Copine A is required for cytokinesis, contractile vacuole function, and development in Dictyostelium.

Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA. Our previous localization studies of green fluorescent protein-tagged CpnA in Dictyostelium suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a cpnA- knockout strain, and here we report the initial characterization of the mutant phenotype. The cpnA- cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions, cpnA- cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water, cpnA- cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the cpnA- cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes except cpnF showed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators of Dictyostelium development.

O-acylation of hydroxyproline residues: effect on peptide-bond isomerization and collagen stability.

In collagen, strands of the sequence XaaYaaGly form a triple-helical structure. The Yaa residue is often (2S,4R)-4-hydroxyproline (Hyp). The inductive effect of the hydroxyl group of Hyp residues greatly increases collagen stability. Here, electron withdrawal by the hydroxyl group in Hyp and its 4S diastereomer (hyp) is increased by the addition of an acetyl group or trifluoroacetyl group. The crystalline structures of AcHyp[C(O)CH3]OMe and Achyp[C(O)CH3]OMe are similar to those of AcHypOMe and AcProOMe, respectively. The O-acylation of AcHypOMe and AchypOMe increases the 13C chemical shift of its Cgamma atom: AcHyp[C(O)CF3]OMe congruent with Achyp[C(O)CF3]OMe > AcHyp[C(O)CH3]OMe congruent with Achyp[C(O)CH3]OMe > or = AcHypOMe congruent with AchypOMe. This increased inductive effect is not apparent in the thermodynamics or kinetics of amide bond isomerization. Despite apparently unfavorable steric interactions, (ProHypGly)(10), which is O-acylated with 10 acetyl groups, forms a triple helix that has intermediate stability: (ProHypGly)(10) > {ProHyp[C(O)CH3]Gly}(10) >> (ProProGly)(10). Thus, the benefit to collagen stability endowed by the hydroxyl group of Hyp residues is largely retained by an acetoxyl group.

Solvent effects on barrier to rotation of enaminonitriles using inversion transfer (1)H NMR spectroscopy and FTIR spectroscopy.

The barrier to rotation and hydrogen bonding interactions of 2,2-dicyano-1-(N,N'-dimethylamino)vinylbenzene (1) were studied in a range of solvents. The barrier to rotation of 1 in chloroform was 14.8 kcal/mol and increased by 1.7 kcal/mol in a protic solvent, trifluoroethanol. FTIR studies showed a shift in the cyano stretch of 1 to a higher wavenumber in trifluoroethanol, which is consistent with 1 participating in a hydrogen bonding interaction at the vinyl carbon (C(v)) of the enaminonitrile group.

Inductive Effects on the Energetics of Prolyl Peptide Bond Isomerization: Implications for Collagen Folding and Stability.

The hydroxylation of proline residues in collagen enhances the stability of the collagen triple helix. Previous X-ray diffraction analyses had demonstrated that the presence of an electron-withdrawing substituent on the pyrrolidine ring of proline residues has significant structural consequences [Panasik, N., Jr.; Eberhardt, E. S.; Edison, A. S.; Powell, D. R.; Raines, R. T. Int. J. Pept. Protein Res.1994, 44, 262-269]. Here, NMR and FTIR spectroscopy were used to ascertain kinetic and thermodynamic properties of N-acetyl-[ß,γ-(13)C]D,L-proline methylester (1); N-acetyl-4(R)-hydroxy-L-proline [(13)C]methylester (2); and N-acetyl-4(R)-fluoro-L-proline methylester (3). The pK(a)'s of the nitrogen atom in the parent amino acids decrease in the order: proline (10.8) > 4(R)-hydroxy-L-proline (9.68) > 4(R)-fluoro-L-proline (9.23). In water or dioxane, amide I vibrational modes decrease in the order: 1 > 2 > 3. At 37 °C in dioxane, the rate constants for amide bond isomerization are greater for 3 than 1. Each of these results is consistent with the traditional picture of amide resonance coupled with an inductive effect that results in a higher bond order in the amide C=O bond and a lower bond order in the amide C-N bond. Further, at 37 °C in water or dioxane equilibrium concentrations of the trans isomer increase in the order: 1 < 2 < 3. Inductive effects may therefore have a significant impact on the folding and stability of collagen, which has a preponderance of hydroxyproline residues, all with peptide bonds in the trans conformation.

Amide-Amide and Amide-Water Hydrogen Bonds: Implications for Protein Folding and Stability.

Amide-amide hydrogen bonds have been implicated in directing protein folding and enhancing protein stability. Inversion transfer (13)C NMR spectroscopy and IR spectroscopy were used to compare the ability of various amide solvents and of water to alter the rate of the cis-trans isomerization of the prolyl peptide bond of Ac-Gly-[ß,δ-(13)C]Pro-OMe and the amide I vibrational mode of [(13)C=O]Ac-Pro-OMe. The results indicate that secondary amides are significantly weaker hydrogen bond donors than is formamide or water. These results are most consistent with models for protein folding in which the formation of secondary structure is a cooperative process that follows hydrophobic collapse. These results also suggest that a hydrogen bond between a main-chain oxygen and an asparagine or glutamine sidechain may contribute more to protein stability than does a main-chain-main-chain hydrogen bond.

Thermodynamic Origin of Prolyl Peptide Bond Isomers.

The thermodynamic preference for the trans isomer of prolyl peptide bonds arises almost entirely from enthalpy in aqueous buffer and in toluene.

Solvent Effects on the Energetics of Prolyl Peptide Bond Isomerization.

Racemic Ac-Gly-[ß,δ-(13)C]Pro-OMe was synthesized, and the kinetics and thermodynamics of the isomerization of its prolyl peptide bond were determined in nine solvents by using NMR and IR spectroscopy. The free energy of activation is 1.3 kcal/mol larger in water than in aprotic solvents, and correlates with the ability of a solvent to donate a hydrogen bond but not with solvent polarity. These results are consistent with conventional pictures of amide resonance, which require transfer of charge between oxygen and nitrogen during isomerization. Similar medium effects may modulate the stability of planar peptide bonds in the active site of peptidyl-prolyl cis-trans isomerases (PPIases) and during the folding, function, or lysis of proteins.