E40K and E54K mutations in alpha-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser alpha-tropomyosin (AS-alpha-Tm) expressed in Escherichia coli, that both mutations decrease Ca(2+) sensitivity. E40K also reduces V(max) of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-alpha-Tm, and the two dilated cardiomyopathy-causing mutants. AS-alpha-Tm has a lower cooperative unit size (6.5) than native alpha-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the K(T) value was the same for all actin-Tm species; however, the K(T) value of actin-Tm-troponin at pCa 5 was 50% less for AS-alpha-Tm E40K than for AS-alpha-Tm and AS-alpha-Tm E54K. K(b), the "closed" to "blocked" equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-alpha-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-alpha-Tm E54K, we observed a unique new domain at 55 degrees C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.