Modeling the troponin core domain on thin filaments using data from cryoelectron microscopy and fluorescence approaches.

Abstract

Our understanding of thin filament structure has been greatly enhanced by the publication of a number of models derived from cryo-electron microscopy studies (Yamada et al. (2020) and then later confirmed by Risi et al. (2021)). These studies elucidated atomic level details of the calcium-dependent activation of the thin filament leading to force development in cardiac muscle. However, while cryo-EM reconstruction is best suited to capture the high-resolution organization of static structures, troponin and tropomyosin are dynamic components of the thin filament and corresponding disorder is not always easily recorded and classified by the method. Thus, in order to better model these structures, it may be necessary to generate small ensembles of structures to incorporate known sources of disorder as guided by information derived from other biophysical techniques. One example is for troponin subunit C, which binds calcium and the troponin subunit I switch peptide during thin filament activation. While the cryo-EM structure has offered a single computed conformation of troponin C, polarized fluorescence of the troponin core domain labeled with bifunctional rhodamine indicates that in cardiac muscle troponin C is very dynamic, and its N-terminal lobe takes up multiple distinct orientations (Sevrieva et al., 2014). In this study, we incorporate data derived both from structural methodologies to build a set of unique models that satisfy the cryo-EM and the fluorescence data. Analysis of these structures shows the details of how the dynamic nature of the N-lobe of troponin C is important for its function in thin filament regulation.