Design and Optimization of Tropomyosin Fragments for Tropomodulin Interaction Studies

Abstract

Monomeric actin, or G-actin, polymerizes into filamentous actin, or F-actin which is an important component of cellular structures controlling many processes including vesicle trafficking, cell motility, inter and intracellular transport, and muscle contraction. Actin filaments are composed of a pointed end (slow growing) and a barbed end (fast growing). Interactions at each end enables control of the length of actin filaments. Tropomodulin (Tmod1-4) modulates actin filament length by interacting with tropomyosin (Tpm) and actin at the pointed end of the filament. Tmod binds two Tpm molecules through two distinct Tpm binding sites. Establishing the structure of each Tmod-Tpm binding interface is critical to understand the thin filament length regulation mechanism of Tmod. To facilitate NMR structure determination, we have established the optimal size of Tpm fragment necessary to form respective stable complexes. The four fragments we designed were residues 1-43, 1-33, 1-26 or 1-19 of Tpm1.8 fused with a GCN4 sequence promoting coiled-coil formation. We used NMR to determine and compare spectral effects between the optimized Tpm fragments and Tpm-binding sites of Tmod2. A significant change in spectra occurred when length of Tpm sequence changed from 19 to 26 residues and with almost no change with further increase in Tpm sequence length. Stability of the designed Tpm fragments and their affinity for Tpm-binding sites on Tmod2 were evaluated by CD spectroscopy. Our results revealed that Tpm1.8 fragments containing residues 1-19 and 1-43 were more stable with a melting temperature of 44 and 52oC, respectively, however, the 1-19 fragment was not long enough to represent the whole binding interface. The calculated dissociation constants showed strong affinity of Tmod2 for the Tpm1.8 1-43. Therefore, Tpm1.8-1-43 fragment is the most optimal for use in NMR studies to further study its interaction with Tmod.