α-Tropomyosin with a D175N or E180G Mutation in Only One Chain Differs from Tropomyosin with Mutations in Both Chains

Abstract

α-Tropomyosin (Tm) carrying hypertrophic cardiomyopathy mutation D175N or E180G was expressed in Escherichia coli. We have assembled dimers of two polypeptide chains in vitro that carry one (αα*) or two (α*α*) copies of the mutation. We found that the presence of the mutation has little effect on dimer assembly, thereby predicting that individuals heterozygous for the Tm mutations are likely to express both αα* and α*α* Tm. Depending on the expression level, the heterodimer may be the predominant form in individuals carrying the mutation. Thus, it is important to define differences in the properties of Tm molecules carrying one or two copies of the mutation. We examined the Tm homo- and heterodimer properties: actin affinity, thermal stability, calcium regulation of myosin subfragment 1 binding, and calcium regulation of myofibril force. We report that the properties of the heterodimer may be similar to those of the wild-type homodimer (actin affinity, thermal stability, D175N αα*), similar to those of the mutant homodimer (calcium sensitivity, D175N αα*), intermediate between the two (actin affinity, E180G αα*), or different from both (thermal stability, E180G αα*). Thus, the properties of the homodimer are not a completely reliable guide to the properties of the heterodimer.

Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation

“…4) than the WT due to weaker actin binding in P4 and P5 (see Table 3), in agreement with experimental binding measurements (Bing et al 1997;Boussouf et al 2007;Golitsina et al 1997Golitsina et al , 1999Janco et al 2012;Kremneva et al 2004). E180 is at the e position of the heptad repeat (forming a salt bridge with R182 of another Tpm chain), so it is involved in stabilizing the Tpm coiled coil structure.…”

“…D175 was found to contribute to actin binding in our previous study (Zheng et al 2013). Experimentally, the D175N mutant was shown to bind less strongly to F-actin than the WT in several binding studies (Boussouf et al 2007;Janco et al 2012;Kremneva et al 2004) although the actin-binding affinity was un-affected in other studies (Bing et al 1997;Golitsina et al 1997Golitsina et al , 1999. D175 is at the g position of the heptad repeat, so it is likely involved in stabilizing the Tpm coiled coil structure.…”

The Relaxation Properties of Myofibrils Are Compromised by Amino Acids that Stabilize α-Tropomyosin

“…In particular, aaTm E180G and D175N have shown a similar incomplete relaxation and apparent increase in Ca 2þ sensitivity (52)(53)(54). In these cases, the effects were also explained by a shift in the equilibrium from the blocked state to the closed state, resulting in increased myosin binding to produce the M À -open state.…”

Polymorphism in tropomyosin structure and function

“…The properties of Tm heterodimers carrying a single copy of the mutation had not been explored due to lack of a reliable method for their formation in vitro just as for αβ-Tm. We have used the same tagging method as used for αβ-Tm (Kalyva et al 2012) and optimized it for the formation and purification of the αα*-Tm heterodimer carrying either Asp175Asn or Glu180Gly HCM mutations (Janco et al 2012). The biochemical and biophysical properties of both Asp175Asn and Glu180Gly Tm homodimers have been widely studied and are well established in vitro (Golitsina et al 1997; Kalyva et al 2012; Kremneva et al 2004; Li et al 2012) and key data are summarized in Table 2 together with the more recent heterodimer data.…”

“…b skαα-wt Tm ( black line ) and the homo ( grey line ) and heterodimer ( dashed line ) of the E180G cardiomyopathy mutation. Data from Janco et al 2012. Each plot is the average of three repeated melting curves on the same sample.…”