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

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.

The interchain disulfide cross-linking of tropomyosin alters its regulatory properties and interaction with actin filament.

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein that plays a key role in the Ca2+-regulated contraction of striated muscles. Two chains of Tpm can be cross-linked by formation of a disulfide bond between Cys-190 residues. Normally, the SH-groups of these residues in cardiac muscle are in reduced state but in heart pathologies the interchain cross-linking of Tpm was shown to occur. Previous studies have shown that this cross-linking increases the thermal stability of the C-terminal part of the Tpm molecule. However it was unclear how this affects its functional properties. In the current work, we studied functional features of cross-linked Tpm at the level of isolated proteins. The results have shown that the cross-linking greatly decreases affinity of Tpm for F-actin and stability of the Tpm-F-actin complex. It also increases sliding velocity of regulated thin filaments in an in vitro motility assay. This last effect was mostly pronounced when cardiac isoforms of myosin and troponin were used instead of skeletal ones. The results indicate that cross-linking significantly affects properties of Tpm and actin-myosin interaction and can explain, at least partly, the role of the interchain disulfide cross-linking of cardiac Tpm in human heart diseases.

NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features.

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60-85°Ð¡, and the affinity of AlDHPyr1147 to the NADP+ at 60°Ð¡ is comparable to that for mesophilic analogues at 25°Ð¡. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

Heat-induced conformational changes of TET peptidase from crenarchaeon Desulfurococcus kamchatkensis.

The effects of heating on the structure and stability of multimeric TET aminopeptidase (APDkam589) were studied by differential scanning calorimetry, tryptophan fluorescence quenching, and dynamic light scattering. Thermally induced structural changes in APDkam589 were found to occur in two phases: local conformational changes, which occur below 70 °C and are not associated with thermal denaturation of the protein, and global structural changes (above 70 °C) induced by irreversible thermal unfolding of the protein accompanied by its spontaneous aggregation. These results may explain the bell-shaped temperature dependence with a maximum at ~70 °C previously observed for enzymatic activity of APDkam589. Interestingly, the thermal unfolding of APDkam589 at about 81.2 °C is accompanied by a so-called blue-shift of about 10 nm-a shift of the Trp fluorescence spectrum toward shorter wavelength. From this point of view, APDkam589 is quite different from most proteins, which are characterized by a long wavelength shift of the spectrum ("red-shift") upon denaturation. The blue-shift of the Trp fluorescence spectrum reflects the changes in the environment of Trp residues, which becomes more hydrophobic upon denaturation. The molecular structure of APDkam589 was determined by X-ray diffraction. The monomer of APDkam589 has six Trp residues, five of which are on the external surface of the dodecamer. Therefore, the blue-shift of the Trp fluorescence spectrum can be explained, at least partly, by aggregation of APDkam589, which occurs simultaneously with its thermal denaturation and probably makes the environment of these Trp residues more hydrophobic.

Effects of two stabilizing substitutions, D137L and G126R, in the middle part of α-tropomyosin on the domain structure of its molecule.

We applied differential scanning calorimetry (DSC) to investigate the effects of substitutions D137L and G126R (i.e. replacement of conserved non-canonical residues Asp137 and Gly126 by canonical ones) in the middle part of tropomyosin (Tm), as well as the combined one, D137L/G126R, on the thermal unfolding of Tm. Special approaches (e.g. combination of DSC with measurements of temperature dependences of pyrene excimer fluorescence) were applied to assign separate thermal transitions (calorimetric domains) on the DSC profiles to the certain parts of Tm molecule. The results show that substitutions D137L and G126R (and, especially, the combined one, D137L/G126R) may stabilize not only the middle region of Tm, but also the other parts of its molecule including N- and C-terminal parts. These results suggest that the stabilization of the Tm middle part can be transmitted along the coiled-coil length displaying long-range effects.

Structural and functional effects of two stabilizing substitutions, D137L and G126R, in the middle part of α-tropomyosin molecule.

Tropomyosin (Tm) is an α-helical coiled-coil protein that binds along the length of actin filament and plays an essential role in the regulation of muscle contraction. There are two highly conserved non-canonical residues in the middle part of the Tm molecule, Asp137 and Gly126, which are thought to impart conformational instability (flexibility) to this region of Tm which is considered crucial for its regulatory functions. It was shown previously that replacement of these residues by canonical ones (Leu substitution for Asp137 and Arg substitution for Gly126) results in stabilization of the coiled-coil in the middle of Tm and affects its regulatory function. Here we employed various methods to compare structural and functional features of Tm mutants carrying stabilizing substitutions Arg137Leu and Gly126Arg. Moreover, we for the first time analyzed the properties of Tm carrying both these substitutions within the same molecule. The results show that both substitutions similarly stabilize the Tm coiled-coil structure, and their combined action leads to further significant stabilization of the Tm molecule. This stabilization not only enhances maximal sliding velocity of regulated actin filaments in the in vitro motility assay at high Ca(2+) concentrations but also increases Ca(2+) sensitivity of the actin-myosin interaction underlying this sliding. We propose that the effects of these substitutions on the Ca(2+)-regulated actin-myosin interaction can be accounted for not only by decreased flexibility of actin-bound Tm but also by their influence on the interactions between the middle part of Tm and certain sites of the myosin head.