A Peek into Tropomyosin Unfolding on the Actin Filament

Abstract

Tropomyosin (TM) is a coiled-coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Here we ask, “Does TM unfold in the presence of F-actin as a single unit or in multiple blocks?” We hypothesize that functional binding sites unfold prior to or during dissociation from actin, preceding chain separation. We refer to the seven periodic repeats (Phillips, 1986) as P1-P7. We monitored local unfolding and chain dissociation by fluorescence of pyrenylated TM (Ishii & Lehrer, 1980). We combined fluorescence with light scattering and DSC (Levitsky et al., 2000) to monitor TM unfolding and dissociation from F-actin. We investigated the relationship of specific regions of the molecule to unfolding and dissociation of the entire molecule from F-actin by pyrenylation of Cys190 (in P5), and in TMs engineered to have a single Cys analogous to that of Cys190 in P2 and P3 (“controls”). We previously reported that the destabilizing Ala cluster in P5 is required for its participation as a “strong” binding site (Singh and Hitchcock-DeGregori, 2006). An Ala cluster was introduced in P2 or P3 to mimic P5. Analysis of the “controls” and mutants showed (1) binding to actin stabilizes all TM variants reflected by the Tm of excimer formation, and (2) that locally destabilized regions in P2, P3, and P5 unfold prior to or during dissociation from F-actin. Initial unfolding of the P2 and P3 regions is distinct, but overlaps at higher temperatures indicating the unfolding of these regions does not occur in a single block but in multiple overlapping blocks. This, and previous work, suggests that regions of TM involved in binding actin have a poorly packed interface and are locally stabilized upon binding. Supported by NIH.