Cardiac troponin T forms a tetramer in vitro.

Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction has led to the development of antibody-based assays for measuring cTnT serum concentration. We examined the behavior of cTnT in solution. Recombinant human cTnT3, the dominant isoform in the adult human heart, was used. The protein was pure and functional, as demonstrated by SDS-PAGE and surface plasmon resonance. cTnT3 was found to bind specifically and in a concentration-dependent manner to cTnC. Routine size exclusion chromatography suggested a higher-than-expected MW for cTnT. Using analytical ultracentrifugation, we found cTnT3 in solution to be mainly in the form of a tightly bound tetramer at concentrations as low as 4 micromol/L. Our sedimentation velocity and transmission electron microscopy results indicate that the tetramer's shape is elongated rather than globular. CTnT's self-association in solution is an important consideration in the design and interpretation of experiments with the aim of understanding the biochemical and biophysical properties of cTnT, its isoforms, and its mutants.

Probing the gamma2 calcium-binding site: studies with gammaD298,301A fibrinogen reveal changes in the gamma294-301 loop that alter the integrity of the "a" polymerization site.

To determine the significance of the gamma2 calcium-binding site in fibrin polymerization, we synthesized the fibrinogen variant, gammaD298,301A. We expected these two alanine substitutions to prevent calcium binding in the gamma2 site. We examined the influence of calcium on the polymerization of gammaD298,301A fibrinogen, evaluated its plasmin susceptibility, and solved 2.7 and 2.4 A crystal structures of the variant with the peptide ligands Gly-Pro-Arg-Pro-amide (GPRP) and Gly-His-Arg-Pro-amide (GHRP), respectively. We found that thrombin-catalyzed polymerization of gammaD298,301A fibrinogen was modestly impaired, whereas batroxobin-catalyzed polymerization was significantly impaired relative to normal fibrinogen. Notably, the influence of calcium on polymerization was the same for the variant and for normal fibrinogen. Fibrinogen gammaD298,301A was more susceptible to plasmin proteolysis in the presence of GPRP. This finding suggests structural changes in the near-by "a" polymerization site. Comparisons of the structures revealed minor conformational changes in the gamma294-301 loop that are likely responsible for the weakened "a" site. When considered altogether, the data suggest that the gamma2 calcium-binding site does not significantly modulate polymerization. We cannot, however, rule out the possibility that the weakened "a" polymerization site masks an important role for the gamma2 calcium-binding site in normal polymerization. Somewhat unexpectedly, the structure data showed that GPRP bound to the "b" site and induced the same local conformational changes as GHRP to this site. This structure shows that "A:b" interactions can occur and suggests that these may participate in normal polymerization.

Analysis of engineered fibrinogen variants suggests that an additional site mediates platelet aggregation and that "B-b" interactions have a role in protofibril formation.

The C-terminal domain of the fibrinogen gamma-chain includes multiple functional sites that have been defined in high-resolution structures and biochemical assays. Calcium binds to this domain through the side chains of gammaD318 and gammaD320 and the backbone carbonyls of gammaF322 and gammaG324. We have examined variant fibrinogens with alanine at position gamma318 and/or gamma320 and found that calcium binding, fibrin polymerization, and fibrinogen-mediated platelet aggregation, but not FXIIIa-catalyzed cross-linking, were abnormal. When measured by turbidity, thrombin-catalyzed polymerization was severely reduced, and batroxobin-catalyzed polymerization was completely obliterated. Moreover, thrombin-catalyzed polymerization was abolished by the peptide GHRP, which binds to the polymerization site in the beta-chain but does not inhibit polymerization of normal fibrinogen. ADP-induced platelet aggregation was also severely impaired. In contrast, as measured by SDS-PAGE, FXIIIa introduced cross-links between gamma-chains for all three variants, as expected if the gamma-chain C-terminal sites were normal. In addition, binding of the monoclonal antibody 4A5, which recognizes the C-terminal residues, was not different from normal. These data suggest two specific conclusions: (1) a site in the gamma-module other than the C-terminus is critical for platelet aggregation and (2) "B-b" interactions have a role in protofibril formation.