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We have expressed alpha & beta isoforms of mammalian striated muscle tropomyosin (Tm) and alpha-Tm carrying the D175N or E180G cardiomyopathy mutations. In each case the Tm carries an Ala-Ser N-terminal extension to mimic the acetylation of the native Tm. We show that these Ala-Ser modified proteins are good analogues of the native Tm in the assays used here. We go on to use an in vitro kinetic approach to define the assembly of actin filaments with the Tm isoforms with either a cardiac or a skeletal muscle troponin (cTn, skTn). With skTn the calcium sensitivity of the actin filament is the same for alpha & beta-Tm and there is little change with the mutant Tms. For cTn switching from alpha to beta-Tm causes an increase of calcium sensitivity of 0.2 pCa units. D175N is very similar to the wild type alpha-Tm and E180G shows a small increase in calcium sensitivity of about 0.1 pCa unit. The formation of the switched-off blocked-state of the actin filament is independent of the Tm isoform but does differ for cardiac versus skeletal Tn. The in vitro assays developed here provide a novel, simple and efficient method for assaying the behaviour of expressed thin filament proteins.

There are significant isoform differences between the skeletal and cardiac troponin complexes. Studies of the regulatory properties of these proteins have previously shown only significant differences in the calcium dependence of their regulation. Using a sensitive myosin subfragment 1 (S1) binding assay we show that in the presence of calcium, thin filaments reconstituted with either skeletal or cardiac troponin produce virtually identical S1 binding curves. However in the absence of calcium the S1 binding curves differ considerably. Combined with kinetic measurements, curve fitting to the three-state thin filament regulatory model shows the main difference is that calcium produces a 4-fold change in K(T) (the closed-open equilibrium) for the skeletal system but little change in the cardiac system. The results show a significant difference in the range of regulatory effect between the cardiac and skeletal systems that we interpret as effects upon actin-troponin (Tn)I-TnC binding equilibria. As structural data show that the Ca(2+)-bound TnC structures differ, the additional counter-intuitive result here is that with respect to myosin binding the +Ca(2+) state of the two systems is similar whereas the -Ca(2+) state differs. This shows the regulatory tuning of the troponin complex produced by isoform variation is the net result of a complex series of interactions among all the troponin components.

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex., (Copyright 2001 Academic Press.)

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.

We have reconstituted thin filaments comprising pyrene-labelled actin (pyr-actin), tropomyosin (Tm) and cardiac troponin (cTn). cTn was isolated in two defined phosphorylation states; completely dephosphorylated on all subunits and with only the cTnI subunit bisphosphorylated. The thin filament was saturated with cTn at a pyr-actin/Tm/cTn ratio of 7:1:1. The calcium-dependent binding of S1 to thin filaments was measured in a stopped-flow spectrophotometer and the dependence of the observed rate constant on [Ca2+] fitted to the Hill equation. The only significant difference between the two phosphorylation states of the filaments was a 0.36 decrease in the pCa50 on bisphosphorylation.

Two serine residues located adjacently in the heart-specific N-terminus of cardiac troponin I can be phosphorylated in vivo. Both residues are sequentially phosphorylated and dephosphorylated by cAMP-dependent protein kinase (PKA) and protein phosphatase 2A (PP2A). The concentration changes of the different troponin I species have been determined separately for the phosphorylation and dephosphorylation reaction and approximated by time courses predicted by a reaction model. Dependent on the concentration ratio of active protein kinase/protein phosphatase, four different troponin I species can be generated; one nonphosphorylated, two monophosphorylated and one bisphosphorylated. This pattern generation will be observed in proteins phosphorylated and dephosphorylated by a single protein kinase and phosphatase on more than one site and is a new principle inherent in signal cascades.

Ca2+/Calmodulin-dependent protein kinase II isolated from heart phosphorylates bovine cardiac troponin T present in the holotroponin complex. Thr-190 has been determined as the main phosphorylation site.

Cardiac holotroponin can be phosphorylated at serine 23 and/or 24 in the heart-specific region of bovine troponin I. When isolated freshly it is composed of a mixture of non-, two mono-, and bisphosphorylated species. At neutral pH the monophosphorylated form carrying phosphate at serine 24 yields a resonance signal at 4.6 ppm and that carrying phosphate at serine 23 at 4.4 ppm; the two phosphate groups of the bisphosphorylated form yield only one 31P-NMR signal at 4.2 ppm. From the chemical shift dependence on pH, pKa values have been estimated to be 5.3 and 5.6 for the phosphate groups at serine 24 and serine 23, respectively. Both phosphates of the bisphosphorylated form exhibit very similar pKa values of approximately 5.8. Separation of bisphosphotroponin I from the complex results in a downfield shift and the appearance of two 31P-NMR signals at positions comparable to those of the two monophospho forms. Complex formation of cardiac troponin I with C or T does not alter the spectrum obtained with isolated troponin I; however, the original troponin spectrum is restored by reconstitution of the holocomplex from all three components T, I, and C. Two signals are also observed with a bisphosphorylated synthetic peptide [PVRRRS(P)S(P)ANYR] representing the phosphorylation domain. pKa values of about 5.3 and 5.6 have been determined for serine 7 (corresponding to serine 24 of troponin I) and serine 6 of the peptide (corresponding to serine 23 of troponin I).

Cardiac troponin I contains two adjacent serines in sequence after three arginine residues thus making up a minimally duplicated recognition motif for cAMP-dependent protein kinase. In a synthetic peptide, PVRRRSSANY, the two serine residues are phosphorylated sequentially with the intermediate formation of a monophosphorylated species according to the following reaction sequence: Peptide k1----Peptide-P k2----Peptide-P2. The calculated rat constants are: k1 = 0.435.min-1 and k2 = 0.034.min-1. Sequence analyses of the monophosphopeptide and its tryptic fragments show that the predominant monophosphoform carries phosphate at the second serine.

From rabbit and human cardiac troponin I N-terminal mono and bisphosphorylated peptides were isolated which were obtained from Lys-C proteinase digests. Two adjacent phosphoserine residues could be localized in each phosphopeptide following further tryptic digestion. The previously published sequence of rabbit cardiac troponin I had to be corrected. Two adjacent phosphoserine residues are a common motif in the very similar sequences of bovine, rabbit and human cardiac troponin I. The N-terminal sequences are: AcADRSGGSTAG DTVPAPPPVR RRS(P)S(P)ANYRAY ATEPHAK (bovine), AcADESTDA-AG EARPAPAPVR RRS(P)S(P)ANYRAY ATEPHAK (rabbit), (Ac,A,D/N,G,S,S,D/N,A,A,R) EPRPAPAPIR RRS(P)S(P)-NYRAY ATEPHAK (human).

Bovine cardiac troponin isolated in a highly phosphorylated form shows four 31P-NMR signals [Beier, N., Jaquet, K., Schnackerz, K. & Heilmeyer, L.M.G. Jr (1988) Eur. J. Biochem. 176, 327-334]. Troponin I, which contains phosphate covalently linked to serine-23 and/or -24 [Swiderek, K., Jaquet, K., Meyer, H. E. & Heilmeyer, L. M. G. Jr (1988) Eur. J. Biochem. 176, 335-342], shows three resonances. Mg2(+)-saturation of holotroponin shifts these troponin I resonances to higher fields. Direct binding of Mg2+ to the phosphate groups can be excluded. Both these serine residues of troponin I, 23 and 24, are substrates for cAMP- and cGMP-dependent protein kinases as well as for protein kinase C. Isolated bovine cardiac troponin T contains 1.5 mol phosphoserine/mol protein, indicating that minimally two serine residues are phosphorylated. One phosphoserine residue is located at the N-terminus. An additional phosphoserine is located in the C-terminal cyanogen bromide fragment, CN4, which contains covalently bound phosphate. Protein kinase C phosphorylates serine-194, thus demonstrating exposure of this residue on the surface of holotoponin.

Phosphorylation of the isolated rabbit skeletal muscle holotroponin complex at troponin-T by phosphorylase kinase is unusual in that it shows maxima and minima. These oscillations are due to protein phosphatase activity present in the preparations. Following tryptic digestion two phosphorylated peptides, I and II, can be isolated. Their amino-acid compositions are identical and correspond to that of the tryptic peptide which contains the two known phosphorylatable sites 149/150 and 156/7 of troponin-T. Peptide I is phosphorylated on both sites and peptide II only on one site. During phosphorylation the doubly phosphorylated peptide I appears first; after a short lag phase peptide II is formed containing only one phosphate. These phenomena probably cause the observed oscillations in the degree of the holotroponin phosphorylation.

Bovine cardiac troponin containing approximately 3 mol P/mol protein could be separated into its subunits without loss of phosphate. Troponin I and troponin T each contain about 1.5 mol P/mol protein. In troponin I two phosphorylated serine residues could be localized in the N-terminal region by conversion of phosphoserine to S-ethylcysteine. They are located in adjacent positions in the following sequence: -Arg-Arg-Ser(P)-Ser(P)-Ala-Asn-Tyr-Tyr-Arg-Ala-Tyr-Ala-Thr-Glu-Pro- His-Ala-Lys. This sequence shows that the first phosphoserine residue in bovine cardiac troponin I occupies a homologous position to phosphoserine-20 of rabbit cardiac troponin I.

A modified procedure for isolation of troponin from bovine heart is described, which results in a stable and highly phosphorylated protein. 31P-NMR spectra show up to four phosphoserine signals indicating that at least four serine residues of cardiac troponin are phosphorylated in the intact organ. The hydrodynamic parameters of phosphotroponin are almost identical to those previously published. Characteristically cardiac troponin shows a strong tendency to associate that is dependent on protein concentration. Mg2+ may specifically induce an aggregation, which can be observed during sedimentation. This phenomenon seems to be analogous to the Mg2+-induced dimerization of cardiac troponin C [Jaquet, K. and Heilmeyer, L. M. G., Jr (1987) Biochem. Biophys. Res. Commun. 145, 1390-1396]. Upon Mg2+ saturation a shift of one of the four 31P-NMR signals is observed. The affinity of troponin to Ca2+ is reduced when the protein concentration is enhanced only in the presence of Mg2+. This effect of Mg2+ suggests a model for the regulation of the Ca2+-binding affinity of cardiac troponin.

Isolated bovine cardiac troponin C forms dimers in presence of Mg2+ dependent on the protein concentration which has been determined by sedimentation velocity and sedimentation equilibrium. Dimer formation is correlated with a decrease in Ca2+ affinity. Positive inotropic drugs (benzimidazol derivatives) influence Ca2+ sensitivity either by changing the association state or by affecting the Ca2+ binding properties directly.