In vitro Interaction of Titin and Myosin-Binding Protein С

Abstract

The study of the basic biophysical principles underlying protein aggregation is a relevant area of modern biophysics, which is of great importance for elucidating the disruption of the protein aggregation process in pathologies and age-related changes. In this work, the morphological and structural features of coaggregates of two muscle proteins of the sarcomeric cytoskeleton (titin and myosin-binding protein C) were studied using atomic force microscopy, FTIR spectroscopy, and fluorimetry using the dye thioflavin T and X-ray diffraction. It was found that depending on the ionic strength of the solution coaggregation of these proteins occurs with the formation of structures with different morphologies. In particular, in a solution containing 30 mM KCl, structures resembling a film with multiple ruptures were observed, while in a solution containing 150 mM KCl the studied proteins formed spherical aggregates. When the aggregates of the studied proteins interacted with thioflavin T, the fluorescence intensity of titin and C-protein complexes that formed at lower ionic strength (30 mM KCl) increased, which may indicate the amyloid nature of the studied aggregates. The FT-IR spectrum of titin and C-protein complexes had two broad peaks; the presence of one of these (between 1611 and 1630 cm^–1) is characteristic of amyloid aggregates. However, the reflections related to the cross-β-structure that are characteristic of amyloid protein aggregates were not detected in these complexes by X-ray diffraction. Similar results were obtained by X-ray diffraction method in the study of coaggregates of the studied proteins formed in a solution with higher ionic strength. Thus, depending on the ionic strength of the solution, coaggregates of titin and myosin-binding protein C are formed that are different in morphology and in their ability to bind thioflavin T. The in vitro data expand the understanding of the peculiarities of aggregation of sarcomeric muscle proteins and are important for a better understanding of their interaction in sarcomeres in vivo. The results of the study are relevant in the context of age-related changes in muscle tissue and may be important for the development of approaches aimed at maintaining normal muscle contractile function in the elderly. The interaction of the investigated proteins in the sarcomere can be considered as a potential molecular target for interventions related to physical activity management in aging.