The Second Half of the Fourth Period of Tropomyosin Is a Key Region for Ca 2+ -Dependent Regulation of Striated Muscle Thin Filaments † (original) (raw)
Related papers
Journal of Molecular Biology, 2009
The Ca 2+ -dependent interaction of troponin I (TnI) with actin•tropomyosin (Actin•Tm) in the muscle thin filament is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca 2+ , TnI interacts with Tm as well as actin in the reconstituted muscle thin filament, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction. To obtain direct evidence for this Tm-TnI interaction we performed photochemical crosslinking studies using Tm labeled with 4-maleimidobenzophenone (BPmal) at position 146 or 174 (Tm146* or Tm174*, respectively), reconstituted with actin and troponin (composed of TnI; troponin T, TnT; and troponin C, TnC) or with actin and TnI. After near uv-irradiation, SDS gels of the Tm*146containing thin filament showed 3 new high molecular weight bands determined to be crosslinked products Tm*146-TnI, Tm*146-TnC and Tm*146-TnT using fluorescence-labeled TnI, mass spectrometry and Western blots. While Tm*146-TnI was produced only in the absence of Ca 2+ , the production of the other crosslinked species did not show a Ca 2+ dependence. Tm*174 mainly crosslinked to TnT. In the absence of actin a similar crosslinking pattern was obtained with a much lower yield. A tryptic peptide from Tm*146-TnI of MW 2601.2 Da that was not present in the tryptic peptides of Tm*146 or TnI was identified using HPLC and MALDI-TOF. This was shown, using absorption and fluorescence spectroscopy, to be the BPmal-labeled peptide from Tm crosslinked to TnI peptide 157-163. These data showing that a region in the C-terminal domain of TnI interacts with Tm in the absence of Ca 2+ support the hypothesis that a TnI-Tm interaction maintains Tm at the outer domain of actin, and will help efforts to localize Tn in the actin•Tm muscle thin filament.
Biochemistry, 2000
In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca 2+ and with actin in the absence of Ca 2+ . To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl)-N′-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca 2+ by measuring the extent of acrylamide quenching of fluorescence and lifetimeresolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca 2+ , reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca 2+ , while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca 2+ . We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-{[(dimethylamino)phenyl]azo}phenyl-4′-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca 2+ , the mean distances were 40.2 Å for Cys-96 and 35.2 Å for Cys-117. In the presence of Ca 2+ , Cys-96 moved away from actin Cys-374 by ∼3.6 Å, while Cys-117 moved away by ∼8 Å. This suggests the existence of a flexible "hinge" region near the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnC in a Ca 2+ -independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca 2+ or to TnC in the presence of Ca 2+ . This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.
FEBS Journal, 2014
Tropomyosin (Tm) is an a-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.
A Modulatory Role for the Troponin T Tail Domain in Thin Filament Regulation
Journal of Biological Chemistry, 2002
In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnI-TnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT 1 tail fragment (residues 1-158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT 1 producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C-to M-state equilibrium, K T , for TnT 1 . This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.
Role of the Head-to-Tail Overlap Region in Smooth and Skeletal Muscle β-Tropomyosin †
Biochemistry, 2008
Tropomyosin (Tm) is an R-helical, parallel, two-chain coiled coil which binds along the length of actin filaments in both muscle and non-muscle cells. Smooth and skeletal muscle Tms differ extensively at the C-terminus encoded by exon 9. Replacement of the striated muscle specific exon 9a-encoded C-terminus with that encoded by exon 9d expressed in smooth muscle and non-muscle cells increases the affinity of unacetylated R-SkTm for actin [Cho, Y. J., and Hitchcock-Degregori, S. E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10153-10157]. Here we show that swapping 10 amino acids at the C-terminus of -SkTm with the corresponding 10 amino acids of -SmTm had little effect on the regulation of S1 binding to actin, but Tm viscosity, Tm binding to actin, and troponin T 1 binding to Tm all become like smooth rather than SkTm. -SkTm point mutations show that these properties are largely defined by the amino acids at two positions, 277 and 279. The N279L mutation reduces the viscosity of -SkTm to close to -SmTm values, while both residues contribute to the binding of TnT 1 . We also show that removing the first 11 N-terminal amino acids of -SmTm to make the mutant ∆N-SmTm results in a 10-fold weakening in actin affinity compared to that of -SmTm. CD studies show no difference in thermal unfolding between -SmTm and ∆N-SmTm; however, the viscosity of ∆N-SmTm is much lower than that of the control. The results suggest that ∆N-SmTm was unable to form filaments in solution but can form filaments on actin.
Journal of Biological Chemistry
Tropomyosin (Tm) is a two-stranded α-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle α-Tm either with an Ala residue, which should stabilize the Tm α-helix, or with an Arg residue, which is expected to stabilize both α-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations conf...
Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function
International Journal of Molecular Sciences, 2020
Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated t...
1997
Contraction of vertebrate striated muscle is regulated by the interaction of Ca 2+ with the heterotrimeric protein troponin (Tn), composed of troponin-C (TnC), troponin-I (TnI), and troponin-T (TnT). Although much is known about the Ca 2+ -induced conformational changes in TnC, the Ca 2+binding subunit of Tn, little is known about how TnI, the inhibitory subunit, responds to the binding of Ca 2+ to TnC. In this work, we used resonance energy transfer to measure the distance between probes attached at Cys48 and Cys133 in the N-and C-terminal domains, respectively, of TnI. A mutant rabbit skeletal TnI, TnI 48/133 (C64S), was constructed by converting Cys64 into Ser. The remaining two thiols at Cys48 and Cys133 were labeled with the fluorescent donor 1,5-IAEDANS, and the nonfluorescent acceptor, DAB-Mal. We found an interprobe distance of ∼41 Å for both uncomplexed TnI and TnI in the binary complex with TnC. This distance increased to 51 Å in the ternary Tn complex with TnT. These distances did not change significantly on binding of Ca 2+ to TnC. In the reconstituted thin filament, this distance remained to be 50 Å in the presence of saturating Ca 2+ , but increased to ∼66 Å on removing Ca 2+ with EGTA in the presence of Mg 2+ . Our results indicate firstly that while TnC has only small effects on the global conformation of TnI, the presence of TnT in the ternary Tn complex gives rise to an apparent elongation of TnI. Secondly, whereas there is no detectable Ca 2+ -dependent change in the global conformation of TnI in the Tn complex free in solution, the removal of Ca 2+ caused a substantial separation of the N-and C-terminal TnI regions in the reconstituted thin filament, owing to the interaction between the C-terminal region of TnI and actin in the relaxed state. † Abstract published in AdVance ACS Abstracts, August 15, 1997. 1 Abbreviations: Tn and Tm, rabbit skeletal troponin and tropomyosin, respectively; TnC, TnI, and TnT, the Ca 2+ -binding, inhibitory, and Tm-binding subunits of Tn, respectively; 1,5-IAEDANS, N-iodoacetyl-N ′-(5-sulfo-1-naphthyl)ethylenediamine; DAN, the dansyl moiety of 1,5-IAEDANS; DAB-Mal, 4-dimethylaminophenylazophenyl-4′-maleimide; DAB, the dimethylaminophenylazophenyl moiety of DAB-Mal; TnI 48/133 , mutant rabbit skeletal TnI with Cys64 replaced by Ser, retaining Cys48 and Cys133; d-TnI 48/133 , TnI 48/133 labeled with the fluorescence donor DAN on the cysteines; da-TnI 48/133 , TnI 48/133 labeled with both donor and acceptor (DAB); MES, 2-(N-morpholino)ethanesulfonic acid; HEPES, N-(2-hydroxyethyl)piperazine-N ′-(2ethanesulfonic acid); DTT, dithiothreitol; EDTA, ethlenediaminetetraacetic acid; PMSF, phenylmethanesulfonylfluoride; ATP, adenosine 5′-triphosphate; PCR, polymerase chain reaction; RET, resonance energy transfer; MOM, method-of-moments; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Functional role of the core gap in the middle part of tropomyosin
The FEBS journal, 2017
Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein playing an essential role in the regulation of muscle contraction. The middle part of the Tpm molecule has some specific features, such as the presence of non-canonical residues as well as a substantial gap at the interhelical interface, which are believed to destabilize coiled-coil and impart structural flexibility to this part of the molecule. To study how the gap affects structural and functional properties of α-striated Tpm (Tpm1.1 isoform that is expressed in cardiac and skeletal muscles) we substituted large conserved apolar core residues located at both sides of the gap with smaller ones by mutations M127A/I130A and M141A/Q144A. We found that in contrast to stabilizing substitutions D137L and G126R studied earlier these substitutions have no appreciable influence on thermal unfolding and domain structure of the Tpm molecule. They also do not affect actin-binding properties of Tpm. However, they strongly incre...