In Vitro Formation and Characterization of the Skeletal Muscle α·β Tropomyosin Heterodimers

Abstract

Tropomyosin (Tm) is a dimer made of two alpha helical chains associated into a parallel coiled-coil. In mammalian skeletal and cardiac muscle, the Tm is expressed from two separate genes to give the α- and β-Tm isoforms. These associate in vivo to form homo- (α(2)) and heterodimers (α·β) with little β(2) normally observed. The proportion of α(2) vs α·β varies across species and across muscle types from almost 100% α(2)- to 50% α·β-Tm. The ratio can also vary during development and in disease. The functional significance of the presence of these two isoforms has not been defined because it is difficult to isolate or purify the α·β dimer for functional studies. Here we report an effective method for purifying bacterially expressed Tm as α·β dimers using a cleavable N-terminal tag on one of the two chains. The same method can be used to isolate Tm dimers in which one chain carries a mutation. We go on to show that the α·β dimers differ in key properties (actin affinity, thermal stability) from either the α(2)- or β(2)-Tm. However, the ability to regulate myosin binding when combined with cardiac troponin appears unaffected.

Polymorphism in tropomyosin structure and function

“…But a study of assembly of sk-Tm using E. coli expressed Tm (carrying the Ala–Ser N-terminal extension to mimic acetylation (Monteiro et al 1994)) showed a small preference for homodimer assembly. Chain exchange studies starting with equal amounts of αα and ββ result in the formation of ~20 % αβ heterodimers and 40 % each of αα and ββ at room temperature equilibrium (Kalyva et al 2012). If the level of β expressed is low then random assembly of the more stable αβ will limit the formation of any ββ.…”

“…Isolation of the singly tagged αβ-Tm was then possible from the zero tagged αα- or double tagged ββ-Tm using Ni affinity chromatography. The tag was proteolytically cleaved after purification allowing the study of the properties of native-like Tm (Kalyva et al 2012). …”

“…These assays showed no detectable differences in the way the three Tm dimers influenced myosin binding to actin (Kalyva et al 2012). The results presented in Kalyva’s study showed that it is not simple to define the functional differences between skαα and skαβ, nor is it possible to explain why there are different ratios of αα and αβ in different cells.…”

“…To date, there are 11 HCM and 4 DCM mutations associated with the TPM1, skα-Tm gene (Kalyva et al 2012; Lakdawala et al 2010; Olson et al 2001; Regitz-Zagrosek et al 2000; Wieczorek et al 2008); and none with the TPM2 β-Tm gene. (Although there are skeletal muscle myopathies associated with the TPM2 gene (Mokbel et al 2013; Tajsharghi et al 2012)).…”

“…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.…”