Structural and Functional Peculiarities of Cytoplasmic Tropomyosin Isoforms, the Products of TPM1 and TPM4 Genes

Abstract

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.

Keywords: actin filaments; circular dichroism; cytoplasmic isoforms; differential scanning calorimetry; tropomyosin.

Figure 1

Schematic representation of exon usage for Tpm isoforms used in this study. Variable exons 6 (6a or 6b) and 9 (9a, 9c or 9d) are only shown.

Figure 2

Thermal unfolding of Tpm isoforms Tpm1.6, Tpm1.12, and Tpm4.2 measured by CD. (A) Temperature dependences of α-helix stability measured at 222 nm. (B) First derivative profiles for data shown in (A). All experiments were done at the constant heating rate of 1 °C/min with a protein concentration 1 mg/mL.

Figure 3

Temperature dependences of the excess heat capacity (Cp) monitored by DSC and the results of the deconvolution analysis of the heat sorption curves for LMW Tpm isoforms Tpm4.2 (A) and Tpm1.12 (B). Solid black lines represent the experimental curves after subtraction of instrumental and chemical baselines, and dotted red lines represent the individual thermal transitions (calorimetric domains) obtained from fitting the data to the non-two-state model.

Figure 4

The affinity of various Tpm isoforms for F-actin. Plot (A) shows a representative SDS-PAGE gel for experiment of cosedimentation of Tpm1.7 isoform with F-actin. The numbers 1–6 above the lanes for supernatants and pellets correspond to concentrations of Tpm1.7 (0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, and 6 μM, respectively) added to F-actin before ultracentrifugation. Plots (B) and (C) show the actin affinity of Tpm isoforms Tpm1.5, Tpm1.6, Tpm1.7, and Tpm4.2 (B), and Tpm1.12 (C) determined by the cosedimentation assay and plotted as the fractional saturation of F-actin by Tpm as a function of free Tpm concentration that was found in the supernatant.

Figure 5

Normalized temperature dependences of dissociation of the complexes of F-actin with Tpm isoforms Tpm1.5, Tpm1.6, Tpm1.7, and Tpm4.2 (A), and Tpm1.12 (B). A 100% value corresponds to the difference between the light scattering of Tpm–F-actin complexes measured at 25 °C and that of pure F-actin stabilized by phalloidin, which was temperature-independent within the temperature range used. A decrease in the light-scattering intensity reflects the dissociation of the Tpm–F-actin complex. Samples contained 20 μM F-actin stabilized by 20 μM phalloidin and 10 μM Tpm (A) or 10 μM phalloidin-stabilized F-actin and 120 μM Tpm1.12 (B) in 30 mM Hepes-Na buffer, pH 7.3, containing 100 mM NaCl and 2 mM DTT.