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1.
Dassanayake Mudiyanselage Sudarshi Premawardhana Fang Zhang Jin Xu Matthew J. Gage 《Biochemistry and Biophysics Reports》2020
The muscle protein titin plays a crucial role in passive elasticity and the disordered PEVK region within titin is central to that function. The PEVK region is so named due to its high proline, glutamate, valine and lysine content and the high charge density in this region results in a lack of organized structure within this domain. The PEVK region is highly extensible but the molecular interactions that contribute to the elastic nature of the PEVK still remain poorly described. The PEVK region is formed by two unique sequence motifs. The PPAK motif is a 26 to 28 amino acid sequence that contains a mixture of charged and hydrophobic residues and is the primary building block for the PEVK region. Poly-E sequence motifs vary in length and contain clusters of 3–4 glutamic acids distributed throughout the motif. In this study, we derived two 28-residue peptides from the human titin protein sequence and measured their structural characteristics over a range of pHs. Our results demonstrate that the poly-E peptide undergoes a shift from a more rigid and elongated state to a more collapsed state as pH decreases with the midpoint of this transition being at pH ~5.5. Interestingly, a similar conformational shift is not observed in the PPAK peptide. These results suggest that the poly-E motif might provide a nucleating site for the PEVK when the muscle is not in an extended state. 相似文献
2.
Linke WA Kulke M Li H Fujita-Becker S Neagoe C Manstein DJ Gautel M Fernandez JM 《Journal of structural biology》2002,137(1-2):194-205
The PEVK domain of the giant muscle protein titin is a proline-rich sequence with unknown secondary/tertiary structure. Here we compared the force-extension behavior of cloned cardiac PEVK titin measured by single-molecule atomic force spectroscopy with the extensibility of the PEVK domain measured in intact cardiac muscle sarcomeres. The analysis revealed that cardiac PEVK titin acts as an entropic spring with the properties of a random coil exhibiting mechanical conformations of different flexibility. Since in situ, titin is in close proximity to the thin filaments, we also studied whether the PEVK domain of cardiac or skeletal titin may interact with actin filaments. Interaction was indeed found in the in vitro motility assay, in which recombinant PEVK titin constructs slowed down the sliding velocity of actin filaments over myosin. Skeletal PEVK titin affected the actin sliding to a lesser degree than cardiac PEVK titin. The cardiac PEVK effect was partially suppressed by physiological Ca(2+) concentrations, whereas the skeletal PEVK effect was independent of [Ca(2+)]. Cosedimentation assays confirmed the Ca(2+)-modulated actin-binding propensity of cardiac PEVK titin, but did not detect interaction between actin and skeletal PEVK titin. In myofibrils, the relatively weak actin-PEVK interaction gives rise to a viscous force component opposing filament sliding. Thus, the PEVK domain contributes not only to the extensibility of the sarcomere, but also affects contractile properties. 相似文献
3.
The I-band region of the giant muscle protein titin contains a large domain enriched for the amino acids proline, glutamate, valine, and lysine and is denoted the PEVK domain. The PEVK domain of titin encodes a random coil shown to be an important factor in the passive elasticity of titin. Muscle-specific splicing of 116 PEVK exons encodes this domain. It has been proposed that proline contents determine the elasticity of the PEVK polypeptide, where the individual exons code for "flexibility cassettes." To test this hypothesis, we have measured the elasticity of three distinct polypeptides encoded by individual PEVK exons (161, 120 and 184) that varied greatly in their proline contents (7, 14, and 37% respectively) and total PEVK contents (55, 70, and 87%). We used single molecule atomic force microscopy techniques to measure the persistence length, p, of the engineered PEVK proteins. Surprisingly, all three exons 161, 120, and 184 coded for proteins with similar values of persistence length, p = 0.92 +/- 0.38, 0.89 +/- 0.42, and 0.98 +/- 0.4 nm, respectively. We conclude that the PEVK exons encode polypeptides of similar elastic properties, unrelated to their total PEVK contents. Hence, alternative splicing solely adjusts the length of the PEVK domain of titin. 相似文献
4.
Titin-actin interaction in mouse myocardium: passive tension modulation and its regulation by calcium/S100A1 总被引:7,自引:0,他引:7
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Yamasaki R Berri M Wu Y Trombitás K McNabb M Kellermayer MS Witt C Labeit D Labeit S Greaser M Granzier H 《Biophysical journal》2001,81(4):2297-2313
Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK-actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK-actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin's extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1-PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction. 相似文献
5.
We have used a biosynthetically incorporated fluorescent probe to monitor domain movements involved in ion transport by the sarcoendoplasmic reticulum Ca-ATPase (SERCA) from rabbit fast-twitch skeletal muscle. X-ray crystal structures suggest that the nucleotide-binding (N) and actuator (A) domains of SERCA move apart by several nanometers upon Ca binding. To test this hypothesis, cDNA constructs were created to fuse cyan-fluorescent protein (CFP) to the N terminus of SERCA (A domain). This CFP-SERCA fluorescent fusion protein retained activity when expressed in Sf21 insect cells using the baculovirus system. Fluorescence resonance energy transfer (FRET) was used to monitor the A-N interdomain distance for CFP-SERCA selectively labeled with fluorescein isothiocyanate (FITC) at Lys 515 in the N domain. At low [Ca (2+)] (E2 biochemical state), the measured FRET efficiency between CFP (donor in A domain) and FITC (acceptor in N domain) was 0.34 +/- 0.03, indicating a mean distance of 61.6 +/- 2.0 A between probes on the two domains. An increase of [Ca (2+)] to 0.1 mM (E1-Ca biochemical state) decreased the FRET efficiency by 0.06 +/- 0.03, indicating an increase in the mean distance by 3.0 +/- 1.2 A. Quantitative molecular modeling of dual-labeled SERCA, including an accurate calculation of the orientation factor, shows that the FRET data observed in the absence of Ca is consistent with the E2 crystal structure, but the increase in distance (decrease in FRET) induced by Ca is much less than predicted by the E1 crystal structure. We conclude that the E1 crystal structure does not reflect the predominant structure of SERCA under physiological conditions in a functional membrane bilayer. 相似文献
6.
《Journal of molecular biology》1996,261(1):62-71
Vertebrate striated muscle behaves elastically when stretched and this property is thought to reside primarily within the giant filamentous protein, titin (connectin). The elastic portion of titin comprises two distinct structural motifs, immunoglobulin (Ig) domains and the PEVK titin, which is a novel motif family rich in proline, glutamate, valine and lysine residues. The respective contributions of the titin Ig and the PEVK sequences to the elastic properties of the molecule have been unknown so far. We have measured both the passive tension in single, isolated myofibrils from cardiac and skeletal muscle and the stretch-induced translational movement of I-band titin antibody epitopes following immunofluorescent labelling of sites adjacent to the PEVK and Ig domain regions. We found that with myofibril stretch, I-band titin does not extend homogeneously. The Ig domain region lengthened predominantly during small stretch, but such lengthening did not result in measurable passive tension and might be explained by straightening, rather than by unfolding, of the Ig repeats. At moderate to extreme stretch, the main extensible region was found to be the PEVK segment whose unravelling was correlated with a steady passive tension increase. In turn, PEVK domain transition from a linearly extended to a folded state appears to be principally responsible for the elasticity of muscle fibers. Thus, the length of the PEVK sequence may determine the tissue-specificity of muscle stiffness, whereas the expression of different Ig domain motif lengths may set the characteristic slack sarcomere length of a muscle type. 相似文献
7.
The elasticity of single titin molecules using a two-bead optical tweezers assay 总被引:2,自引:0,他引:2
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Titin is responsible for the passive elasticity of the muscle sarcomere. The mechanical properties of skeletal and cardiac muscle titin were characterized in single molecules using a novel dual optical tweezers assay. Antibody pairs were attached to beads and used to select the whole molecule, I-band, A-band, a tandem-immunoglobulin (Ig) segment, and the PEVK region. A construct from the PEVK region expressing >25% of the full-length skeletal muscle isoform was chemically conjugated to beads and similarly characterized. By elucidating the elasticity of the different regions, we showed directly for the first time, to our knowledge, that two entropic components act in series in the skeletal muscle titin I-band (confirming previous speculations), one associated with tandem-immunoglobulin domains and the other with the PEVK region, with persistence lengths of 2.9 nm and 0.76 nm, respectively (150 mM ionic strength, 22 degrees C). Novel findings were: the persistence length of the PEVK component rose (0.4-2.7 nm) with an increase in ionic strength (15-300 mM) and fell (3.0-0.3 nm) with a temperature increase (10-60 degrees C); stress-relaxation in 10-12-nm steps was observed in the PEVK construct and hysteresis in the native PEVK region. The region may not be a pure random coil, as previously thought, but contains structured elements, possibly with hydrophobic interactions. 相似文献
8.
The equilibrium structural ensemble of a 20-residue polyglutamic acid peptide (E(20)) was studied with FRET, circular dichroism, and molecular dynamics (MD) simulations. A FRET donor, o-aminobenzamide, and acceptor, 3-nitrotyrosine, were introduced at the N- and C-termini, respectively. Circular dichroism, steady state FRET, and time-resolved FRET measurements were employed to characterize the fraction helix and end-to-end distance under different pH conditions: pH 4 (60% alpha-helix), pH 6 (0% alpha-helix), and pH 9 (0% alpha-helix). At pH 4, the end-to-end distance was measured at 24 A and determined to be considerably less than the 31 A predicted for an alpha-helix of the same length. At pH 6 and 9, the end-to-end distance was measured at > 31 and 39 A respectively, both which are determined to be considerably greater than the 27 A predicted for a freely jointed random coil of the same length. To better understand the physical forces underlying the unusual helix-coil transition in this peptide, three theoretical MD models of E(20) were constructed: (1) a pure alpha-helix, (2) an alpha-helix with equivalent attractive intramolecular contacts, and (3) a weak alpha-helix with termini-weighted intramolecular contacts ("sticky ends"). Using MD simulations, the bent helix structure calculated from Model 3 was found to be the closest in agreement with the experimental data. 相似文献
9.
Interaction forces between F-actin and titin PEVK domain measured with optical tweezers 总被引:1,自引:0,他引:1
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Bianco P Nagy A Kengyel A Szatmári D Mártonfalvi Z Huber T Kellermayer MS 《Biophysical journal》2007,93(6):2102-2109
Titin is a giant protein that determines the elasticity of striated muscle and is thought to play important roles in numerous regulatory processes. Previous studies have shown that titin's PEVK domain interacts with F-actin, thereby creating viscous forces of unknown magnitude that may modulate muscle contraction. Here we measured, with optical tweezers, the forces necessary to dissociate F-actin from individual molecules of recombinant PEVK fragments rich either in polyE or PPAK motifs. Rupture forces at a stretch rate of 250 nm/s displayed a wide, nonnormal distribution with a peak at approximately 8 pN in the case of both fragments. Dynamic force spectroscopy experiments revealed low spontaneous off-rates that were increased even by low forces. The loading-rate dependence of rupture force was biphasic for polyE in contrast with the monophasic response observed for PPAK. Analysis of the molecular lengths at which rupture occurred indicated that there are numerous actin-binding regions along the PEVK fragments' contour, suggesting that the PEVK domain is a promiscuous actin-binding partner. The complexity of PEVK-actin interaction points to an adaptable viscoelastic mechanism that safeguards sarcomeric structural integrity in the relaxed state and modulates thixotropic behavior during contraction. 相似文献
10.
Bengt Svensson Tetsuro Oda Florentin R. Nitu Yi Yang Iustin Cornea Ye Chen-Izu James D. Fessenden Donald M. Bers David D. Thomas Razvan L. Cornea 《Biophysical journal》2014
To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca2+ channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460–2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca2+ leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ∼35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP. 相似文献
11.
Ma K Forbes JG Gutierrez-Cruz G Wang K 《The Journal of biological chemistry》2006,281(37):27539-27556
The richness of proline sequences in titins qualifies these giant proteins as the largest source of intrinsically disordered structures in nature. An extensive search and analysis for Src homology domain 3 (SH3) ligand motifs revealed a myriad of broadly distributed SH3 ligand motifs, with the highest density in the PEVK segments of human titin. Besides the canonical class I and II motifs with opposite orientations, novel overlapping motifs consisting of one or more of each canonical motif are abundant. Experimentally, the binding affinity and critical residues of these putative titin-based SH3 ligands toward nebulin SH3 and other SH3-containing proteins in muscle and non-muscle cell extracts were validated with peptide array technology and by the sarcomere distribution of SH3-containing proteins. A 28-mer overlapping motif-containing PEVK module binds to nebulin SH3 in and around the canonical cleft, especially to the acidic residues in the loops, as revealed by NMR titration. Molecular dynamics and molecular docking studies indicated that the overlapping motif can bind in opposite orientations with comparable energy and contact areas and predicts correctly orientation-specific contacts in NMR data. We propose that the overlap ligand motifs are a new class of ligands with innate ability to dictate SH3 domain orientation and to facilitate the rate, strength, and stereospecificity of receptor interactions. Proline-rich sequences of titins are candidates as major hubs of SH3-dependent signaling pathways. The interplay of elasticity and dense clustering of mixed receptor orientations in titin PEVK segment have important implications for the mechanical sensing, force sensitivity, and inter-adapter interactions in signaling pathways. 相似文献
12.
We have created a set of rationally designed peptides that form calcium-dependent hydrogels based on the beta roll peptide domain. In the absence of calcium, the beta roll domain is intrinsically disordered. Upon the addition of calcium, the peptide forms a beta helix secondary structure. We have designed two variations of our beta roll domain. First, we have mutated one face of the beta roll domain to contain leucine residues so that the calcium-dependent structural formation leads to dimerization through hydrophobic interactions. Second, an α-helical leucine zipper domain is appended to the engineered beta roll domain as an additional means of forming intermolecular cross-links. This full peptide construct forms a hydrogel only in calcium-rich environments. The resulting structural and mechanical properties of the supramolecular assemblies are compared with the wild-type domain using several biophysical techniques including circular dichroism, FRET, bis-ANS binding and microrheology. The calcium responsiveness and rheological properties of the leucine beta roll containing construct confirm the potential of this allosterically regulated scaffold to serve as a cross-linking domain for stimulus-responsive biomaterials development. 相似文献
13.
Titin, a family of giant elastic proteins, constitutes an elastic sarcomere matrix in striated muscle. In the I-band region of the sarcomere, the titin PEVK segment acts as a molecular spring to generate elasticity as well as sites of adhesion with parallel thin filaments. Previously, we reported that PEVK consists of tandem repeats of 28 residue modules and that the "polyproline II-coil" motif is the fundamental conformational motif of the PEVK module. In order to characterize the factors that may affect and alter the PPII-coil conformational motifs, we have initiated a systematic study of the interaction with divalent cations (Cu2+, Ca2+, Zn2+, and Ni2+) and a conformational profile of PEVK peptides (a representative 28-mer peptide PR: PEPPKEVVPEKKAPVAPPKKPEVPPVKV and its subfragments PR1: kvPEPPKEVVPE, PR2: VPEKKAPVAPPK, PR3: KPEVPPVKV). UV-Vis absorption difference spectra and CD spectra showed that Cu2+ bound to PR1 with high affinity (20 microM), while its binding to PR2 and PR3 as well as the binding of other cations to all four peptides were of lower affinity (>100 microM). Conformational studies by CD revealed that Cu2+ binding to PR1 resulted in a polyproline II to turn transition up to a 1:2 PR1/Cu2+ ratio and a coil to turn transition at higher Cu2+ concentration. ESI-MS provided the stoichiometry of PEVK peptide-Cu2+ complexes at both low and high ion strength, confirming the specific high affinity binding of Cu2+ to PR1 and PR. Furthermore, NMR and ESI-MS/MS fragmentation analysis elucidated the binding sites of the PEVK peptide-Cu2+ complexes at (-2)KVPE2, 8VPE10, 13APV15, and 22EVP24. A potential application of Cu2+ binding in peptide sequencing by mass spectrometry was also revealed. We conclude that Cu2+ binds and bends PEVK peptides to a beta-turn-like structure at specific sites. The specific targeting of Cu2+ towards PPII is likely to be of significant value in elucidating the roles of PPII in titin elasticity as well as in interactions of proline-rich proteins. 相似文献
14.
The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface. 相似文献
15.
Modular motif, structural folds and affinity profiles of the PEVK segment of human fetal skeletal muscle titin 总被引:2,自引:0,他引:2
The extension of the PEVK segment of the giant elastic protein titin is a key event in the elastic response of striated muscle to passive stretch. PEVK behaves mechanically as an entropic spring and is thought to be a random coil. cDNA sequencing of human fetal skeletal PEVK reveals a modular motif with tandem repeats of modules averaging 28 residues and with superrepeats of seven modules. Conformational studies of bacterially expressed 53-kDa fragment (TP1) by circular dichroism suggest that this soluble protein contains substantial polyproline II (PPII) type left-handed helices. Urea and thermal titrations cause gradual and reversible decrease in PPII content. The absence of sharp melting in urea and thermal titrations suggests that there is no long range cooperativity among the PPII helices. Studies with solid phase and surface plasmon resonance assays indicate that TP1 interacts with actin and some but not all cloned nebulin fragments with high affinity. Interestingly, Ca(2+)/calmodulin and Ca(2+)/S100 abolish nebulin/PEVK interaction. We suggest that in aqueous solution, PEVK is an open and flexible chain of relatively stable structural folds of the polyproline II type. PEVK region of titin may be involved in interfilament association with thin filaments in a calcium/calmodulin-sensitive manner. This adhesion may modulate titin extensibility and elasticity. 相似文献
16.
A recent publication in Biophysical Journal by Bianco et al. (“Interaction forces between F-actin and titin PEVK domain measured with optical tweezers”) shows that the PEVK domain of titin molecules interacts with F-actin. This newly discovered behavior could influence the mechanical properties of striated muscles, and Bianco et al. suggest that the interactions between actin and titin could modulate thixotropic behavior. In this Comment to the Editor, we suggest that the thixotropic properties of striated muscles in vivo are more likely to reflect dynamic changes in the proportion of myosin cross-bridges bound between the myofilaments. 相似文献
17.
Peter Nagy László Bene William C Hyun Gy?rgy Vereb Manuela Braun Christof Antz Jacques Paysan Sándor Damjanovich John W Park János Sz?llsi 《Cytometry. Part A》2005,67(2):86-96
BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry. 相似文献
18.
Linke WA 《Histology and histopathology》2000,15(3):799-811
Titin, the giant protein of striated muscle, provides a continuous link between the Z-disk and the M-line of a sarcomere. The elastic I-band section of titin comprises two main structural elements, stretches of immunoglobulin-like domains and a unique sequence, the PEVK segment. Both elements contribute to the extensibility and passive force development of nonactivated muscle. Extensibility of the titin segments in skeletal muscle has been determined by immunofluorescence/immunoelectron microscopy of sarcomeres stained with sequence-assigned titin antibodies. The force developed upon stretch of titin has been measured on isolated molecules or recombinant titin fragments with the help of optical tweezers and the atomic force microscope. Force has also been measured in single isolated myofibrils. The force-extension relation of titin could be readily fitted with models of biopolymer elasticity. For physiologically relevant extensions, the elasticity of the titin segments was largely explainable by an entropic-spring mechanism. The modelling explains why during stretch of titin, the Ig-domain regions (with folded modules) extend before the PEVK domain. In cardiac muscle, I-band titin is expressed in different isoforms, termed N2-A and N2-B. The N2-A isoform resembles that of skeletal muscle, whereas N2-B titin is shorter and is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. Examination of N2-B titin extensibility revealed that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at lower stretch and, in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains. However, unfolding of a very small number of Ig domains remains a possibility. 相似文献
19.
Nicolas Niederl?nder Fabrice Raynaud Catherine Astier Patrick Chaussepied 《European journal of biochemistry》2004,271(22):4572-4581
Titin is known to interact with actin thin filaments within the I-band region of striated muscle sarcomeres. In this study, we have used a titin fragment of 800 kDa (T800) purified from striated skeletal muscle to measure the effect of this interaction on the functional properties of the actin-myosin complex. MALDI-TOF MS revealed that T800 contains the entire titin PEVK (Pro, Glu, Val, Lys-rich) domain. In the presence of tropomyosin-troponin, T800 increased the sliding velocity (both average and maximum values) of actin filaments on heavy-meromyosin (HMM)-coated surfaces and dramatically decreased the number of stationary filaments. These results were correlated with a 30% reduction in actin-activated HMM ATPase activity and with an inhibition of HMM binding to actin N-terminal residues as shown by chemical cross-linking. At the same time, T800 did not affect the efficiency of the Ca(2+)-controlled on/off switch, nor did it alter the overall binding energetics of HMM to actin, as revealed by cosedimentation experiments. These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the actin-HMM interface. They also suggest that titin may participate in the regulation of the active tension generated by the actin-myosin complex. 相似文献
20.
To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA+CaMKII sites). FRET increased with protein concentration up to a maximum (FRET(max)) that was taken to represent the intrinsic FRET of the bound complex. The concentration dependence of FRET yielded dissociation constants (K(D)) for the PLB-PLB and PLB-SERCA interactions. PLB-PLB FRET data suggest pseudo-phosphorylation of PLB increased oligomerization of PLB but did not alter PLB pentamer quaternary structure. PLB-SERCA FRET experiments showed an apparent decrease in binding of PLB to SERCA and an increase in the apparent PLB-SERCA binding cooperativity. It is likely that these changes are secondary effects of increased oligomerization of PLB; a change in the inherent affinity of monomeric PLB for SERCA was not detected. In addition, PLB-SERCA complex FRET(max) was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation. 相似文献