolding profiles of each protein. Fig. 3B shows that the unfolding and refolding profiles of Tm(1–131) wild type overlay, indicating that the sample was in thermodynamic equilibrium. The unfolding and refolding profiles of Tm(1–131) L110A and A109L overlapped similarly (data not shown) and indicated that the unfolding of all of our samples was reversible. We proceeded to compare the thermal stabilities of the Tm(1–131) proteins evaluated by CD and observed novel results in which the L110A and A109L mutations completely altered the stability of this N-terminal domain in very different ways. Fig. 4 shows their melting profiles and associated non-linear least squares fit according to the Gibbs-Helmholtz equation (Equation 3). Relative to wild type, L110A destabilized the Tm(1–131) protein by more than 6 °C (Fig. 4, A and B, and Table 1). However, A109L simultaneously stabilized 55% of the molecule by more than 11 °C and destabilized 45% of the molecule by more than 12 °C, relative to wild type, based on the fractions of total ellipticity associated with each of two apparent transitions (Fig. 4, A and C, and Table 1). We had expected A109L to increase the stability of the entire protein, but instead Tm(1–131) was divided into two domains of stability, as is clearly shown in Fig. 4D.

Bottom Line: The single mutation L110A destabilizes the entire Tm(1-131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction.We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect.We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.

View Article: PubMed Central - PubMed

Affiliation: From the Program in Structural Biology and Biophysics, Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, Colorado 80045.

ABSTRACT
Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97-118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1-131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1-131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.

Show MeSH
Related in: MedlinePlus