Effects of two familial hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on the thermal unfolding of actin-bound tropomyosin

Abstract

Differential scanning calorimetry was used to investigate the thermal unfolding of native alpha-tropomyosin (Tm), wild-type alpha-Tm expressed in Escherichia coli and the wild-type alpha-Tm carrying either of two missense mutations associated with familial hypertrophic cardiomyopathy, D175N or E180G. Recombinant alpha-Tm was expressed with an N-terminal Ala-Ser extension to substitute for the essential N-terminal acetylation of the native Tm. Native and Ala-Ser-Tm were indistinguishable in our assays. In the absence of F-actin, the thermal unfolding of Tm was reversible and the heat sorption curve of Tm with Cys-190 reduced was decomposed into two separate calorimetric domains with maxima at approximately 42 and 51 degrees C. In the presence of phalloidin-stabilized F-actin, a new cooperative transition appears at 46-47 degrees C and completely disappears after the irreversible denaturation of F-actin. A good correlation was found to exist between the maximum of this peak and the temperature of half-maximal dissociation of the F-actin/Tm complex as determined by light scattering experiments. We conclude that Tm thermal denaturation only occurs upon its dissociation from F-actin. In the presence of F-actin, D175N alpha-Tm shows a melting profile and temperature dependence of dissociation from F-actin similar to those for wild-type alpha-Tm. The actin-induced stabilization of E180G alpha-Tm is significantly less than for wild-type alpha-Tm and D175N alpha-Tm, and this property could contribute to the more severe myopathy phenotype reported for this mutation.

FIGURE 1

SDS-PAGE analysis under nonreducing conditions (in the absence of BME) of C190S α-Tm (1 and 2), wt α-Tm (3 and 4), D175N α-Tm (5 and 6), and E180G α-Tm (7 and 8). Lanes 1, 3, 5, and 7 represent the samples without any special treatments. Lanes 2, 4, 6, and 8 represent the Tm samples subjected to reduction procedure (incubation to 60°C for 1 h in the presence of 20 mM BME). The monomer bands are labeled as α, and the disulfide cross-linked Tm homodimers are labeled as α-α.

FIGURE 2

Temperature dependence of the excess heat capacity (C_p) and deconvolution analysis of the heat sorption curves of cross-linked (A) or reduced (B) recombinant α-Tm and of mutant C190S α-Tm (C), which is insensitive to reduction procedure (Fig. 1). The protein concentration was 20 μM. Other conditions: 20 mM Hepes, pH 7.3, 100 mM KCl, and 2 mM MgCl₂. The heating rate was 1 K/min. The curves were analyzed according to the non-two-state model. Solid lines represent the experimental curves after subtraction of instrumental and chemical baselines, and dotted lines represent the individual thermal transitions (calorimetric domains 1, 2, and 3) obtained from fitting the data to the non-two-state model.

FIGURE 3

Temperature dependence of the excess heat capacity (C_p) of D175N α-Tm (A and B) and E180G α-Tm (C and D) in the cross-linked state (A and C) or in the reduced state (B and D). Solid lines represent the experimental curves after subtraction of chemical baselines, and dotted lines represent the individual thermal transitions (calorimetric domains M, 1, 2, and 3) obtained from fitting the data to the non-two-state model. Protein concentrations were 20 μM. Other conditions were the same as in Fig. 2.

FIGURE 4

Affinity of the α-Tm constructs to actin, plotted as the fractional saturation of actin by Tm as a function of free Tm concentration. The binding of reduced recombinant wt α-Tm (□), D175N α-Tm (○), and E180G α-Tm (Δ) was carried out at 20°C in the same buffer conditions as the DSC experiments. The K_50% values, corresponding to the Tm concentration at which half of actin becomes saturated, are 0.18 μM, 0.51 μM, and 0.68 μM for recombinant α-Tm, D175N, and E180G α-Tm, respectively.

FIGURE 5

Thermal denaturation of the complex of reduced α-Tm with phalloidin stabilized F-actin. For comparison, the thermal unfolding of α-Tm in the absence of F-actin is also shown. The actin-free data shown as a dotted line were obtained by heating a corresponding sample without actin to that represented by a solid line. Conditions: 30 μM α-Tm, 46 μM F-actin, 70 μM phalloidin in 20 mM Hepes, pH 7.3, 2 mM MgCl₂, 100 mM KCl, and 1 mM BME. The vertical bar corresponds to 10 μW.

FIGURE 6

Deconvolution analysis of calorimetric curves obtained from the first (A) and the third (B) heating of the complex of reduced recombinant wt α-Tm with phalloidin-stabilized F-actin. The results of the second heating to 90°C, performed for complete irreversible denaturation of phalloidin-stabilized F-actin, were identical to those obtained from the first heating. Solid lines represent the experimental curves after subtraction of instrumental and chemical baselines, and dotted lines represent the individual thermal transitions (calorimetric domains) obtained from fitting the data to the non-two-state model. Domains 1, 2, and 3, as well as transition AT, are described in the text. Conditions: 15 μM α-Tm, 46 μM F-actin, 70 μM phalloidin, 20 mM Hepes, pH 7.3, 2 mM MgCl₂, 100 mM KCl, and 1 mM BME.

FIGURE 7

Deconvolution analysis of calorimetric curves obtained from the first heating (A) and the third heating (B) of the complex of D175N α-Tm with phalloidin-stabilized F-actin. The results of the second heating were identical to those obtained from the first heating. Concentration of D175N α-Tm was 7.5 μM. Other conditions and symbols: same as for Fig. 6.

FIGURE 8

Deconvolution analysis of the DSC curves obtained from the first heating (A) and from the third heating (B) of the complex of E180G α-Tm with phalloidin-stabilized F-actin. The results of the second heating were identical to those obtained from the first heating. Concentration of E180G α-Tm was 7.5 μM. Other conditions and symbols: same as for Fig. 6.

FIGURE 9

(A) Temperature dependence of light scattering for the complex of wt α-Tm with F-actin stabilized by phalloidin (curve a), for phalloidin-stabilized F-actin alone (curve b), and for actin-free Tm (curve d). Curve c was obtained by subtraction of curve b from curve a. A decrease in the light-scattering intensity reflects dissociation of the Tm–F-actin complex. Arrows indicate the temperature region from 35 to 50°C where dissociation occurs; 100% corresponds to the difference between light scattering of the Tm–F-actin complex and that of the mixture of F-actin with denatured Tm obtained after dissociation of the complex. T_diss is the temperature of half-maximal dissociation of the Tm–F-actin complex, i.e., the temperature at which a 50% decrease in light scattering occurs. (B–D) Normalized temperature dependence of dissociation of the F-actin complexes with reduced wt α-Tm (B), D175N α-Tm (C), and E180G α-Tm (D) obtained at various molar ratios Tm/actin. For ease of viewing, only the fitted curves are shown. Actin concentration 46 μM; concentration of Tm is indicated for each curve. Conditions were the same as for DSC experiments presented in Figs. 6–8. Heating rate was 1°C/min.