Kinetic Mechanism of the Ca2+-Dependent Switch-On and Switch-Off of Cardiac Troponin in Myofibrils

The kinetics of Ca2+-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca2+] or the Ca2+-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca2+] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s−1 and ∼330 s−1, respectively. At low [Ca2+] (pCa 6.6), the slower rate was reduced to ∼25 s−1, but was still ∼50-fold higher than the rate constant of Ca2+-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca2+] from pCa 5.0–7.9 induced a fluorescence decay with a rate constant of 39 s−1, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca2+-binding (kB ≈ 120 μM−1 s−1) and dissociation (kD ≈ 550 s−1); and 2), slower switch-on (kon = 390s−1) and switch-off (koff = 36s−1) kinetics. At high [Ca2+], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation

Charakterisierung der Tat-abhängigen Proteintranslokation in Gram-negativen Bakterien

Tat-dependent protein translocation was investigated in Escherichia coli and Z. mobilis with regard to species specific differences . E. coli is unable to export the authentic Tat-substrate glucose-fructose oxidoreductase (GFOR) of Zymombnas mobilis. The replacement of the Z. mobilis GFOR signal peptide by the E. coli Tat signal peptide of the Trimethylamin N-oxide (TMAO) reductase (TorA) leads to an efficient strictly Tat-dependent export of the mature GFOR in E. coli. This result shows that the GFOR signal sequence is not recognised by the E. coli Tat-apparatus, whereas the folded mature part of GFOR is compatible with Tatdependent translocation in E. coli. The tatABC operon of Z. mobilis was cloned . The single Tat proteins TatA, TatB and TatC of Z. mobilis are functional in E. coli and at least one of these proteins is involved in the specific recognition of the GFOR signal sequence (Tat signal peptide receptor) . Therefore, a specific recognition event between Tat substrate and Tat receptor takes place that goes beyond the recognition of the conserved general features found in all Tat signal peptides. The species specificity of signal sequence recognition in the Tat pathway is in marked contrast to the situation that is known to exist for the Sec pathway. In E. coli, only four different components of the Tat pathway are known so far. Therefore, a screening assay that is suitable for the identification of so far unknown tat-genes was established. In this assay, a TorA-MalE fusion protein was proved to be an ideal reporter protein for the Tat-pathway . Due to a strong Sec avoidance motive in the C-region of the TorA signal sequence, export of the fusion protein is strictly Tat-dependent. Export is essential for maltose metabolism in a maIE-negative E. coli strain and easy to detect on maltose-containing indicator plates. After mutagenesis of an E. coli Tat wildtype strain, mutants which are unable to metabolise maltose due to a mutation in genes of the Tat pathway or maltose metabolism, were selected from the indicator plates. The tat-mutants were identified by means of their inability to grow under certain anaerobic growth conditions. In a first test of the sceening assay, several mutants in the already known tatABC-operon were selectively isolated

Genetic Analysis of Pathway Specificity during Posttranslational Protein Translocation across the Escherichia coli Plasma Membrane

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the “Sec avoidance signal,” the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway