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It is now known that the flexibility of the troponin T (TnT) tail determines thin filament conformation and hence cross-bridge cycling properties, expanding the classic structural role of TnT to a dynamic role regulating sarcomere function. Here, using transgenic mice bearing R-92W and R-92L missense mutations in cardiac TnT known to alter the flexibility of the TnT tropomyosin-binding domain, we found mutation-specific differences in the cost of contraction at the whole heart level. Compared to age- and gender-matched sibling hearts, mutant hearts demonstrate greater ATP utilization measured using (31)P NMR spectroscopy as decreases in [ATP] and [PCr] and |DeltaG(~ATP)| at all workloads and profound systolic and diastolic dysfunction at all energetic states. R-92W hearts showed more severe energetic abnormalities and greater contractile dysfunction than R-92L hearts. The cost of increasing contraction was abnormally high when [Ca(2+)] was used to increase work in mutant hearts but was normalized with supply of the beta-adrenergic agonist dobutamine. These results show that R-92L and R-92W mutations in the TM-binding domain of cardiac TnT alter thin filament structure and flexibility sufficiently to cause severe defects in both whole heart energetics and contractile performance, and that the magnitude of these changes is mutation specific.

The thin filament protein cardiac troponin T (cTnT) is an important regulator of myofilament activation. Here we report a significant change in cardiac energetics in transgenic mice bearing the missense mutation R92Q within the tropomyosin-binding domain of cTnT, a mutation associated with a clinically severe form of familial hypertrophic cardiomyopathy. This functional domain of cTnT has recently been shown to be a crucial modulator of contractile function despite the fact that it does not directly interact with the ATP hydrolysis site in the myosin head. Simultaneous measurements of cardiac energetics using 31P NMR spectroscopy and contractile performance of the intact beating heart revealed both a decrease in the free energy of ATP hydrolysis available to support contractile work and a marked inability to increase contractile performance upon acute inotropic challenge in hearts from R92Q mice. These results show that alterations in thin filament protein structure and function can lead to significant defects in myocardial energetics and contractile reserve.

Activation of protein kinase C (PKC) protects the heart from ischemic injury; however, its mechanism of action is unknown, in part because no model for chronic activation of PKC has been available. To test whether chronic, mild elevation of PKC activity in adult mouse hearts results in myocardial protection during ischemia or reperfusion, hearts isolated from transgenic mice expressing a low level of activated PKCbeta throughout adulthood (beta-Tx) were compared with control hearts before ischemia, during 12 or 28 min of no-flow ischemia, and during reperfusion. Left-ventricular-developed pressure in isolated isovolumic hearts, normalized to heart weight, was similar in the two groups at baseline. However, recovery of contractile function was markedly improved in beta-Tx hearts after either 12 (97 +/- 3% vs. 69 +/- 4%) or 28 min of ischemia (76 +/- 8% vs. 48 +/- 3%). Chelerythrine, a PKC inhibitor, abolished the difference between the two groups, indicating that the beneficial effect was PKC-mediated. (31)P NMR spectroscopy was used to test whether modification of intracellular pH and/or preservation of high-energy phosphate levels during ischemia contributed to the cardioprotection in beta-Tx hearts. No difference in intracellular pH or high-energy phosphate levels was found between the beta-Tx and control hearts at baseline or during ischemia. Thus, long-term modest increase in PKC activity in adult mouse hearts did not alter baseline function but did lead to improved postischemic recovery. Furthermore, our results suggest that mechanisms other than reduced acidification and preservation of high-energy phosphate levels during ischemia contribute to the improved recovery.

The nature and distribution of amino acids in the helix interfaces of four polytopic membrane proteins (cytochrome c oxidase, bacteriorhodopsin, the photosynthetic reaction center of Rhodobacter sphaeroides, and the potassium channel of Streptomyces lividans) are studied to address the role of glycine in transmembrane helix packing. In contrast to soluble proteins where glycine is a noted helix breaker, the backbone dihedral angles of glycine in transmembrane helices largely fall in the standard alpha-helical region of a Ramachandran plot. An analysis of helix packing reveals that glycine residues in the transmembrane region of these proteins are predominantly oriented toward helix-helix interfaces and have a high occurrence at helix crossing points. Moreover, packing voids are generally not formed at the position of glycine in folded protein structures. This suggests that transmembrane glycine residues mediate helix-helix interactions in polytopic membrane proteins in a fashion similar to that seen in oligomers of membrane proteins with single membrane-spanning helices. The picture that emerges is one where glycine residues serve as molecular notches for orienting multiple helices in a folded protein complex.

Hydrophobic interactions are responsible for stabilizing leucine zippers in peptides containing heptad repeats. The effects of substituting leucine by phenylalanine and alanine by glycine on the self-assembly of coiled-coils were examined in minimalist antimicrobial peptides designed to form amphipathic alpha-helices. The secondary structure of these peptides was monitored in solution and in diphosphocholine (DPC) micelles using circular dichroism spectroscopy. The leucine peptides (KLAKLAK)3 and (KLAKKLA)n (n = 3, 4) become alpha-helical with increasing concentrations of salt, peptide, and DPC. The aggregation state and equilibrium constant for self-association of the peptides were measured by sedimentation equilibrium. The glycine peptide (KLGKKLG)3 does not self-associate. The leucine peptides and phenylalanine peptides (KFAKFAK)3 and (KFAKKFA)n (n = 3, 4) are in a monomer-tetramer equilibrium in solution, with the phenylalanine zippers being 2-4 kcal/mol less stable than the equivalent leucine zippers. Thermodynamic parameters for the association reaction were calculated from the temperature dependence of the association constants. Leucine zipper formation has DeltaCp = 0, whereas phenylalanine zipper formation has a small negative DeltaCp, presumably due to the removal of the larger surface area of phenylalanine from water. Self-association of the peptides is coupled to formation of a hydrophobic core as detected using 1-anilino-naphthalene-8-sulfonate fluorescence. Carboxyfluorescein-labeled peptides were used to determine the aggregation state of (KLAKKLA)3 and (KLGKKLG)3 in DPC micelles. (KLAKKLA)3 forms dimers, and (KLGKKLG)3 is a monomer. Aggregation appears to correlate with the cytotoxicity of these peptides.

De novo antimicrobial peptides with the sequences: (KLAKKLA)n, (KLAKLAK)n (where n = 1,2,3), (KALKALK)3, (KLGKKLG)n, and (KAAKKAA)n (where n = 2,3), were prepared as the C-terminus amides. These peptides were designed to be perfectly amphipathic in helical conformations. Peptide antibacterial activity was tested against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Peptide cytotoxicity was tested against human erythrocytes and 3T3 mouse fibroblasts. The 3T3 cell testing was a much more sensitive test of cytotoxicity. The peptides were much less lytic toward human erythrocytes than 3T3 cells. Peptide secondary structure in aqueous solution, sodium dodecylsulfate micelles, and phospholipid vesicles was estimated using circular dichroism spectroscopy. The leucine/alanine-containing 21-mers were bacteriostatic at 3-8 microM and cytotoxic to 3T3 cells at about 10 microM concentrations. The leucine/alanine- or leucine/glycine-containing 14-mers and the leucine/glycine 21-mer were bacteriostatic at 6-22 microM but had much lower cytotoxicity toward 3T3 cells and higher selectivities than the natural antimicrobial peptides magainin 2 amide and cecropin B amide. The 7-mer peptides are devoid of biological activity and of secondary structure in membrane mimetic environments. The 14-mer peptides and the glycine-containing 21-mer show modest levels of helicity in model membranes. The leucine/alanine-containing 21-mer peptides have substantial helicity in model membranes. The propensity to alpha-helical conformation of the peptides in amphipathic media is proportional to their 3T3 cell cytotoxicity.