Investigation of immunological relationships among myosin light chains and troponin C.

Two classes of myosin light chains can be distinguished functionally: those that restore calcium regulation to "desensitized" scallop myofibrils, and those that do not (Kendrick-Jones, J., et al. (1976), J. Mol. Biol. 104, 747--775). Despite this functional classification, chemical analyses reveal few patterns unique to regulatory light chains, and, indeed, sequence comparisons suggest structural similarities between both classes of myosin subunits (Collins, J. H. (1977), Nature (London) 259, 699--700; Kendrick-Jones, J., and Jakes, R. (1977), in International Symposium on Myocardial Failure at Tegernsee, Riecker, G., and Boehringer, Ed., Munich, West Germany, Springer-Verlag, pp. 28--40). Immunological assays using antisera to regulatory and to nonregulatory light chains showed no correlation between antigenic activity and the presence or absence of regulatory function. Weak cross-reactivity was observed, however, among myosin light chains and troponin C, consistent with the suggestion made on the basis of sequence homologies that these subunits contain similar structural domains (Weeds, A. G., and McLachlan, A. D. (1974), Nature (London) 252, 646--649). Unexpectedly, the strongest cross-reactivity observed was that between the vertebrate myosin alkali 1 and DTNB light chains.     

Interaction of troponin C and troponin C fragments with troponin I and the troponin I inhibitory peptide.

We have quantitated the interactions of two rabbit skeletal troponin C fragments with troponin I and the troponin I inhibitory peptide. The calcium binding properties of the fragments and the ability of the fragments to exert control in the regulated actomyosin ATPase assay have also been studied. The N- and C-terminal divalent metal binding domains of rabbit skeletal troponin C, residues 1-97 and residues 98-159, respectively, were prepared by specific cleavage at cysteine-98 and separation by gel exclusion chromatography. Both of the troponin C fragments bind calcium. The calcium affinity of the weak sites within the N-terminal fragment is about an order of magnitude greater than is reported for these sites in troponin C, suggesting interaction between the calcium-saturated strong sites and the weak sites. Stoichiometric binding (1:1) of the troponin I inhibitory peptide to each fragment and to troponin C increased the calcium affinities of the fragments and troponin C. Complex formation was detected by fluorescence quenching or enhancement using dansyl-labeled troponin C (and fragments) or tryptophan-labeled troponin I inhibitory peptide. The troponin C fragments bind to troponin I with 1:1 stoichiometry and approximately equal affinities (1.6 x 10(6) M-1) which are decreased 4-fold in the presence of magnesium versus calcium. These calcium effects are much smaller than is observed for troponin C. The summed free energies for the binding of the troponin C fragments to troponin I are much larger than the free energy of binding troponin C. This suggests a large positive interaction free energy for troponin C binding to troponin I relative to the fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proximity relationships between residue 6 of troponin I and residues in troponin C: further evidence for extended conformation of troponin C in the troponin complex

Skeletal muscle troponin C (TnC) adopts an extended conformation when crystallized alone and a compact one when crystallized with an N-terminal troponin I (TnI) peptide, TnI(1-47) [Vassylyev et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. The N-terminal region of TnI (residues 1-40) was suggested to play a functional role of facilitating the movement of TnI's inhibitory region between TnC and actin [Tripet et al. (1997) J. Mol. Biol. 271, 728-750]. To test this hypothesis and to investigate the conformation of TnC in the intact troponin complex and in solution, we attached fluorescence and photo-cross-linking probes to a mutant TnI with a single cysteine at residue 6. Distances from this residue to residues of TnC were measured by the fluorescence resonance energy transfer technique, and the sites of photo-cross-linking in TnC were determined by microsequencing and mass spectrometry following enzymatic digestions. Our results show that in the troponin complex neither the distance between TnI residue 6 and TnC residue 89 nor the photo-cross-linking site in TnC, Ser133, changes with Ca(2+), in support of the notion that this region plays mainly a structural rather than a regulatory role. The distances to residues 12 and 41 in TnC's N-domain are both considerably longer than those predicted by the crystal structure of TnC.TnI(1-47), supporting an extended rather than a compact conformation of TnC. In the binary TnC.TnI complex and the presence of Ca(2+), Met43 in TnC's N-domain was identified as the photo-cross-linking site, and multiple distances between TnI residue 6 and TnC residue 41 were detected. This was taken to indicate increased flexibility in TnC's central helix and that TnC dynamically changes between a compact and an extended conformation when troponin T (TnT) is absent. Our results further emphasize the difference between the binary TnC.TnI and the ternary troponin complexes and the importance of using intact proteins in the study of structure-function relationships of troponin.     

Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

New grass phylogeny resolves deep evolutionary relationships and discovers C4 origins

? Grasses rank among the world's most ecologically and economically important plants. Repeated evolution of the C(4) syndrome has made photosynthesis highly efficient in many grasses, inspiring intensive efforts to engineer the pathway into C(3) crops. However, comparative biology has been of limited use to this endeavor because of uncertainty in the number and phylogenetic placement of C(4) origins. ? We built the most comprehensive and robust molecular phylogeny for grasses to date, expanding sampling efforts of a previous working group from 62 to 531 taxa, emphasizing the C(4)-rich PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae and Danthonioideae) clade. Our final matrix comprises c. 5700 bp and is > 93% complete. ? For the first time, we present strong support for relationships among all the major grass lineages. Several new C(4) lineages are identified, and previously inferred origins confirmed. C(3)/C(4) evolutionary transitions have been highly asymmetrical, with 22-24 inferred origins of the C(4) pathway and only one potential reversal. ? Our backbone tree clarifies major outstanding systematic questions and highlights C(3) and C(4) sister taxa for comparative studies. Two lineages have emerged as hotbeds of C(4) evolution. Future work in these lineages will be instrumental in understanding the evolution of this complex trait.     

Rotational and translational motion of troponin C.

Time resolved fluorescence anisotropy and sedimentation velocity has been used to study the rotational and translational hydrodynamic behavior of two mutants of chicken skeletal troponin C bearing a single tryptophan residue at position 78 or 154 in the metal-free-, metal-bound-, and troponin I peptide (residues 96-116 of troponin I)-ligated states. The fluorescence anisotropy data of both mutants were adequately described by two rotational correlation times, and these are compared with the theoretically expected values based on the rotational diffusion of an idealized dumbbell. These data imply that the motion of the N- and C-terminal domains of troponin C are independent. They also suggest that in the metal-free, calcium-saturated and calcium-saturated troponin I peptide-bound states, troponin C is elongated, having an axial ratio of 4-5. Calcium or magnesium binding to the high affinity sites alone reduces the axial ratio to approximately 3. However, with calcium bound to sites III and IV and in the presence of a 1:1 molar ratio of the troponin I peptide, troponin C is approximately spherical. The metal ion and troponin I peptide-induced length changes in troponin C may play a role in the mechanism by which the regulatory function of troponin C is effected.     

Periodic binding of troponin C.I and troponin I to tropomyosin-actin filaments

We investigated the distribution of troponin C.I and troponin I along tropomyosin-actin filaments by immunoelectron microscopy and found that anti-troponin I antibody formed transverse striations at 38 nm intervals along the bundle of filaments of both troponin C.I-tropomyosin-actin and troponin I-tropomyosin-actin. Since the length of 38 nm corresponds to the repeating period of filamentous tropomyosin along actin double strands, the present study indicates that troponin I is located at a specific region of each tropomyosin, suggesting that a specific interaction between troponin I and tropomyosin is involved in determining the periodic distribution of troponin I along tropomyosin-actin filaments.     

Functional relationship between bovine brain phosphodiesterase activator protein and bovine heart troponin C

Phosphodiesterase activator protein has been purified from bovine brain and its properties compared with that of bovine heart troponin C. While both proteins activate ‘activator depleted phosphodiesterase’ in the presence of Ca²⁺, a 200-fold greater concentration of troponin C was necessary and the maximal effect was less than that with the activator protein. The activator protein formed a Ca²⁺ -dependent complex with bovine heart troponin I during electrophoresis in 6 M urea-polyacrylamide gel. However, the mobility of this complex was different from that of troponin C · troponin I complex and the affinity between troponin C and troponin I was much stronger than that between the activator protein and troponin I. Ca²⁺ induced changes in the electrophoretic mobility of activator protein and the pattern of its elution during gel filtration which were similar to the Ca²⁺-dependent conformational changes observed with troponin C. Bovine heart troponin I reduced basal, troponin C and the activator protein stimulation of phosphodiesterase activity. These results are compatible with the concept that phosphodiesterase activator protein and troponin C might have a functional relationship.     

The taxonomic position and evolutionary relationships of Trypanosoma rangeli.

This paper presents a re-evaluation of the taxonomic position and evolutionary relationships of Trypanosoma (Herpetosoma) rangeli based on the phylogenetic analysis of ssrRNA sequences of 64 Trypanosoma species and comparison of mini-exon sequences. All five isolates of T. rangeli grouped together in a clade containing Trypanosoma (Schizotrypanum) cruzi and a range of closely related trypanosome species from bats [Trypanosoma (Schizotrypanum) dionisii, Trypanosoma (Schizotrypanum) vespertilionis] and other South American mammals [Trypanosoma (Herpetosoma) leeuwenhoeki, Trypanosoma (Megatrypanum) minasense, Trypanosoma (Megatrypanum) conorhini] and an as yet unidentified species of trypanosome from an Australian kangaroo. Significantly T. rangeli failed to group with (a) species of subgenus Herpetosoma, other than those which are probably synonyms of T. rangeli, or (b) species transmitted via the salivarian route, although either of these outcomes would have been more consistent with the current taxonomic and biological status of T. rangeli. We propose that use of the names Herpetosoma and Megatrypanum should be discontinued, since these subgenera are clearly polyphyletic and lack evolutionary and taxonomic relevance. We hypothesise that T. rangeli and T. cruzi represent a group of mammalian trypanosomes which completed their early evolution and diversification in South America.     

Structure-function relationships in cardiac troponin T

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.     

Functional morphology and evolutionary biology

In this study the relationship between functional morpholoy and evolutionary biology is analysed by confronting the main concepts in both disciplines.Rather than only discussing this connection theoretically, the analysis is carried out by introducing important practical and experimental studies, which use aspects from both disciplines. The mentioned investigations are methodologically analysed and the consequences for extensions of the relationship are worked out. It can be shown that both disciplines have a large domain of their own and also share a large common ground. Many disagreements among evolutionary biologists can be reduced to differences in general philosophy (idealism vs. realism), selection of phenomenona (structure vs. function), definition of concepts (natural selection) and the position of the concept theory as an explaining factor (neutralists vs. selectionists, random variation, determinate selection, etc.).The significance of functional morphology for evolutionary biology, and vice versa depends on these differences. For a neo-Darwinian evolutionary theory, contributions from functional and ecological morphology are indispensable. Of ultimate importance are the notions of internal selection and constraints in the constructions determining further development. In this context the concepts of random variation and natural selection need more detailed definition.The study ends with a recommendation for future research founded in a system-theoretical or structuralistic conception.     

Functional morphology and evolutionary ecology

Functional morphology studies physico-chemical properties and biological roles of organs. If applied to fossils this approach is connected with specific methodological problems. The fields of application of functional morphology in paleontology are illustrated with examples. In a working concept it is shown how the results of functional morphology can be integrated into an account of the evolutionary ecology of fossil taxa.     

The mobility of troponin C and troponin I in muscle

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca²⁺ concentration. The binding of Ca²⁺ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τ_r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg²⁺ by Ca²⁺ at these sites (τ_r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca²⁺ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd.     

Amino acid compositions and evolutionary relationships with protein families.

We have examined the merits of the three functions based on amino acid compositions which have been proposed to indicate the similarity in amino acid sequences of two proteins; the difference index, the composition divergence and the composition coefficient. We have taken the amino acid compositions and sequences of 41 cytochrome c's and used the 820 values from all possible comparisons in the evaluation. We conclude that the functions do have a limited value in predicting proteins which are closely related in sequence and that the three functions are equivalent in this predictive ability. We have used the composition divergence values obtained from available pyruvate kinase amino acid compositions to generate a phylogenetic tree for this glycolytic enzyme.     

Drug binding to cardiac troponin C.

Compounds that sensitize cardiac muscle to Ca(2+) by intervening at the level of regulatory thin filament proteins would have potential therapeutic benefit in the treatment of myocardial infarctions. Two putative Ca(2+) sensitizers, EMD 57033 and levosimendan, are reported to bind to cardiac troponin C (cTnC). In this study, we use heteronuclear NMR techniques to study drug binding to [methyl-(13)C]methionine-labeled cTnC when free or when complexed with cardiac troponin I (cTnI). In the absence of Ca(2+), neither drug interacted with cTnC. In the presence of Ca(2+), one molecule of EMD 57033 bound specifically to the C-terminal domain of free cTnC. NMR and equilibrium dialysis failed to demonstrate binding of levosimendan to free cTnC, and the presence of levosimendan had no apparent effect on the Ca(2+) binding affinity of cTnC. Changes in the N-terminal methionine methyl chemical shifts in cTnC upon association with cTnI suggest that cTnI associates with the A-B helical interface and the N terminus of the central helix in cTnC. NMR experiments failed to show evidence of binding of levosimendan to the cTnC.cTnI complex. However, levosimendan covalently bound to a small percentage of free cTnC after prolonged incubation with the protein. These findings suggest that levosimendan exerts its positive inotropic effect by mechanisms that do not involve binding to cTnC.