Ca2+ -Induced Structural Changes In Tn: A Multi-Site Fret Study Combining Tcspc With Single Filament Imaging

Troponin is the allosteric sensor for calcium in cardiac muscle. During activation, calcium binds to troponin (Tn), causing a conformational change leading to myofilament activation. We seek to understand the energetics and design principle of the regulated actin (TnTmA7) switch and the basis for cooperative activation in cardiac muscle contraction. To study Ca2+-induced structural changes in Tn, we have engineered single Cys-mutants within Tn that enable the measurement of 36 unique inter-probe distances by time-resolved FRET (trFRET). Regulated actin (rAc) was reconstituted from dye-labeled Tn, tropomyosin (Tm) and f-actin (A7), immobilized on glass coverslips. Epifluorescence images revealed actin filaments with no TnTm bound, actin filaments fully decorated with TnTm, bundles of rAc, and random points of fluorescence from Tn and Tm not bound to actin. Video microscopy demonstrated Brownian movement in non-immobilized segments of rAc. To obtain reliable trFRET data from Tn within non-bundled rAc filaments, we first imaged partially immobilized rAc. Isolated filaments were identified, and TCSPC was performed on a single point within the filament for 1 min, yielding 20,000 photons from approximately 50 Tn molecules within the 1 μm3 confocal volume. The inter-dye distance was obtained under Mg2+- and Ca2+-saturating conditions in 36 FRET constructs.