A Functional and Structural Study of Troponin C Mutations Related to Hypertrophic Cardiomyopathy
2009; Elsevier BV; Volume: 284; Issue: 28 Linguagem: Inglês
10.1074/jbc.m109.007021
ISSN1083-351X
AutoresJosé R. Pinto, Michelle S. Parvatiyar, Michelle Jones, Jingsheng Liang, Michael J. Ackerman, James D. Potter,
Tópico(s)Trypanosoma species research and implications
ResumoRecently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca2+ sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281–288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca2+] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca2+ sensitivity with only A8V demonstrating lowered Ca2+ sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca2+. None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca2+ affinity of site II measured by 2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca2+. Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca2+ sensitivity of the myofilament; 2) the effects of the mutations on the Ca2+ affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca2+ sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca2+ binding to cTnC. Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca2+ sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281–288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca2+] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca2+ sensitivity with only A8V demonstrating lowered Ca2+ sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca2+. None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca2+ affinity of site II measured by 2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca2+. Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca2+ sensitivity of the myofilament; 2) the effects of the mutations on the Ca2+ affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca2+ sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca2+ binding to cTnC. Hypertrophic cardiomyopathy (HCM) 3The abbreviations used are: HCMhypertrophic cardiomyopathyDTTdithiothreitolIAANS2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acidMOPS3-(N-morpholino)propanesulfonic acidWTwild typecTncardiac troponinTntroponinTmtropomyosinCDcircular dichroismHcTnChuman cTnCMREmean residue ellipticity.3The abbreviations used are: HCMhypertrophic cardiomyopathyDTTdithiothreitolIAANS2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acidMOPS3-(N-morpholino)propanesulfonic acidWTwild typecTncardiac troponinTntroponinTmtropomyosinCDcircular dichroismHcTnChuman cTnCMREmean residue ellipticity. is typically inherited as an autosomal dominant disease that is caused by mutations in sarcomeric genes and is the most prevalent cause of sudden death in athletes and young people (1.Marian A.J. Roberts R. J. Cardiovasc. Electrophysiol. 1998; 9: 88-99Crossref PubMed Scopus (90) Google Scholar, 2.Maron B.J. Card. Electrophysiol. Rev. 2002; 6: 100-103Crossref PubMed Scopus (35) Google Scholar). The clinical hallmark of HCM is an increased thickness of the left ventricular wall. Myocyte disarray, fibrosis, septal hypertrophy, and abnormal diastolic function can also be present in HCM patients (3.Maron B.J. J. Am. Med. Assoc. 2002; 287: 1308-1320Crossref PubMed Scopus (0) Google Scholar). HCM mutations have been reported in 13 myofilament-related genes; however, the cardiac troponin C (cTnC) gene remained excluded from this list (4.Towbin J.A. Bowles N.E. Nature. 2002; 415: 227-233Crossref PubMed Scopus (446) Google Scholar, 5.Liew C.C. Dzau V.J. Nat. Rev. Genet. 2004; 5: 811-825Crossref PubMed Scopus (137) Google Scholar, 6.Van Driest S.L. Ellsworth E.G. Ommen S.R. Tajik A.J. Gersh B.J. Ackerman M.J. Circulation. 2003; 108: 445-451Crossref PubMed Scopus (195) Google Scholar, 7.Alcalai R. Seidman J.G. Seidman C.E. J. Cardiovasc. Electrophysiol. 2008; 19: 104-110PubMed Google Scholar). The clinical and functional phenotypes may vary according to the gene and the location of the mutation (8.Gomes A.V. Potter J.D. Ann. N.Y. Acad. Sci. 2004; 1015: 214-224Crossref PubMed Scopus (91) Google Scholar). Recently our group has reported evidence that brings cTnC into focus as an HCM susceptibility gene (9.Landstrom A.P. Parvatiyar M.S. Pinto J.R. Marquardt M.L. Bos J.M. Tester D.J. Ommen S.R. Potter J.D. Ackerman M.J. J. Mol. Cell. Cardiol. 2008; 45: 281-288Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Interestingly the prevalence for cTnC HCM mutations was the same as other well characterized genes (i.e. actin and tropomyosin) (6.Van Driest S.L. Ellsworth E.G. Ommen S.R. Tajik A.J. Gersh B.J. Ackerman M.J. Circulation. 2003; 108: 445-451Crossref PubMed Scopus (195) Google Scholar). To date, prior to our recent report, only one mutation in cTnC (L29Q) had been linked to HCM (10.Hoffmann B. Schmidt-Traub H. Perrot A. Osterziel K.J. Gessner R. Hum. Mutat. 2001; 17: 524Crossref PubMed Scopus (118) Google Scholar). In vitro and in situ studies demonstrating changes in the functional parameters of cardiac muscle regulation suggest that this mutation is causative of the disease (11.Schmidtmann A. Lindow C. Villard S. Heuser A. Mügge A. Gessner R. Granier C. Jaquet K. FEBS J. 2005; 272: 6087-6097Crossref PubMed Scopus (50) Google Scholar, 12.Liang B. Chung F. Qu Y. Pavlov D. Gillis T.E. Tikunova S.B. Davis J.P. Tibbits G.F. Physiol. Genomics. 2008; 33: 257-266Crossref PubMed Scopus (45) Google Scholar). hypertrophic cardiomyopathy dithiothreitol 2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid 3-(N-morpholino)propanesulfonic acid wild type cardiac troponin troponin tropomyosin circular dichroism human cTnC mean residue ellipticity. hypertrophic cardiomyopathy dithiothreitol 2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid 3-(N-morpholino)propanesulfonic acid wild type cardiac troponin troponin tropomyosin circular dichroism human cTnC mean residue ellipticity. Analysis of a cohort of 1025 HCM patients from the Mayo Clinic revealed four new cTnC mutations (A8V, C84Y, E134D, and D145E) (9.Landstrom A.P. Parvatiyar M.S. Pinto J.R. Marquardt M.L. Bos J.M. Tester D.J. Ommen S.R. Potter J.D. Ackerman M.J. J. Mol. Cell. Cardiol. 2008; 45: 281-288Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The clinical records showed that the patients displayed left ventricle hypertrophy and significant left ventricular outflow obstruction managed by surgical myectomy. Symptoms such as dyspnea, syncope, and chest pain were also present. A8V, C84Y, and E134D patients did not present a familial history of HCM indicating that these were likely sporadic de novo mutations. The D145E mutation was observed in six family members suggesting genetic linkage. Functional analysis performed in skinned fibers showed increased Ca2+ sensitivity of force development (a characteristic of troponin (Tn) mutations related to HCM) for three of the four mutations. Additionally the A8V and D145E mutations that are located in different domains caused increases in maximal force in this system. These data strongly suggest that HCM mutations in distinct regions of cTnC can result in a similar functional phenotype (9.Landstrom A.P. Parvatiyar M.S. Pinto J.R. Marquardt M.L. Bos J.M. Tester D.J. Ommen S.R. Potter J.D. Ackerman M.J. J. Mol. Cell. Cardiol. 2008; 45: 281-288Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In cardiac muscle, the tropomyosin (Tm)·Tn complex, located in the thin filament, is responsible for muscle regulation (13.Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1314) Google Scholar, 14.Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (472) Google Scholar). Three Tn subunits are involved in this process: troponin T (TnT), which connects the Tn complex to the thin filament and is responsible for actomyosin ATPase activation in the presence of Ca2+ (8.Gomes A.V. Potter J.D. Ann. N.Y. Acad. Sci. 2004; 1015: 214-224Crossref PubMed Scopus (91) Google Scholar, 15.Potter J.D. Sheng Z. Pan B.S. Zhao J. J. Biol. Chem. 1995; 270: 2557-2562Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar); troponin I (TnI) is the subunit that binds to both TnT and TnC, inhibits muscle contraction, and is also implicated in HCM and restrictive cardiomyopathy (16.Gomes A.V. Potter J.D. Mol. Cell. Biochem. 2004; 263: 99-114Crossref PubMed Scopus (55) Google Scholar); and TnC, a subunit that plays a crucial function in muscle regulation triggering contraction upon binding Ca2+ and is also considered an important intracellular Ca2+ buffer (17.Bers D.M. Excitation-Contraction Coupling and Cardiac Contractile Force. 2nd Ed. Kluwer Academic Publishers, London2001: 39-56Google Scholar, 18.Holroyde M.J. Robertson S.P. Johnson J.D. Solaro R.J. Potter J.D. J. Biol. Chem. 1980; 255: 11688-11693Abstract Full Text PDF PubMed Google Scholar). In the absence of Ca2+ binding to site II of cTnC, its N terminus is detached from the C terminus of cTnI, which under these conditions is bound to actin and inhibits muscle contraction. As Ca2+ binds to site II of cTnC, its N terminus binds to the C terminus of cTnI causing it to dissociate from actin. This is accompanied by the movement of cardiac Tm out of its inhibitory position on actin, thus relieving the inhibition of contraction (19.Gordon A.M. Regnier M. Homsher E. News Physiol. Sci. 2001; 16: 49-55PubMed Google Scholar, 20.Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (381) Google Scholar, 21.Takeda S. Yamashita A. Maeda K. Maéda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (629) Google Scholar). The dynamics of the interactions between Tn subunits and the thin filament that regulate contraction have been extensively studied (22.Lindhout D.A. Boyko R.F. Corson D.C. Li M.X. Sykes B.D. Biochemistry. 2005; 44: 14750-14759Crossref PubMed Scopus (19) Google Scholar, 23.Hoffman R.M. Blumenschein T.M. Sykes B.D. J. Mol. Biol. 2006; 361: 625-633Crossref PubMed Scopus (53) Google Scholar, 24.Murakami K. Yumoto F. Ohki S.Y. Yasunaga T. Tanokura M. Wakabayashi T. J. Mol. Biol. 2005; 352: 178-201Crossref PubMed Scopus (93) Google Scholar). TnC consists of two globular regions that are connected by a long central helix (25.Herzberg O. James M.N. Nature. 1985; 313: 653-659Crossref PubMed Scopus (482) Google Scholar). It is well known that cTnC has two EF-hands containing high affinity Ca2+ binding sites III and IV (∼107m−1) in the C terminus and only one functional low affinity Ca2+ binding site II (∼105m−1) in the N terminus (18.Holroyde M.J. Robertson S.P. Johnson J.D. Solaro R.J. Potter J.D. J. Biol. Chem. 1980; 255: 11688-11693Abstract Full Text PDF PubMed Google Scholar). An additional feature of helix-loop-helix Ca2+-binding proteins is the presence of short segments of antiparallel β-sheets between the Ca2+ binding loops of each domain (25.Herzberg O. James M.N. Nature. 1985; 313: 653-659Crossref PubMed Scopus (482) Google Scholar, 26.Moews P.C. Kretsinger R.H. J. Mol. Biol. 1975; 91: 201-225Crossref PubMed Scopus (383) Google Scholar). The C-terminal domain of cTnC can also bind Mg2+ competitively (∼103m−1) and is termed the structural domain because it is essential to keep it bound to the thin filament. The N terminus is considered the regulatory domain because Ca2+ binding to site II initiates muscle contraction. When TnC is in the Tn complex, the Ca2+ binding affinity at all sites is increased by ∼10-fold (18.Holroyde M.J. Robertson S.P. Johnson J.D. Solaro R.J. Potter J.D. J. Biol. Chem. 1980; 255: 11688-11693Abstract Full Text PDF PubMed Google Scholar, 27.Putkey J.A. Liu W. Lin X. Ahmed S. Zhang M. Potter J.D. Kerrick W.G. Biochemistry. 1997; 36: 970-978Crossref PubMed Scopus (41) Google Scholar, 28.Johnson J.D. Collins J.H. Robertson S.P. Potter J.D. J. Biol. Chem. 1980; 255: 9635-9640Abstract Full Text PDF PubMed Google Scholar). Several studies have shown that there is coupling between TnC and actomyosin ATPase. For example, bepridil and calmidazolium, two known Ca2+ sensitizers that bind to cTnC and enhance its Ca2+ binding affinity, also stimulate myofibrillar ATPase activity (29.Solaro R.J. Bousquet P. Johnson J.D. J. Pharmacol. Exp. Ther. 1986; 238: 502-507PubMed Google Scholar, 30.el-Saleh S.C. Solaro R.J. J. Biol. Chem. 1987; 262: 17240-17246Abstract Full Text PDF PubMed Google Scholar). In addition, deletion of the N-helix of the TnC N-domain diminishes activation of regulated actomyosin ATPase activity (31.Chandra M. da Silva E.F. Sorenson M.M. Ferro J.A. Pearlstone J.R. Nash B.E. Borgford T. Kay C.M. Smillie L.B. J. Biol. Chem. 1994; 269: 14988-14994Abstract Full Text PDF PubMed Google Scholar, 32.Smith L. Greenfield N.J. Hitchcock-DeGregori S.E. J. Biol. Chem. 1994; 269: 9857-9863Abstract Full Text PDF PubMed Google Scholar). The purpose of this study was to determine the functional effects of the four newly discovered HCM cTnC mutations not previously addressed and to investigate possible changes in their structure and Ca2+ binding properties. To answer these questions we performed reconstituted ATPase activity, co-sedimentation, and spectroscopy assays. In the presence of 100% HCM mutant or wild type (WT) cTnC, the ATPase activity rate measured by increasing the Ca2+ concentration in an actomyosin·Tm·Tn reconstituted complex showed increases in Ca2+ sensitivity similar to those obtained previously with cardiac skinned fibers (9.Landstrom A.P. Parvatiyar M.S. Pinto J.R. Marquardt M.L. Bos J.M. Tester D.J. Ommen S.R. Potter J.D. Ackerman M.J. J. Mol. Cell. Cardiol. 2008; 45: 281-288Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). At a ratio of 50% mutant to 50% WT, only A8V had a diminished Ca2+ sensitivity. We also evaluated the ability of the Tn HCM mutants to activate and inhibit the ATPase activity in the presence and absence of Ca2+. Only cTnC-D145E showed higher levels of ATPase activation. Co-sedimentation did not show changes in the ability of the Tn complex containing the cTnC mutants to bind to actin·Tm. The Ca2+ binding properties of the regulatory site II of cTnC as estimated from fluorescence and measured at cTnC and cTn levels did not match the apparent affinity of this site in the fiber and reconstituted filaments. However, D145E showed increased Ca2+ affinity in the isolated and cTn states that was minimally affected in the presence of the thin filament (TF). In the presence of the TF, A8V was the only mutant that showed an increase in Ca2+ affinity that more closely approached the Ca2+ sensitivity measured in the fiber. However, the circular dichroism (CD) measurements suggest that significant structural changes exist in the secondary structure of the cTnC mutants A8V, C84Y, and D145E compared with wild type. All of these results considered together with the PyMol illustrations suggest that structural changes are present in at least three TnC HCM mutants that are likely to be crucial for protein-protein interactions but unable to affect the Ca2+ binding properties of TnC at the different levels of TF complexity. Here we show for the first time that the thick filament is probably essential to completely recreate the increased Ca2+ sensitivity produced by HCM TnCs and observed in ATPase and skinned fiber assays. cDNAs cloned in our laboratory from human cardiac tissue were used for the expression and purification of cTnI (33.Zhang R. Zhao J. Potter J.D. J. Biol. Chem. 1995; 270: 30773-30780Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and cTnT (34.Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Human cTnC (HcTnC) was used as the template for PCR using primers designed to introduce the mutations of interest that result in the TnC HCM mutants (HcTnC-A8V, HcTnC-C84Y, HcTnC-E134D, HcTnC-D145E, HcTnC-C35S, HcTnC-A8V/C35S, HcTnC-E134D/C35S, and HcTnC-D145E/C35S). All subcloned DNA sequences were inserted into the pET3d expression plasmid and sequenced for verification prior to expression and purification. Standard methods previously used in this laboratory were utilized for expression and purification of the HcTnCs (35.Pinto J.R. Parvatiyar M.S. Jones M.A. Liang J. Potter J.D. J. Biol. Chem. 2008; 283: 2156-2166Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Briefly the cTnC WT and cTnC HCM mutants were expressed in Escherichia coli BL21 (DE3) CodonPlus bacterial cells (Stratagene). The bacterially expressed TnCs were passed over a Q-Sepharose column and eluted with a linear gradient of 0–0.6 m KCl (4 °C) in a buffer containing 50 mm Tris-HCl (pH 7.8), 6 m urea, 1 mm EDTA, 1 mm DTT. The purest TnC fractions were dialyzed into 50 mm Tris-HCl (pH 7.5), 1 mm CaCl2, 1 mm MgCl2, 50 mm NaCl, 1 mm DTT; loaded onto a phenyl-Sepharose column after the addition of 0.5 m (NH4)2SO4; and eluted with Ca2+-free buffer containing 50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm DTT at room temperature. Collected fractions were resolved by SDS-PAGE, and the fractions containing the purest, most concentrated fractions were pooled yielding >97% pure protein, dialyzed exhaustively against 5 mm NH4HCO3, and then lyophilized. All steps were performed at 4 °C unless otherwise indicated. The individual purified troponin subunits were first dialyzed against 3 m urea, 1 m KCl, 10 mm MOPS, 1 mm DTT, 0.1 mm phenylmethanesulfonyl fluoride and then twice against the same buffer excluding urea. The protein concentration of the individual subunits was determined using the Coomassie Plus kit (Pierce), and then the subunits were combined in a 1.3:1.3:1 TnT:TnI:TnC molar ratio. After 1 h, the complexes were successively dialyzed against decreasing concentrations of KCl (0.7, 0.5, 0.3, 0.1, 0.05, and 0.025 m). The excess precipitated TnT and TnI that formed during complex formation was removed by centrifugation. Proper stoichiometry was verified by SDS-PAGE before storage of troponin complexes at −80 °C. Porcine cardiac myosin, rabbit skeletal F-actin, and porcine cardiac Tm were prepared as described previously (36.Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The protein concentrations used for actomyosin ATPase assays were 0.6 μm porcine cardiac myosin, 3.5 μm rabbit skeletal F-actin, 1 μm porcine cardiac tropomyosin, and 0–2 μm preformed Tn complexes as described above. The buffer conditions for the proteins were as follows: myosin in 10 mm MOPS (pH 7.0), 0.4 m KCl, 1 mm DTT; actin in 10 mm MOPS (pH 7.0), 40 mm KCl; tropomyosin in 10 mm (pH 7.0), 300 mm KCl, 1 mm DTT. The final ionic strength of the reactions was ∼75 mm when considering the ionic contributions from the various protein buffers. The ATPase inhibitory assay was performed in a 0.1-ml reaction mixture of 3.4 mm MgCl2, 0.13 μm CaCl2, 1.5 mm EGTA, 3.5 mm ATP, 1 mm DTT, 11.5 mm MOPS (pH 7.0) at 25 °C. The ATPase activation assay was performed using the same 0.1-ml buffer mixture with 3.3 mm MgCl2 and 1.7 mm CaCl2. The ATPase reaction was initiated with the addition of ATP and quenched after 20 min using trichloroacetic acid to a final concentration of 35%. The precipitated assay proteins were removed by centrifugation, and the inorganic phosphate concentration in the supernatant that was released by ATP hydrolysis was determined according to the method of Fiske and Subbarow (37.Fiske C.H. Subbarow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar). pCa curves were performed using 1 μm preformed Tn complex with the same concentrations of myosin, actin, and tropomyosin as described above. For the experiments of 50% mutant and 50% WT the preformed Tn complexes were combined to a final concentration of 1 μm. The conditions of the assay (11.55 mm MOPS, 2 mm EGTA, 1 mm NTA, 3.7 mm MgCl2 (pH 7.0 at 25 °C)) changed slightly in varied pCa solutions: pCa 8.0 (0.113 mm CaCl2), pCa 7.0 (0.8997 mm CaCl2), pCa 6.5 (1.915 mm CaCl2), pCa 6.0 (2.982 mm CaCl2), pCa 5.5 (3.639 mm CaCl2), pCa 5.0 (3.971 mm CaCl2), pCa 4.5 (4.278 mm CaCl2), and pCa 4.0 (4.913 mm CaCl2) calculated according to methods established in our laboratory (38.Dweck D. Reyes-Alfonso Jr., A. Potter J.D. Anal. Biochem. 2005; 347: 303-315Crossref PubMed Scopus (65) Google Scholar). The thin filament constituents, F-actin at a concentration of 20 μm, and Tn/Tm at a concentration of 2.86 μm were used to obtain a molar ratio of 7:1:1 and were co-sedimented using an air-driven ultracentrifuge (Beckman Airfuge; rotor A100) for 30 min at 28 p.s.i. (∼120,000 × g). The buffer conditions were 20 mm imidazole (pH 7.0), 60 mm NaCl, 3 mm MgCl2, 0.5 mm CaCl2, 2 mm β-mercaptoethanol. For isolated cTnC and cTn measurements the cTnC WT and mutants were labeled with IAANS at Cys-35 and Cys-84 except for cTnC-C84Y, which was only labeled at Cys-35. C84S was used as a control for the C84Y mutant. For the thin filament measurements the cTnC WT and mutants A8V, E134D, and D145E had Cys-35 mutated to Ser and were labeled with IAANS only at Cys-84. IAANS was obtained from Molecular Probes, Plano, TX. Fluorescent labeling and purification of cTnC were performed according to established methods (27.Putkey J.A. Liu W. Lin X. Ahmed S. Zhang M. Potter J.D. Kerrick W.G. Biochemistry. 1997; 36: 970-978Crossref PubMed Scopus (41) Google Scholar, 28.Johnson J.D. Collins J.H. Robertson S.P. Potter J.D. J. Biol. Chem. 1980; 255: 9635-9640Abstract Full Text PDF PubMed Google Scholar). Isolated IAANS-labeled HcTnCs (HcTnC mutants and WT) were dialyzed into fluorescence buffer containing 2 mm EGTA, 5 mm nitrilotriacetic acid, 120 mm MOPS, 90 mm KCl. Before each titration 1.25 mm MgCl2 and 1 mm freshly prepared DTT were added. cTn complexes were made as described above ("Formation of Troponin Complexes"); however, the complexes underwent final dialysis in the fluorescence buffer containing 1.25 mm MgCl2. Thin filaments were constructed using the protocol established in our laboratory (39.Dweck D. Hus N. Potter J.D. J. Biol. Chem. 2008; 283: 33119-33128Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The cTnCs used in the thin filaments were mutated at Cys-35 to Ser and were labeled with IAANS at Cys-84. For C84Y the label was placed at Cys-35; however, the thin filament did not show spectral changes from pCa 8 to 4. Steady state fluorescence measurements were performed in a Jasco 6500 spectrofluorometer where IAANS fluorescence was excited at 330 nm and emission was detected at 450 nm. The protein concentrations used for isolated cTnC, cTn, and thin filaments were 10 μm, 0.5 μm, and 0.05 mg/ml, respectively. Fluorescence spectral changes were recorded during the titration of microliter amounts of CaCl2. The concentration of free Ca2+ and amounts of titrated Ca2+ were calculated using the pCa calculator program developed by our laboratory (38.Dweck D. Reyes-Alfonso Jr., A. Potter J.D. Anal. Biochem. 2005; 347: 303-315Crossref PubMed Scopus (65) Google Scholar). The program made corrections for dilution effects that occur during titration of Ca2+. The data were fitted to a version of the Hill equation that accounted for the spectral changes that occur at a low Ca2+ concentration and plotted using SigmaPlot 10.0. The apparent Ca2+ affinities are reported as pCa50 values ±S.E. Far-UV CD spectra were collected using a 1-mm-path quartz cell in a Jasco J-720 spectropolarimeter. Spectra were recorded at 195–250 nm with a bandwidth of 1 nm at a speed of 50 nm/min at a resolution of 0.5 nm at room temperature (∼20 °C). Ten scans were averaged, and no numerical smoothing was applied. The optical activity of the buffer was subtracted from relevant protein spectra. Mean residue ellipticity ([θ]MRE in degree·cm2/dmol) for the spectra were calculated utilizing the same Jasco system software using the following equation: [θ]MRE = [θ]/(10 × Cr × l) where [θ] is the measured ellipticity in millidegrees, Cr is the mean residue molar concentration, and l is the path length in cm (40.Szczesna D. Ghosh D. Li Q. Gomes A.V. Guzman G. Arana C. Zhi G. Stull J.T. Potter J.D. J. Biol. Chem. 2001; 276: 7086-7092Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Protein concentrations were determined by the biuret reaction using TnC as a standard that was calibrated by determining its protein nitrogen content. The CD experiments were performed using three different conditions: for apo, 1 mm EGTA, 20 mm MOPS, 100 mm KCl (pH 7.0); for Mg2+, 1 mm EGTA, 20 mm MOPS, 100 mm KCl, 2.075 mm MgCl2 (pH 7.0) yielding a free [Mg2+] of 2 mm); and for Ca2+, 1 mm EGTA, 20 mm MOPS, 100 mm KCl, 2.075 mm MgCl2, 1.096 mm CaCl2 (pH 7.0) yielding a free [Ca2+] of 10−4m and free [Mg2+] of 2 mm). The experimental protein concentration for the WT and each mutation was 0.2 mg/ml. PyMol is an open source molecular visualization program that allows the user to download Protein Data Bank files that contain molecular coordinates from x-ray crystallography- or nuclear magnetic resonance (NMR)-solved structures that were deposited in the Protein Data Bank. The program allows the known structures to be manipulated for example mutagenesis of selected residues, details of potential side chain interactions, and potential for polar contacts such as hydrogen bonding due to the nature and proximity of the side chains. The experimental results are reported as mean ± S.E. and were analyzed for significance using Student's t test at p < 0.05 (paired or unpaired depending on the experimental design). To assess the Ca2+ sensitivity of the newly discovered HCM cTnC mutants at a 100 and 50% ratio in a reconstituted system, actomyosin ATPase assays were evaluated as a function of increasing Ca2+ concentration. This system is more sensitive than the skinned fiber assays because of the lack of organization between thin and thick filaments; however, the Ca2+ sensitivity measured from the actomyosin ATPase correlates to the changes found by isometric tension (41.Zot A.S. Potter J.D. Biochemistry. 1989; 28: 6751-6756Crossref PubMed Scopus (43) Google Scholar, 42.Güth K. Potter J.D. J. Biol. Chem. 1987; 262: 13627-13635Abstract Full Text PDF PubMed Google Scholar). At 100% mutant reconstitution, A8V and C84Y generated the largest increase in Ca2+ sensitivity of the reconstituted myofilament with a leftward shift of +0.51 (pCa50 = 6.20 ± 0.03) and +0.56 (pCa50 = 6.25 ± 0.03) pCa units compared with the WT (pCa50 = 5.69 ± 0.05), respectively (Fig. 1A). D145E also generated a leftward shift with a ΔpCa50 of +0.25. At the 50% ratio only A8V showed a corresponding decrease in pCa50 compared with the amount of mutant protein present (Fig. 1B). In skinned fibers the leftward shift was ∼0.4 and ∼0.3 pCa units for A8V and C84Y, respectively (9.Landstrom A.P. Parvatiyar M.S. Pinto J.R. Marquardt M.L. Bos J.M. Tester D.J. Ommen S.R. Potter J.D. Ackerman M.J. J. Mol. Cell. Cardiol. 2008; 45: 281-288Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The D145E mutant (pCa50 = 5.94 ± 0.03) showed an intermediate increase in Ca2+ sensitivity with a 0.25 pCa unit leftward shift compared with WT in both ATPase and force measurements. At both ratios, the E134D (pCa50 = 5.72 ± 0.03 at 100%) mutant showed no significant change
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