Mutant Tropomyosin Research Articles

Persistence Length of Tropomyosin Heterodimers Formed from Wild-Type and Mutant Chains

Mutations in many contractile proteins, including coiled-coil tropomyosin, can lead to hypertrophic cardiomyopathy (HCM) and heart failure. In the case of HCM-linked αα-tropomyosin, affected individuals may be heterozygous for wild-type and mutant protein; hence, heterodimers are likely to form. Indeed, HCM-causing mutant D175N and E180G tropomyosins both readily dimerize with wild-type tropomyosin in vitro and the heterodimers formed then can be purified(see Kalyva et al., 2012). Here, we have studied the material properties of the heterodimers containing these mutant tropomyosins. Electron microscopy was carried out on tropomyosin molecules following adsorption and rotary-shadowing of the molecules on untreated, freshly cleaved mica. Images of gently curved heterodimers showing no obvious kinking are qualitatively similar to ones of wild-type and mutant homodimers previously examined (Li et al., 2010, 2012). The new EMs were skeletonized by cubic interpolation using the MatLab PCHIP program as described in Li et al. (2010) and then persistence length (PL) determined by the tangent correlation method (ibid). PL for the heterodimer containing D175N and wild-type chains (89 nm) was found to be slightly lower than that for homodimeric wild-type molecules (107 nm), but the value for the E180G - wild-type heterodimer (69 nm) was much lower, suggesting that the E180G - wild-type heterodimer is more curved or more flexible than wild-type tropomyosin. These results agree with the lower thermal stability recorded for the E180G heterodimer than for the D175N heterodimer or the wild-type homodimer and may explain the increased phenotypic severity manifested by the E180G than by the D175N mutation.

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca2+, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T HC (high Ca2+ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T a, ±SEM, N = 34; E54K: 1.24±0.07 T a, N = 28), a significantly lower T LC (low- Ca2+ tension at pCa 7.0) (E40K: 0.07±0.02 T a, N = 34; E54K: 0.06±0.02 T a, N = 28), and a significantly lower T act (Ca2+ activatable tension) (T act = T HC–TLC, E40K: 1.15±0.08 T a, N = 34; E54K: 1.18±0.06 T a, N = 28) than WT (T HC = 1.53±0.07 T a, T LC = 0.12±0.01 T a, T act = 1.40±0.07 T a, N = 25). All tensions were normalized to T a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca2+ sensitivity (pCa50) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.

Congenital myopathy-causing tropomyosin mutations induce thin filament dysfunction via distinct physiological mechanisms

In humans, congenital myopathy-linked tropomyosin mutations lead to skeletal muscle dysfunction, but the cellular and molecular mechanisms underlying such dysfunction remain obscure. Recent studies have suggested a unifying mechanism by which tropomyosin mutations partially inhibit thin filament activation and prevent proper formation and cycling of myosin cross-bridges, inducing force deficits at the fiber and whole-muscle levels. Here, we aimed to verify this mechanism using single membrane-permeabilized fibers from patients with three tropomyosin mutations (TPM2-null, TPM3-R167H and TPM2-E181K) and measuring a broad range of parameters. Interestingly, we identified two divergent, mutation-specific pathophysiological mechanisms. (i) The TPM2-null and TPM3-R167H mutations both decreased cooperative thin filament activation in combination with reductions in the myosin cross-bridge number and force production. The TPM3-R167H mutation also induced a concomitant reduction in thin filament length. (ii) In contrast, the TPM2-E181K mutation increased thin filament activation, cross-bridge binding and force generation. In the former mechanism, modulating thin filament activation by administering troponin activators (CK-1909178 and EMD 57033) to single membrane-permeabilized fibers carrying tropomyosin mutations rescued the thin filament activation defect associated with the pathophysiology. Therefore, administration of troponin activators may constitute a promising therapeutic approach in the future.

The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy

Point mutations targeting muscle thin filament proteins are the cause of a number of cardiomyopathies. In many cases, biological effects of the mutations are well-documented, whereas their structural and mechanical impact on filament assembly and regulatory function is lacking. In order to elucidate molecular defects leading to cardiac dysfunction, we have examined the structural mechanics of two tropomyosin mutants, E180G and D175N, which are associated with hypertrophic cardiomyopathy (HCM). Tropomyosin is an α-helical coiled-coil dimer which polymerizes end-to-end to create an elongated superhelix that wraps around F-actin filaments of muscle and non-muscle cells, thus modulating the binding of other actin-binding proteins. Here, we study how flexibility changes in the E180G and D175N mutants might affect tropomyosin binding and regulatory motion on F-actin. Electron microscopy and Molecular Dynamics simulations show that E180G and D175N mutations cause an increase in bending flexibility of tropomyosin both locally and globally. This excess flexibility is likely to increase accessibility of the myosin-binding sites on F-actin, thus destabilizing the low-Ca2+ relaxed-state of cardiac muscle. The resulting imbalance in the on–off switching mechanism of the mutants will shift the regulatory equilibrium towards Ca2+-activation of cardiac muscle, as is observed in affected muscle, accompanied by enhanced systolic activity, diastolic dysfunction, and cardiac compensations associated with HCM and heart failure.

Functional effects of congenital myopathy-related mutations in gamma-tropomyosin gene

Missense mutations in human TPM3 gene encoding γ-tropomyosin expressed in slow muscle type 1 fibers, were associated with three types of congenital myopathies—nemaline myopathy, cap disease and congenital fiber type disproportion. Functional effects of the following substitutions: Leu100Met, Ala156Thr, Arg168His, Arg168Cys, Arg168Gly, Lys169Glu, and Arg245Gly, were examined in biochemical assays using recombinant tropomyosin mutants and native proteins isolated from skeletal muscle. Most, but not all, mutations decreased the affinity of tropomyosin for actin alone and in complex with troponin (±Ca2+). All studied tropomyosin mutants reduced Ca-induced activation but had no effect on the inhibition of actomyosin cross-bridges. Ca2+-sensitivity of the actomyosin interactions, as well as cooperativity of myosin-induced activation of the thin filament was affected by individual tropomyosin mutants with various degrees. Decreased motility of the reconstructed thin filaments was a result of combined functional defects caused by myopathy-related tropomyosin mutants. We conclude that muscle weakness and structural abnormalities observed in TPM3-related congenital myopathies result from reduced capability of the thin filament to fully activate actin–myosin cross-bridges.

DCM-Linked D230N Tropomyosin Mutation Results in Early Dilatation and Systolic Dysfunction in Mice

Recently, a study in two large multi-generational families described a familial dilated cardiomyopathy (DCM) caused by a single amino acid substitution Asp230Asn (D230N) in tropomyosin. These families demonstrated a unique bimodal disease distribution in which infants presented with a severe form of DCM, while adults presented with a mild to moderate clinical phenotype. To determine the biophysical consequences of this mutation on tropomyosin and its effects on regulatory function in the sarcomere, we employed circular dichroism and the regulated in vitro motility assay. We found that while this mutation does not affect overall thermal stability of tropomyosin, it has a profound effect on regulatory function. As previously shown in solution, the presence of the D230N mutation decreases the maximal velocity of filament sliding and calcium sensitivity of thin filament activation compared to wild type filaments. Additionally, the D230N mutation increases the cooperativity of myofilament activation. In order to further explore our biophysical observations and the physiologic effects of the D230N mutation, we created a transgenic murine model. In mice carrying the D230N tropomyosin mutation we found evidence of early dilatation and systolic dysfunction by echocardiogram in the absence of histological changes such as fibrosis or inflammatory cell invasion. Ultrastructural analysis of transgenic left ventricular tissue demonstrated z-disk alterations. Finally, preliminary studies on isolated myocytes from transgenic mice loaded with fura-2AM demonstrate no discernible differences in calcium transients compared to non-transgenic siblings suggesting that functional impairments are not due to calcium handling defects. Collectively, these studies suggest that the D230N mutation in tropomyosin is responsible for alterations in structure and function of the thin filament that result in a primary dilatation of the cardiac left ventricle. This work is supported by funding from the Children's Cardiomyopathy Foundation.

Do We Know the Complete Story of TPM1κ Expression in Vertebrate Hearts?

Copyright: © 2012 Siddiqui MT. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Tropomyosin (TM) is a component of myofibrils, the contractile apparati of striated muscle cells. The assembly of a myofibril involves the precise ordering of several proteins into a linear array of sarcomeres. The process is complicated further because most of these proteins have multiple isoforms. Recent demonstration of the association of TM mutations with Hypertrophic Cardiomyopathy (HCM)/Familial Hypertrophic Cardiomyopathy (FHC) [1-3] or other cardiomyopathies such as Dilated Cardiomyopathy (DCM) [4,5] and nemalin skeletal myopathy [6] in humans have spurred renewed interest in the structural/functional relationships of TM. In vertebrate striated muscle, the thin filament consists largely of actin, TM, the Troponin (Tn) complex (Tn-I, Tn-C and Tn-T), Tropomodulin (Tmod), and Leiomodin (Lmod). It is responsible for mediating Ca+2 control of contraction and relaxation. With the influx of Ca+2 in muscle cells, troponin-C binds with Ca+2 and undergoes a rapid conformational change, which causes additional conformation changes in the TM-Tn complex exposing the myosin associated ATPase activity. As a result, the free myosin associated ATPase activity hydrolyzes ATP with the release of energy that helps muscle to contract. There are four genes (designated as TPM1, TPM2, TPM3, and TPM4) for TM in vertebrates [7,8] except for zebrafish where there are six TM genes [9], which generate a multitude of tissue and developmental specific isoforms through the use of different promoters, alternative mRNA splicing, and tissue specific translational control [10]. The functional significance of this diversity and how it may affect interactions with other sarcomeric proteins is not fully understood. Sarcomeric isoforms for both TPM1 and TPM2 are expressed in large mammals, including humans [11]. The protein of the sarcomeric isoform of TPM3 is not expressed in mammalian hearts. In mammals TPM4 is truncated relative to the gene found in avian [12] and amphibian species [13].

Abstract P248: The DCM-Causing D230N Tropomyosin Mutation Produces an Age-Dependent Phenotype in Mice that Is Influenced by Transgene Dosage

A recent study in two large multigenerational families demonstrated a novel mutation in tropomyosin (Tm), Asp230Asn (D230N) that caused DCM with a unique natural history resulting in a striking bimodal disease distribution. Infants and young children with the D230N Tm mutation presented with a severe, often fatal DCM while adults developed a mild to moderate clinical phenotype. Position 230 in tropomyosin is proximal to the C-terminal unwinding of tropomyosin that is essential for the Tm-Tm overlap and for the Tm-cardiac troponin T (cTnT) interaction. We hypothesized that the age-dependent remodeling is a result of temporal isoform switches in the closely linked Tm-binding partner cTnT. To directly address this hypothesis, we used the regulated in vitro motility assay to determine the biophysical effects of the D230N Tm mutation as well as the effects of cTnT isoform changes on the regulatory function of thin filaments. We have found that both wild type and D230N Tm filaments inhibit filament sliding at low calcium indicating that the D230N mutation does not completely disrupt regulation. However in the presence of high calcium the D230N Tm mutation significantly decreases the maximal velocity of filament sliding (3.790 ± 0.1333 microns/sec, n=98) as compared to the wild type filaments (4.900 ± 0.1044, microns/sec n=76). These findings support an altered Tm-cTnT interaction because at maximal calcium concentrations, the myofilament activation at the level of cardiac troponin is not properly transmitted to tropomyosin thus decreasing the activity of myosin-ATPase. In order to extend our biophysical observations and to provide translational insight, we generated a D230N Tm transgenic mouse model. In preliminary studies we demonstrate that at two months the D230N transgenic mice have evidence for early dilatation. In addition, the differences in ventricular remodeling were highly dependent on transgene dose. At six months, we observed dilatation of the ventricle and thinning of the walls compared to non-transgenic siblings. However, the differences were not as marked as those at the two-month time point. Collectively, these findings support an age-dependent DCM phenotype that is modulated by transgene dosage.

Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into “ghost muscle fibers”. The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.

Several cardiomyopathy causing mutations on tropomyosin either destabilize the active state of actomyosin or alter the binding properties of tropomyosin

We examined the cardiomyopathy-causing tropomyosin mutations E180G, D175N, and V95A to determine their effects on actomyosin regulation. V95A reduced the ATPase rate when filaments were saturated with regulatory proteins both in the presence and absence of calcium, indicating either a stabilization of the inactive state or an inability to fully populate the active state. Effects of E180G and D175N were more complex. These two mutations increased ATPase rates at sub-saturating concentrations of troponin and tropomyosin as compared to wild type tropomyosin. At higher concentrations of regulatory proteins, ATPase rates became similar to wild type. Normal activation was achieved with the tight-binding myosin analog N-ethylmaleimide-S1, at saturating regulatory protein concentrations. These results suggest that the E180G and D175N mutations reduce the affinity of tropomyosin for actin and also destabilize troponin binding to the actin thin filaments.

Monitoring the Real-Time Binding of Tropomyosin to Actin using Total Internal Reflection Fluorescence Microscopy

Tropomyosin, an elongated actin binding protein, regulates the access of numerous other proteins onto the thin filament in virtually all cell types. This association, in turn, regulates various functions. Tropomyosin monomers assemble end-to-end to form a continuous polymeric chain that lies along the length of the actin filament. A number of different tropomyosin isoforms are present in all three muscle types: viz. smooth, cardiac, and skeletal. In cardiac and skeletal muscle, tropomyosin and troponin are the primary regulators of contraction. A plethora of tropomyosin isoforms are also present in non-muscle cells where they associate with cytoskeletal actin and play a role in numerous cellular processes including lamellipodia formation and actin-based cell motility. Moreover, various mutations in tropomyosin are associated with diseases such as congenital fiber type distortion (CFTD), ulcerative colitis, nemaline myopathy, and various cardiomyopathies. Tropomyosin is a modular protein, and individually charged residues within its repeating domains are thought to be critical for actin binding. However, the mechanism of tropomyosin binding to actin is still uncertain. In the present study, we have acquired real-time videos of skeletal and smooth tropomyosin isoforms binding to actin filaments using total internal reflection fluorescence microscopy (TIRF). The videos suggest that weak monomer binding facilitates the gradual formation of small “nuclei” of tropomyosin monomers and/or oligomers. Once the nucleus is formed, the affinity of tropomyosin for actin increases exponentially and additional monomers are able to rapidly add to either end of the growing tropomyosin chain lying on F-actin.

Enhanced Active Cross-Bridges during Diastole: Molecular Pathogenesis of Tropomyosin's HCM Mutations

Three HCM-causing tropomyosin (Tm) mutants (V95A, D175N, and E180G) were examined using the thin-filament extraction and reconstitution technique. The effects of Ca 2+, ATP, phosphate, and ADP concentrations on cross-bridge kinetics in myocardium reconstituted with each of these mutants were studied at 25°C, and compared to wild-type (WT) Tm at physiological ionic strength (200 mM). All three mutants showed significantly higher (2–3.5 fold) low Ca 2+ tension ( T LC ) and stiffness than WT at pCa 8.0. High Ca 2+ tension ( T HC ) was significantly higher for E180G than that for WT, whereas T HC of V95A and D175N was similar to WT; high Ca 2+ stiffness ( Y HC ) had the same trend. The Ca 2+ sensitivity of isometric force was significantly greater for V95A and E180G than for WT, whereas that of D175N remained the same as for WT; for all mutants, cooperativity was lower than for WT. Nine kinetic constants and the cross-bridge distribution were deduced using sinusoidal analysis. The number of force-generating cross bridges was similar among the D175N, E180G, and WT Tm forms, but it was significantly larger in the case of V95A than WT. We conclude that the increased number of actively cycling cross bridges at pCa 8 is the major cause of Tm mutation-related HCM pathogenesis, which may result in diastolic dysfunction. Decreased contractility ( T act ) in V95A and D175N may further contribute to the severity of myocyte hypertrophy and related prognosis of the disease.

Functional effects of a tropomyosin mutation linked to FHC contribute to maladaptation during acidosis

Familial hypertrophic cardiomyopathy (FHC) is a leading cause of sudden cardiac death among young athletes but the functional effects of the myofilament mutations during FHC-associated ischemia and acidosis, due in part to increased extravascular compressive forces and microvascular dysfunction, are not well characterized. We tested the hypothesis that the FHC-linked tropomyosin (Tm) mutation Tm-E180G alters the contractile response to acidosis via increased myofilament Ca2+ sensitivity. Intact papillary muscles from transgenic (TG) mice expressing Tm-E180G and exposed to acidic conditions (pH 6.9) exhibited a significantly smaller decrease in normalized isometric tension compared to non-transgenic (NTG) preparations. Times to peak tension and to 90% of twitch force relaxation in TG papillary muscles were significantly prolonged. Intact single ventricular TG myocytes demonstrated significantly less inhibition of unloaded shortening during moderate acidosis (pH 7.1) than NTG myocytes. The peak Ca2+ transients were not different for TG or NTG at any pH tested. The time constant of re-lengthening was slower in TG myocytes, but not the rate of Ca2+ decline. TG detergent-extracted fibers demonstrated increased Ca2+ sensitivity of force and maximal tension compared to NTG at both normal and acidic pH (pH 6.5). Tm phosphorylation was not different between TG and NTG muscles at either pH. Our data indicate that acidic pH diminished developed force in hearts of TG mice less than in NTG due to their inherently increased myofilament Ca2+ sensitivity, thus potentially contributing to altered energy demands and increased propensity for contractile dysfunction.

A myopathy-linked tropomyosin mutation severely alters thin filament conformational changes during activation

Human point mutations in beta- and gamma-tropomyosin induce contractile deregulation, skeletal muscle weakness, and congenital myopathies. The aim of the present study was to elucidate the hitherto unknown underlying molecular mechanisms. Hence, we recorded and analyzed the X-ray diffraction patterns of human membrane-permeabilized muscle cells expressing a particular beta-tropomyosin mutation (R133W) associated with a loss in cell force production, in vivo muscle weakness, and distal arthrogryposis. Upon addition of calcium, we notably observed less intensified changes, compared with controls, (i) in the second (1/19 nm(-1)), sixth (1/5.9 nm(-1)), and seventh (1/5.1 nm(-1)) actin layer lines of cells set at a sarcomere length, allowing an optimal thin-thick filament overlap; and (ii) in the second actin layer line of overstretched cells. Collectively, these results directly prove that during activation, switching of a positive to a neutral charge at position 133 in the protein partially hinders both calcium- and myosin-induced tropomyosin movement over the thin filament, blocking actin conformational changes and consequently decreasing the number of cross-bridges and subsequent force production.

Initial Polarized Bud Growth by Endocytic Recycling in the Absence of Actin Cable–dependent Vesicle Transport in Yeast

The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast.

Combinatorial effects of double cardiomyopathy mutant alleles in rodent myocytes: a predictive cellular model of myofilament dysregulation in disease.

Inherited cardiomyopathy (CM) represents a diverse group of cardiac muscle diseases that present with a broad spectrum of symptoms ranging from benign to highly malignant. Contributing to this genetic complexity and clinical heterogeneity is the emergence of a cohort of patients that are double or compound heterozygotes who have inherited two different CM mutant alleles in the same or different sarcomeric gene. These patients typically have early disease onset with worse clinical outcomes. Little experimental attention has been directed towards elucidating the physiologic basis of double CM mutations at the cellular-molecular level. Here, dual gene transfer to isolated adult rat cardiac myocytes was used to determine the primary effects of co-expressing two different CM-linked mutant proteins on intact cardiac myocyte contractile physiology. Dual expression of two CM mutants, that alone moderately increase myofilament activation, tropomyosin mutant A63V and cardiac troponin mutant R146G, were shown to additively slow myocyte relaxation beyond either mutant studied in isolation. These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles αTmA63V and cTnI R193H, which approached a functional threshold. Interestingly, a combination of a CM myofilament deactivating mutant, troponin C G159D, together with an activating mutant, cTnIR193H, produced a hybrid phenotype that blunted the strong activating phenotype of cTnIR193H alone. This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination. Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients.

Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

An Evolutionary Structural Bioinformatics Analysis of the Actin Binding Protein, Tropomyosin

Tropomyosin is a two-chained, α-helical coiled-coil protein that associates end-to-end to form a continuous strand along actin filaments and regulates the functions and stability of actin. Mutations in tropomyosin cause skeletal and cardiomyopathies. We have carried out a phylogenetic analysis of tropomyosin to identify its conserved residues and to elucidate their importance for binding actin. Actin is a highly conserved protein and our hypothesis is that the actin binding sites of tropomyosin have been conserved through evolution to retain its actin-binding function. Phylogenetic trees of 60 coding sequences were constructed from tropomyosin genes from 19 species within the phyla Chordata, Hemichordata and Echinoderm. The rates of substitution (ω) at amino acid sites (codons) in the protein sequence were calculated to identify the most conserved sites (lowest ω) using CODEML in PAML 4.1. A total of 103 out of 284 residues were identified as highly conserved (ω ≤ 0.015), of which 24 are in a and d positions of the heptad repeat involved in the hydrophobic coiled coil interface, 38 are in potential interhelical e-g position salt bridges important for folding of the coiled coil, and 41 are in b, c, or f surface positions available for binding to other proteins, such as actin. The conserved residues at the b, c, and f surface positions were selected for initial mutagenesis studies with mutations to alanine. Preliminary actin binding co-sedimentation assays carried out for three tropomyosin mutants showed a 2- to 3-fold decrease in actin-binding affinity compared to the wild-type protein. Further analysis of mutations at other conserved surface positions and their effect on actin-binding affinity and stability of the mutant proteins are in progress. Supported by Muscle Dystrophy Association.

Studies of the Mid Region of Tropomyosin Using an Incorporated Tryptophan Analogue

Striated muscle contraction is controlled by highly sensitive alterations in thin filament conformation that involve a Ca2+-dependent interaction between troponin I (TnI) and actin-tropomyosin (Tm). To study these interactions, 5-OH Tryptophan (OHW) was incorporated into striated muscle alpha-tropomyosin. A Single Trp tropomyosin mutant, Q135W, was generated and expressed in Trp auxotrophs. OHW incorporation was 76%. Thin filaments containing Q135W retained excellent function: 12-fold Ca2+ regulation in myosin S1-thin filament MgATPase assays. Fluorescence emission titrations with increasing amounts of Ca2+ were performed (emission max 338nm) in presence of thin filaments comprised of cardiac troponin, F-actin, and Q135W tropomyosin. Similar titrations using troponin, actin, and wt Trp-free tropomyosin produced low fluorescence (ex wavelength 312 nm). Ca2+ decreased the OHW fluorescence of Q135W-containing thin filaments by 7.0 +/− 0.4 %. This transition occurred with K = 1.8 +/− 0.3 uM −1, consistent with the affinity of the cardiac thin filament regulatory calcium site on TnC. OHW Q135W tropomyosin is a novel tool for monitoring thin filament behavior, in particular for detecting Ca2+ binding to troponin. Troponin has been viewed as binding to the C-terminus of tropomyosin, which does not include the mid-tropomyosin residue 135 that is found in the current study to be Ca2+-sensitive. However, the findings may be related to recent results, suggesting that the TnI C-terminus of troponin bridges from one actin strand to the other, and contacts tropomyosin near residue 146. Galinska-Rakoczy et al, JMB 2008. Mudalige et al, JMB2009. Finally, human cardiac troponins with defective inhibitory activity due to alterations in the TnI inhibitory region have been generated. They are currently under study using this tropomyosin.

α-트로포마이오신의 276 또는 277 아미노산 잔기가 단일 시스테인 잔기로 치환된 돌연변이 트로포마이오신의 액틴친화력

화학적 변형 방식에 의한 트로포마이오신과 액틴의 상호작용을 규명하기 위하여 액틴결합에 중요한 역할을 하는 C-말단부위의 아미노산 잔기 276 또는 277을 단일 시스테인 잔기로 치환한 돌연변이 트로포마이오신을 제조하여 대장균에서 대량 발현시킨 후 액틴 결합력을 측정하였다. 잔기277을 시스테인 잔기로 치환시킨 TM24(QC) 및 TM29(HC)는 액틴 결합 성질을 잃어버렸을 뿐만 아니라 트로포닌 존재 하에서도 액틴결합력이 증가하지 않았다. 이 결과는 잔기 277이 트로포마이신 기능에 중요한 역할을 한다는 것을 제시한다. 반면 잔기 276을 시스테인 잔기로 치환한 TM22(CT) 및 TM23(CA)는 액틴과 비교적 잘 결합하였을 뿐만 아니라 트로포닌 존재 하에서 액틴결합력이 증가하였다. 따라서 TM23(CA)는 시스테인 잔기를 도입하여도 트로포마이오신의 기능을 유지하였으며 향후 화학적 변형 연구를 위한 도구로 중요하게 사용될 수 있을 것이다. It has been previously reported that the carboxyl terminal residues 276 and 277 of <TEX>${\alpha}$</TEX>-tropomyosin are important for actin affinity. In order to investigate actin affinities of these two residues of skeletal (HA) and smooth (QT) muscle <TEX>${\alpha}$</TEX>-tropomyosins, a series of mutant tropomyosins were constructed in which residues at either 276 or 277 were individually replaced with a cysteine residue for chemical modification. These mutants were overexpressed in E. coli as unacetylated and Ala-Ser (AS) dipeptide fusion forms. While actin affinities of unacetylated tropomyosins were considerably low, those of AS/TMs were remarkably higher than those of corresponding unacetylated tropomyosins. However, actin affinities of AS/TM24 (QC) and AS/TM29 (HC) were dramatically lower than those of other AS/TMs and were close to those of unacetylated tropomyosins. In addition, actin affinities of unacetylated TM24 (QC) and TM29 (HC) failed to be restored in the presence of troponin, unlike unacetylated TM10 (HA) and TM23 (CA). These results indicated that the presence of a cysteine residue at 277 caused a drastic decrease in actin affinity, and also that the residue 277 is important for actin affinity of <TEX>${\alpha}$</TEX>-tropomyosin. Since TM23 (CA) showed high actin affinity, it may serve as a valuable tool for chemical modification studies for investigating the interaction of the carboxyl terminal residues of <TEX>${\alpha}$</TEX>-tropomyosin with actin and/or troponin.