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

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 alpha- and beta-Tm isoforms. These associate in vivo to form homo- (alpha(2)) and heterodimers (alpha center dot beta) with little beta(2) normally observed. The proportion of alpha(2) vs alpha center dot beta varies across species and across muscle types from almost 100% alpha(2)- to 50% alpha center dot beta-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 alpha center dot beta dimer for functional studies. Here we report an effective method for purifying bacterially expressed Tm alpha center dot beta 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 alpha center dot beta dimers differ in key properties (actin affinity, thermal stability) from either the alpha(2)- or beta(2)-Tm. However, the ability to regulate myosin binding when combined with cardiac troponin appears unaffected.