Functional alpha-tropomyosin produced in Escherichia coli. A dipeptide extension can substitute the amino-terminal acetyl group.

Monteiro, P. B.; Lataro, Renata Cristina; Ferro, J. A.; Reinach, Fernando de Castro

doi:10.1016/s0021-9258(17)34082-6

Cited by 218 publications

(64 citation statements)

References 38 publications

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“…Interactions of human tropomyosin isoforms Tpm1.1 and Tpm3.12, specific for fast and slow skeletal muscle fibers, with F-actin were analyzed in vitro by using a co-sedimentation assay. Both isoforms were recombinant proteins carrying Ala-Ser N-terminal extension, which compensated for the lack of the N-terminal acetylation, typical for proteins expressed in bacterial cells [35]. Binding of both isoforms to actin was analyzed before, but under different salt concentrations [36,37].…”

Section: Binding Of Human Tpm11 and Tpm312 To F-actinmentioning

confidence: 99%

“…Human Tpm1.1 and Tpm3.12 isoforms were expressed in BL21 (DE3) cells (Novagen Inc, Affiliate of Merck KGaA, Darmstadt, Germany) and purified as described before [37,39]. The sequence of both isoforms was identical with the human Tpm1.1 and Tpm3.12, except for two amino acid extension (Ala-Ser), which is routinely used to compensate for the lack of the N-terminal acetylation in the recombinant proteins expressed in bacterial cells [35]. Protein concentration was determined spectrophotometrically at 280 nm using molar extinction coefficient 17,880.…”

Section: Preparation Of Recombinant Tropomyosin Isoforms and Cofilin-2mentioning

confidence: 99%

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Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin

Robaszkiewicz

Śliwińska

Moraczewska

2020

IJMS

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In striated muscle the extent of the overlap between actin and myosin filaments contributes to the development of force. In slow twitch muscle fibers actin filaments are longer than in fast twitch fibers, but the mechanism which determines this difference is not well understood. We hypothesized that tropomyosin isoforms Tpm1.1 and Tpm3.12, the actin regulatory proteins, which are specific respectively for fast and slow muscle fibers, differently stabilize actin filaments and regulate severing of the filaments by cofilin-2. Using in vitro assays, we showed that Tpm3.12 bound to F-actin with almost 2-fold higher apparent binding constant (Kapp) than Tpm1.1. Cofilin2 reduced Kapp of both tropomyosin isoforms. In the presence of Tpm1.1 and Tpm3.12 the filaments were longer than unregulated F-actin by 25% and 40%, respectively. None of the tropomyosins affected the affinity of cofilin-2 for F-actin, but according to the linear lattice model both isoforms increased cofilin-2 binding to an isolated site and reduced binding cooperativity. The filaments decorated with Tpm1.1 and Tpm3.12 were severed by cofilin-2 more often than unregulated filaments, but depolymerization of the severed filaments was inhibited. The stabilization of the filaments by Tpm3.12 was more efficient, which can be attributed to lower dynamics of Tpm3.12 binding to actin.

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Section: Binding Of Human Tpm11 and Tpm312 To F-actinmentioning

confidence: 99%

Section: Preparation Of Recombinant Tropomyosin Isoforms and Cofilin-2mentioning

confidence: 99%

Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin

Robaszkiewicz

Śliwińska

Moraczewska

2020

IJMS

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“…Another possible explanation for very low actin affinity of the Tpm1.12 was that its C-terminal residues for some reason could not normally interact with non-acetylated N-terminus. To check this assumption, we produced recombinant Tpm1.12 that had an Ala-Ser N-terminal extension mimicking N-terminal acetylation of Tpm [40]. It was shown earlier that recombinant α-Tpm (Tpm1.1) expressed in E. coli loses N-terminal acetylation and cannot bind actin, while Ala-Ser N-terminal extension fully restores the Tpm ability to interact with actin [40].…”

Section: 1mentioning

confidence: 99%

“…To check this assumption, we produced recombinant Tpm1.12 that had an Ala-Ser N-terminal extension mimicking N-terminal acetylation of Tpm [40]. It was shown earlier that recombinant α-Tpm (Tpm1.1) expressed in E. coli loses N-terminal acetylation and cannot bind actin, while Ala-Ser N-terminal extension fully restores the Tpm ability to interact with actin [40]. We confirmed, using MALDI-TOF mass-spectrometry, the presence of Ala-Ser N-terminal extension in this Tpm.12 sample and performed cosedimentation experiments to investigate its affinity for actin.…”

Section: 1mentioning

confidence: 99%

“…All Tpm species used in this work were recombinant proteins whose coding sequences were commercially synthesized in Evrogen company (Moscow, Russia) and cloned into pet23a + vector. The HMW Tpm isoforms (Tpm1.5, Tpm1.6, and Tpm1.7) had an Ala-Ser N-terminal extension to mimic natural N-terminal acetylation of native Tpm [40]. The construct of the LMW Tpm1.12 isoform with the Ala-Ser N-terminal extension was also produced by the same method, especially to check the reason for its very low actin affinity (see Section 3.2 in Discussion).…”

Section: Protein Preparationsmentioning

confidence: 99%

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

Marchenko

Nefedova

Artemova

et al. 2021

IJMS

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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.

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Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end‐to‐end overlaps

et al. 2011

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Tropomyosins are dimeric rod-like proteins which polymerize along actin filaments and regulate interactions with other actin-binding proteins. Homologous sequences responsible for the binding of tropomyosin to consecutive actin monomers repeat along tropomyosin and are called actin-binding periods. In this work, the localization of tropomyosin isoforms on actin alone and on actin–myosin complex was evaluated by measuring Förster resonance energy transfer (FRET) distances between a donor (AEDANS) attached to either the N-terminal actin-binding period 1 or to the central actin-binding period 5 and an acceptor (DABMI) bound to actin's Cys374. The recombinant -tropomyosin isoforms–TM2, TM5a, and TM1b9a, used in this study, had various amino acid sequences of the N- and C-termini forming the end-to-end overlap. Although the sequences of actin-binding period 5 of the three isoforms were identical, the donor–acceptor distances calculated for each isoform varied between 38.6 and 41.5 Å. Differences in FRET distances between the three tropomyosin isoforms labeled in actin-binding period 1 varied between 34.8 and 40.2 Å. Rigor binding of myosin heads to actin increased all measured distances. The degree and cooperativity of myosin-induced shift was different for each of the isoforms and actin-binding periods. The structural differences correlate with cooperative regulation of actin-activated S1 ATPase by the three tropomyosins. The results indicate that amino acid sequences of the end-to-end overlap determine specific orientation of tropomyosin isoform on actin. This can be important for steric and cooperative regulation of the actin filament and determine functional specificity of multiple tropomyosin isoforms present in eucaryotic cells.

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Functional alpha-tropomyosin produced in Escherichia coli. A dipeptide extension can substitute the amino-terminal acetyl group.

Cited by 218 publications

References 38 publications

Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin

Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin

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

Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end‐to‐end overlaps

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