Folding and Function of the Troponin Tail Domain
2002; Elsevier BV; Volume: 278; Issue: 1 Linguagem: Inglês
10.1074/jbc.m209194200
ISSN1083-351X
AutoresAshley Hinkle, Larry S. Tobacman,
Tópico(s)Viral Infections and Immunology Research
ResumoTroponin contains a globular Ca2+-binding domain and an elongated tail domain composed of the N terminus of subunit troponin T (TnT). The tail domain anchors troponin to tropomyosin and actin, modulates myosin function, and is a site of cardiomyopathy-inducing mutations. Critical interactions between tropomyosin and troponin are proposed to depend on tail domain residues 112–136, which are highly conserved across phyla. Most cardiomyopathy mutations in TnT flank this region. Three such mutations were examined and had contrasting effects on peptide TnT-(1–156), promoting folding and thermal stability assessed by circular dichroism (F110I) or weakening folding and stability (T104V and to a small extent R92Q). Folding of both TnT-(1–156) and whole troponin was promoted by replacing bovine TnT Thr-104 with human TnT Ala-104, further indicating the importance of this cardiomyopathy site residue for protein folding. Mutation F110I markedly stabilized the troponin tail but weakened binding of holo-troponin to actin-tropomyosin 8-fold, suggesting that loss of flexibility impairs troponin tail function. The effect of the F110I mutation on troponin-tropomyosin binding to actin was much less, indicating this flexibility is particularly important for the interactions of troponin with tropomyosin. We suggest that most cardiomyopathic mutations in the troponin tail alter muscle function indirectly, by perturbing interactions between troponin and tropomyosin requisite for the complex effects of these proteins on myosin. Troponin contains a globular Ca2+-binding domain and an elongated tail domain composed of the N terminus of subunit troponin T (TnT). The tail domain anchors troponin to tropomyosin and actin, modulates myosin function, and is a site of cardiomyopathy-inducing mutations. Critical interactions between tropomyosin and troponin are proposed to depend on tail domain residues 112–136, which are highly conserved across phyla. Most cardiomyopathy mutations in TnT flank this region. Three such mutations were examined and had contrasting effects on peptide TnT-(1–156), promoting folding and thermal stability assessed by circular dichroism (F110I) or weakening folding and stability (T104V and to a small extent R92Q). Folding of both TnT-(1–156) and whole troponin was promoted by replacing bovine TnT Thr-104 with human TnT Ala-104, further indicating the importance of this cardiomyopathy site residue for protein folding. Mutation F110I markedly stabilized the troponin tail but weakened binding of holo-troponin to actin-tropomyosin 8-fold, suggesting that loss of flexibility impairs troponin tail function. The effect of the F110I mutation on troponin-tropomyosin binding to actin was much less, indicating this flexibility is particularly important for the interactions of troponin with tropomyosin. We suggest that most cardiomyopathic mutations in the troponin tail alter muscle function indirectly, by perturbing interactions between troponin and tropomyosin requisite for the complex effects of these proteins on myosin. troponin T familial hypertrophic cardiomyopathy In striated muscles, including the heart and skeletal muscle, contraction is tightly regulated by the reversible binding of Ca2+ to the thin filament protein troponin. Tight and specific attachment of troponin to the thin filament is mediated by the troponin tail domain, which is composed of the N-terminal portion of TnT1 and interacts with the tropomyosin C terminus. Hydrodynamic studies (1Byers D.M. McCubbin W.D. Kay C.M. FEBS Lett. 1979; 104: 106-110Crossref PubMed Scopus (18) Google Scholar), rotary-shadowed electron micrographs of troponin (2Flicker P.F. Phillips G.N., Jr. Cohen C. J. Mol. Biol. 1982; 162: 495-501Crossref PubMed Scopus (137) Google Scholar), and intermediate resolution studies of both troponin-tropomyosin (3Phillips G.N., Jr. Lattman E.E. Cummins P. Lee K.Y. Cohen C. Nature. 1979; 278: 413-417Crossref PubMed Scopus (110) Google Scholar) and TnT-tropomyosin co-crystals (4Cabral-Lilly D. Tobacman L.S. Mehegan J.P. Cohen C. Biophys. J. 1996; 73: 1763-1770Abstract Full Text PDF Scopus (34) Google Scholar) indicate that the tail domain is highly asymmetric and ∼160 Å in length. Electron microscopy of tropomyosin-TnT co-crystals suggests that a long region of the tropomyosin C terminus may interact with the troponin tail (4Cabral-Lilly D. Tobacman L.S. Mehegan J.P. Cohen C. Biophys. J. 1996; 73: 1763-1770Abstract Full Text PDF Scopus (34) Google Scholar). However, most of this extended interaction may be very weak, because a variety of other evidence suggests that only the C terminus of tropomyosin binds strongly to troponin (reviewed in Ref. 5Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (461) Google Scholar). Recently, an x-ray crystallographic study of the tropomyosin C terminus identified and determined the structure of an 18-residue tropomyosin region that comprises a critical TnT-binding site (6Li Y. Mui S. Brown J.H. Strand J. Reshetnikova L. Tobacman L.S. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7378-7383Crossref PubMed Scopus (84) Google Scholar). The TnT element that binds to this tropomyosin region is unknown. A new approach to these important interactions has been provided by the discovery that any of several mutations in the troponin tail region can cause the autosomal dominant disorder, familial hypertrophic cardiomyopathy (FHC). Regardless whether because of mutations in thick filament or thin filament components of the cardiac sarcomere, the characteristic finding in FHC patients is missense or mild truncation mutations (7Geisterfer-Lowrance A.A. Kass S. Tanigawa G. Vosberg H.P. McKenna W. Seidman C.E. Seidman J.G. Cell. 1990; 62: 999-1006Abstract Full Text PDF PubMed Scopus (1054) Google Scholar, 8Thierfelder L. Watkins H. MacRae C. Lamas R. McKenna W. Vosberg H.-P. Seidman J.G. Seidman C.E. Cell. 1994; 77: 701-712Abstract Full Text PDF PubMed Scopus (864) Google Scholar, 9Watkins H. Conner D. Thierfelder L. Jarcho J.A. MacRae C. McKenna W.J. Maron B.J. Seidman J.G. Seidman C.E. Nat. Genet. 1995; 11: 434-437Crossref PubMed Scopus (479) Google Scholar, 10Bonne G. Carrier L. Bercovici J. Cruaud C. Richard P. Hainque B. Gautel M. Labeit S. James M. Beckman J. Weissenbach J. Vosberg H.-P. Fiszman M. Komajda M. Schwartz K. Nat. Genet. 1995; 11: 438-440Crossref PubMed Scopus (367) Google Scholar, 11Watkins H. 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The disease phenotype has high penetrance, including myofibrillar disarray and risk of sudden death by arrhythmia. Therefore, troponin tail function must be altered and protein function studies, if sufficiently sensitive, are likely to identify abnormalities. Indeed, beginning in 1996 (14Lin D. Bobkova A. Homsher E. Tobacman L.S. J. Clin. Invest. 1996; 97: 2842-2848Crossref PubMed Scopus (103) Google Scholar), numerous studies have described effects on unloaded sliding speed, force, Ca2+ affinity, Ca2+ sensitivity, and cooperativity (15Sweeney H.L. Feng H.S. Yang Z. Watkins H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14406-14410Crossref PubMed Scopus (134) Google Scholar, 16Morimoto S. Yanaga F. Minakami R. Ohtsuki I. Am. J. Physiol. 1998; 44: C200-C207Crossref Google Scholar, 17Homsher E. Lee D.M. Morris C. Pavlov D. Tobacman L.S. J. Physiol. ( Lond. ). 2000; 524: 233-243Crossref PubMed Scopus (78) Google Scholar, 18Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. 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J. Biol. Chem. 2001; 276: 3743-3755Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). These effects support the view that the troponin tail has subtle effects on myosin, in addition to its critical function as an anchoring domain. The present work concerns three FHC-inducing TnT mutations that are of interest because of their location in the important but poorly understood troponin tail region, and because their unexpected effects on troponin solubility suggested alterations in protein folding. In an earlier publication (18Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), we found that mutations R92Q and F110I (11Watkins H. McKenna W.J. Thierfelder L. Suk H.J. Anan R. O'Donoghue A. Spirito P. Matsumori A. Moravec C.S. Seidman J.G. Seidman C.E. N. Engl. J. Med. 1995; 332: 1058-1064Crossref PubMed Scopus (782) Google Scholar) greatly impaired the solubility of bovine cardiac troponin. (Bovine TnT amino acids are designated in the present report, unlike some of our previous work (18Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 25Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274: 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 26Tobacman L.S. Nihli M. Butters C. Heller M. Hatch V. Craig R. Lehman W. Homsher E. J. Biol. Chem. 2002; 277: 27636-27642Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), by the sequence positions of homologous human cardiac TnT residues (27Mesnard L. Samson F. Espinasse I. Durand J. Neveux J.-Y. Mercadier J.-J. FEBS Lett. 1993; 328: 139-144Crossref PubMed Scopus (56) Google Scholar).) Although it was possible to reconstitute troponin complexes containing these mutant TnTs by gradually dialyzing away denaturant under high salt conditions, R92Q troponin precipitated when the ionic strength was decreased below 0.5m, and F110I troponin precipitated unless 10% glycerol was added in addition to a high ionic strength buffer. Insoluble proteins can cause human disease (28Dobson C.M. Nature. 2002; 418: 729-730Crossref PubMed Scopus (303) Google Scholar, 29Taylor J.P. Hardy J. Fischbeck K.H. Science. 2002; 296: 1991-1995Crossref PubMed Scopus (1023) Google Scholar), but there was no precedent for protein insolubility in FHC. Therefore, it seemed plausible that the observations were due to the use of bovine rather than human troponin. In this regard, it is notable that between residues 70 and 141, human and bovine TnTs are identical except at one position: bovine Thr-104 is Ala in the human sequence. Because this residue is located near both positions 92 and 110, the sites of mutations causing poor solubility, we created bovine TnT double mutants R92Q/T104A and F110I/T104A. As shown below, this maneuver corrected the insolubility and permitted reconstitution of functional troponins, suggesting the importance of residue 104 in proper folding of the troponin tail. Interestingly, there is a family with FHC due to the TnT mutation T104V (30Nakajima-Taniguchi C. Matusi H. Fujio Y. Nagata S. Kishimoto T. Yamauchi-Takihara K. J. Mol. Cell. Cardiol. 1997; 29: 839-843Abstract Full Text PDF PubMed Scopus (54) Google Scholar), reinforcing the significance of this residue for folding and/or function. The results below detail the effects of R92Q, T104A, T104V, and F110I mutations on the folding of the troponin tail region and on its function as an anchoring domain. The respective mutation sites either had little effect, inhibited, or strongly promoted folding stability of the troponin tail. Mutation F110I weakened the affinity of troponin for the thin filament, despite promoting folding, and overall the data suggest that troponin tail flexibility is particularly important for the interactions of troponin with tropomyosin. In addition, multiple vertebrate and invertebrate TnTs were subjected to amino acid sequence alignment, revealing very high conservation of residues 112–136. In consideration of this analysis as well as previous deletional studies, we suggest this region is a critical anchoring element of TnT. Numerous cardiomyopathy-inducing mutations flank this TnT sequence. Bovine cardiac tropomyosin, native troponin, TnI, and TnC were purified as described previously (31Tobacman L.S. Adelstein R.S. Biochemistry. 1986; 25: 798-802Crossref PubMed Scopus (62) Google Scholar, 32Tobacman L.S. Lee R. J. Biol. Chem. 1987; 262: 4059-4064Abstract Full Text PDF PubMed Google Scholar) from a heart muscle ether powder. Actin was obtained from an acetone powder of rabbit fast skeletal muscle (33Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). Whole troponin was reconstituted from denatured subunits mixed in a 1:1:1 ratio, followed by stepwise dialysis and size exclusion column chromatography (32Tobacman L.S. Lee R. J. Biol. Chem. 1987; 262: 4059-4064Abstract Full Text PDF PubMed Google Scholar). Recombinant control and mutant bovine whole TnT and TnT-(1–156) were expressed using pET3d in DE3 cells and purified to homogeneity as described (25Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274: 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). T104V or T104A mutations were introduced into bovine cardiac TnT cDNA (18Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) by the same PCR-based approach used previously to create the R92Q and F110I mutations (18Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The same method was also used to introduce the T104A mutation into TnT R92Q and F110I. To create the various TnT-(1–156) constructs, the corresponding DNA fragment was amplified by PCR from the various full-length plasmids and inserted into the NcoI/BamHI sites of pET3d. All coding sequences in expression plasmids were confirmed by automated DNA sequencing at the University of Iowa DNA Facility. The ellipticity of TnT-(1–156) was monitored as a function of temperature using an Aviv DS65 circular dichroism spectrometer, recording from −5 to 80 °C. Two sets of protein preparations gave similar results. Conditions are as follows: 0.1 mg/ml TnT-(1–156), 300 mm KCl, 15 mm NaH2PO4 (pH 6.5). Similar results were also obtained for one of the sets of protein preparations, examined in the presence of 150 mm KCl. Data were fit to a two-state, temperature (T)-dependent transition, with −RT lnK = ΔG = ΔH m(1 − T/T m). The ellipticity of the unfolded state was taken as constant, but satisfactory fits required that the ellipticity of the folded state be assumed to vary linearly with temperature. This procedure should be considered as semi-empirical, because the unfolding process is not two-state as assumed in the modeling. Differential scanning calorimetry of wt TnT-(1–156), F110I TnT-(1–156), and of F110I/T104A TnT-(1–156) demonstrated that the heat of unfolding was in fact gradual and could not be described accurately with models including as many as four states (three transitions) (data not shown). Binding of radiolabeled tropomyosin to actin was measured by cosedimentation with a TLA100 rotor in a Beckman TL100 centrifuge (34Hill L.E. Mehegan J.P. Butters C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: 16106-16113Abstract Full Text PDF PubMed Google Scholar). The tropomyosin was stoichiometrically labeled under denaturing conditions on Cys-190 with [3H]iodoacetic acid (34Hill L.E. Mehegan J.P. Butters C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: 16106-16113Abstract Full Text PDF PubMed Google Scholar). Binding was calculated from the decrease in supernatant radioactivity following sedimentation. As described in the figures, conditions were chosen in which the tropomyosin bound negligibly to actin unless troponin or a troponin fragment was added. Troponin binds very tightly to the thin filament, making its affinity problematic to measure by sedimentation. Therefore, the relative affinity of troponin for actin-tropomyosin was measured by its ability to displace a control, 3H-labeled troponin from the thin filament. Bound and free [3H]troponin were separated by ultracentrifugation as described previously (25Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274: 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). By fitting the data to Equation 1 from Ref. 25Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274: 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, the affinity of troponin for actin-tropomyosin was measured, relative to the affinity of the3H-labeled troponin. Conditions are as follows: 25 °C, 7 μm actin, 3 μm tropomyosin, 1 μm3H-labeled troponin, 10 mmTris (pH 7.5), 300 mm KCl, 3 mmMgCl2, 0.2 mm dithiothreitol, 0.3 mg/ml bovine serum albumin, and 0.5 mm EGTA. The strongest interaction between troponin and tropomyosin involves the TnT N terminus and a tropomyosin C-terminal region found specifically in tropomyosins that bind to troponin (reviewed in Ref. 6Li Y. Mui S. Brown J.H. Strand J. Reshetnikova L. Tobacman L.S. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7378-7383Crossref PubMed Scopus (84) Google Scholar). Moreover, a newly reported crystallographic study of tropomyosin shows that conserved C-terminal residues of mammalian striated muscle tropomyosin effect a distinctive structure, a troponin T recognition site. This structural and sequence conservation in tropomyosin suggests a similar conservation in the protein target, i.e. in the troponin tail. To help identify this region, a multiple sequence alignment of 15 TnTs was performed (Fig. 1). In the figure, chordate (mammals, birds, and tunicate) TnT conservation is indicated in yellow, and blue indicates conservation among three invertebrate phyla (nematode, mollusks, and insect). Regions conserved among both vertebrate and invertebrate TnTs are indicated in green and are readily apparent. Fig. 1 shows that no TnT region is more conserved than residues 112–136 of the troponin tail domain. These residues are 70% homologous across the analyzed sequences. Significantly, engineered cardiac troponin constructs bind tightly to the thin filament only if they contain this entire region (see "Discussion"), regardless whether the construct is a troponin tail fragment containing only the TnT N terminus or instead is a truncated ternary troponin complex with an N-terminal deletion (25Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274: 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 34Hill L.E. Mehegan J.P. Butters C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: 16106-16113Abstract Full Text PDF PubMed Google Scholar, 35Fisher D. Wang G. Tobacman L.S. J. Biol. Chem. 1995; 270: 25455-25460Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The present sequence alignment and these earlier deletional experiments together suggest that residues 112–136 comprise a tropomyosin-binding element. Also, note that this region is flanked by many hypertrophic cardiomyopathy alleles, including (# in Fig. 1) sites Arg-92, Ala-104, and Phe-110. Although not the focus of the present study, the sequence alignment in Fig. 1 should also be considered in relationship to a preliminary, high resolution structure of a troponin complex containing TnC, most of TnI, and TnT fragment 188–288 (36Takeda S. Yamashita A. Maeda K. Maeda Y. Biophys. J. 2002; 82 (abstr.): 170Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Of the 101 TnT residues crystallized, 70 (residues 202–271) are ordered and identifiable in the x-ray structure. As can be seen in the figure, these boundaries correspond to those residues of the C-terminal half of TnT that are evolutionarily conserved. Within this region there is particular conservation of amino acids 226–271, which form a coiled coil with subunit TnI and also interact with TnC (36Takeda S. Yamashita A. Maeda K. Maeda Y. Biophys. J. 2002; 82 (abstr.): 170Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In an earlier study, we reported that TnT R92Q and F110I mutations interfered with the ability of bovine cardiac troponin subunits to reconstitute into a soluble troponin complex (18Tobacman L.S. Lin D. Butters C.A. Landis C.A. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). To investigate this folding abnormality quantitatively, we isolated troponin tail fragments TnT-(1–156), finding them to be soluble regardless of mutation. No precipitation was observed in low ionic strength buffer at protein concentrations studied (∼1.5 mg/ml). Troponin tail fragment TnT-(1–156) was predominantly α-helical as assessed by circular dichroism (data not shown), as expected (37Pearlstone J.R. Smillie L.B. Can. J. Biochem. 1977; 55: 1032-1038Crossref PubMed Scopus (47) Google Scholar, 38Tanokura M. Tawada Y. Ono A. Ohtsuki I. J. Biochem. ( Tokyo ). 1983; 93: 331-337Crossref PubMed Scopus (81) Google Scholar). When ellipticity was examined as a function of temperature, the mutations caused large but disparate effects (Fig. 2 A and TableI). TnT T104V altered the ellipticity in the manner expected for a destabilizing mutation, decreasing the temperature of the unfolding curve midpoint (T m) by 4.2 °C and also decreasing the absolute magnitude of the maximal ellipticity observed at the lowest temperature examined (−5 °C). Cardiomyopathy causing mutation R92Q similarly decreased both the ellipticity magnitude at low temperature and the T m, but to lesser extents. Interestingly, TnT mutation F110I had the opposite consequences, markedly shifting the circular dichroism results in the direction expected for a stabilizing rather than a de-stabilizing mutation.Table IThermal denaturation midpoint of bovine cardiac troponin tail peptide TnT-(1–156) containing cardiomyopathy mutations and/or bovine versus human difference T104ACardiomyopathy mutationBovine Thr-104 (control and single mutants)Human Ala-104 (control and double mutants)°CNone (control)29.5 ± 0.333.2 ± 0.2R92Q27.7 ± 0.533.1 ± 0.2F110I32.3 ± 0.338.8 ± 0.1T104V25.3 ± 0.5NACircular dichroism results in Fig. 2 were analyzed to determine values for T m, the temperature corresponding to 50% thermal denaturation. Cardiomyopathic mutation T104V decreased thermal stability; F110I increased thermal stability, and R92Q had little effect. Thermal stability was increased when human Ala-104 was inserted into the bovine sequence, in either the absence ("none") or the presence of the R92Q and F110I mutations. NA indicates not applicable. Open table in a new tab Circular dichroism results in Fig. 2 were analyzed to determine values for T m, the temperature corresponding to 50% thermal denaturation. Cardiomyopathic mutation T104V decreased thermal stability; F110I increased thermal stability, and R92Q had little effect. Thermal stability was increased when human Ala-104 was inserted into the bovine sequence, in either the absence ("none") or the presence of the R92Q and F110I mutations. NA indicates not applicable. Human cardiac and bovine cardiac TnT residues 70–141 are identical except at amino acid 104, which is alanine in the former and threonine in the latter. Alanine is among the most helix-promoting amino acids, and as described below, the solubility of bovine troponins containing human cardiomyopathy mutations R92Q or F110I was restored by incorporating Ala at position 104 of bovine TnT. Ala-104 improved the folding of the troponin tail fragments (Table I and Fig. 2 B), raising the melting temperature by 4–6 °C regardless of the presence of the R92Q mutation, the F110I mutation, or neither. This suggests the importance of residue 104 for troponin tail folding stability, and this implication is reinforced by the results with the cardiomyopathy-causing valine at this position. T104V TnT-(1–156) had the lowest stability of any of the tail fragments tested. Fig. 2 B shows that bovine TnT-(1–156) containing the double mutation T104A/F110I was particularly stable, with aT m even higher than that of the T104A peptide, by 5.6 °C. In contrast, cardiomyopathic mutation R92Q had no effect on the melting curve in the presence of the T104A mutation (Fig. 2 B, circles versus triangles). The CD data overall demonstrate that cardiomyopathy-causing mutations at the three positions, 92, 104, and 110, had strikingly disparate effects on the folding of the troponin tail domain. While this work was in preparation, qualitatively similar results were reported using human cardiac TnT fragment 70–170 (39Palm T. Graboski S. Hitchcock-DeGregori S.E. Greenfield N.J. Biophys. J. 2001; 81: 2827-2837Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) (see "Discussion"). Despite substantial differences in the N and C termini of the peptides studied in the two reports, and differences in T m values and ellipticity at 0 °C, the effects of the mutations on apparent stability were very similar. Like whole troponin, troponin tail peptide TnT-(1–156) promotes the binding of tropomyosin to actin (25Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274: 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 26Tobacman L.S. Nihli M. Butters C. Heller M. Hatch V. Craig R. Lehman W. Homsher E. J. Biol. Chem. 2002; 277: 27636-27642Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), an effect most conveniently studied under high ionic strength conditions that weaken tropomyosin-actin affinity. In the presence of 300 mm KCl and sub-micromolar tropomyosin concentrations, negligible tropomyosin binds to actin unless either troponin or the troponin tail peptide is added (Fig. 3). The effect of TnT-(1–156) was independent of the bovineversus human Thr-104 versus Ala-104 substitution but was significantly impaired by each of the cardiomyopathy mutations, T104V, F110I, or R92Q. Thus, function was lost not only by mutations that impaired folding (T104V and R92Q), but also by a mutation that strengthened folding (F110I). When the R92Q mutation was combined with the T104A substitution (construct R92Q/T104A), troponin tail function was restored. Because folding was also improved (Table I), this is explainable by a higher fraction of the protein being folded and therefore binding competent at 25 °C. However, the F110I/T104A troponin tail fragment is particularly stable according to the data in Fig. 2, but nevertheless had no effect on tropomyosin binding to actin. This suggests that normal interactions of the troponin tail with its target (actin and/or tropomyosin) require sufficient flexibility, lacking as a consequence of the mutation. TnT R92Q and F110I mutations adversely affect bovine cardiac whole troponin solubility (25Hinkle A. Goranson A. Butters C.A. Tobacman L.S. J. Biol. Chem. 1999; 274: 7157-7164Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), complicating assessment of the properties of these molecules. Similarly, repeated efforts in the present study to reconstitute bovine troponin containing the T104V mutation produced a soluble complex only when the ionic strength was maintained very high, with more than 1 m KCl. However, no such problem was observed for troponins with the TnT double mutants R92Q/T104A or F110I/T104A. In the context of the human Ala-104, which promotes folding (Fig. 2), the cardiomyopathy mutations did not affect troponin solubility, and ternary troponin complexes were routinely isolated at concentrations of ∼1.5 mg/ml in low ionic strength buffer. Fig. 4 shows the same experiment as in Fig. 3, except binding of tropomyosin to actin is promoted by whole troponin with or without cardiomyopathy mutations, instead of by troponin tail fragments. T104A troponin behaved similarly to control troponin, as did R92Q/T104A troponin. Both the maximal tropomyosin-actin binding and the troponin concentration dependence of the effect were indistinguishable from data for control troponin
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