Copine A is Required for Cytokinesis, Contractile Vacuole Function, and Development in Dictyostelium
Author(s): Damer, C.K., Bayeva, M., Kim, P.S., Ho L.K., Eberhardt, E.S., Socec, C.I., Lee, J.S., Bruce, E.A., Goldman-Yassen, A.E., Naliboff, L.C.
ISSN: 15359778
CODEN: ECUEA
DOI: 10.1128/EC.00322-06
PubMed ID: 17259548
Document Type: Article
Source Type: Journal
Year: 2007
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 thecpnA− 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
Author(s): Jenkins, C.L., McCloskey, A.I., Guzei, I.A., Eberhardt, E.S., Raines, R.T.
ISSN: 00063525
CODEN: BPSSF
DOI: 10.1002/bip.20164
PubMed ID: 15558658
Document Type: Article
Source Type: Journal
Year: 2005
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)CH 3]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 Cγ atom: AcHyp[C(O)CF3]OMe ≅ Achyp[C(O)CF3]OMe > AcHyp[C(O)CH3]OMe ≅ Achyp[C(O)CH3]OMe ≥ AcHypOMe ≅ 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. © 2004 Wiley Periodicals, Inc.
Preparing Undergraduates to Participate in the Post-Genome Era: A Capstone Laboratory Experience in Proteomics
Author(s): Eric S. Eberhardt, Johanna Hansen, Luke Riservato, Melissa Cole, Brandon Smaglo, Paul Szaniawski
ISSN: 14708175
CODEN: BMBEC
DOI: 10.1002/bmb.2003.494031060291
Document Type: Review
Source Type: Journal
Year: 2003
Proteomics is one of the important new disciplines to emerge from the genome sequencing projects of the last decade. In order to introduce our students to the techniques and promise of this emerging field, a capstone laboratory experience has been developed. The exercise involves multiple aspects of proteomics research including microbial culturing methods, two-dimensional gel electrophoresis techniques, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, and database mining. Over a 12-week semester, students design their own experiments and apply a proteomic approach to investigate the heat shock response in Escherichia coli. In the trial presented in this article, students successfully identified several major heat shock proteins. The laboratory outlined here can be readily adapted to explore a wide variety of responses in metabolic pathways or responses resulting from other environmental insults or stresses. Additionally, the laboratory can be modified to explore the proteomes of organelles, tissues, and other model organisms.
Microtubule-associated Protein 2 (MAP2) Associates with the NMDA Receptor and is Spatially Redistributed Within Rat Hippocampal Neurons After Oxygen-glucose Deprivation
Author(s): Buddle, M., Eberhardt, E., Ciminello, L.H., Levin, T,. Wing, R., DiPasquale, K., Raley-Susman, K.M.
ISSN: 00068993
CODEN: BRREA
DOI: 10.1016/S0006-8993(03)02758-6
PubMed ID: 12834896
Document Type: Article
Source Type: Journal
Year: 2003
MAP2 (microtubule-associated protein 2) is a cytoskeletal phosphoprotein that regulates the dynamic assembly characteristics of microtubules and appears to provide scaffolding for organelle distribution into the dendrites and for the localization of signal transduction apparatus in dendrites, particularly near spines. MAP2 is degraded after ischemia and other metabolic insults, but the time course and initial triggers of that breakdown are not fully understood. This study determined that MAP2 resides in a complex with the NMDA receptor, suggesting that spatially localized changes may be important in the mechanism of MAP2 redistribution and breakdown after oxygen-glucose deprivation (OGD). Using OGD in the adult rat hippocampal slice as a model system, this study demonstrated that MAP2 breakdown occurs very early after OGD, with the first statistical decrease in MAP2 levels within the first 30 min after the insult. There is a dramatic redistribution of MAP2 to the somata of pyramidal neurons, particularly neurons at the CA1-subiculum border. Free radicals and nitric oxide are not involved in the damage to MAP2. NMDA-receptor activation plays a prominent role in the MAP2 breakdown. In direct response to NMDA receptor activation, calcium influx, likely through the receptor ion channel complex, as well as release of calcium from the mitochondria through activation of the 2Na+-Ca2+ exchanger of mitochondria, triggers MAP2 degradation. The proteolysis of MAP2 is limited by endogenous calpain activity, likely via the spatial access of calpain to MAP2. © 2003 Elsevier Science B.V. All rights reserved.
Solvent Effects on Barrier to Rotation of Enaminonitriles Using Inversion Transfer 1H NMR Spectroscopy and FTIR Spectroscopy
Author(s): Kaur, S., Eberhardt, E.S., Doucette, A., Chase, A., Dalby, C.
ISSN: 00223263
CODEN: JOCEA
DOI: 10.1021/jo025516a
PubMed ID: 12027720
Document Type: Article
Source Type: Journal
Year: 2002
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 (Cv) of the enaminonitrile group.
Inductive Effects on the Energetics of Prolyl Peptide Bond Isomerization: Implications for Collagen Folding and Stability
Author(s): Eberhardt, E.S., Panasik Jr. N., Raines, R.T.
ISSN: 00027863
CODEN: JACSA
DOI: 10.1021/ja9623119
Document Type: Article
Source Type: Journal
Year: 1996
The hydroxylation of proline residues in collagen increases 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-[β,γ-13C]D,L-proline methyl ester (1); N-acetyl-4(R)-hydroxy-L-proline [13C]methyl ester (2); and N-acetyl-4(R)-fluoro-L-proline methyl ester (3). The pK(a)’s of the nitrogen atom in the parent amino acids decrease in the following 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 following 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 accelerate the folding and enhance the stability of collagen, which has a preponderance of hydroxyproline residues, all with peptide bonds in the trans conformation.
Contribution of a Tyrosine Side Chain to Ribonuclease A Catalysis and Stability
Author(s): Eberhardt, E.S., Wittmayer, P.K., Templer, B.M., Raines, R.T.
ISSN: 09618368
CODEN: PRCIE
PubMed ID: 8844858
Document Type: Article
Source Type: Journal
Year: 1996
An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of k(cat) being perturbed more significantly than that of K(m). The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate- limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr → Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41- Pro 42 peptide bond.
Amide-amide and Amide-water Hydrogen Bonds: Implications for Protein Folding and Stability
Author(s): Eberhardt, E.S., Raines, R.T.
ISSN: 00027863
CODEN: JACSA
Document Type: Article
Source Type: Journal
Year: 1994
Inductive Effects on the Structure of Proline Residues
Author(s): Panasik Jr., N., Eberhardt, E.S., Edison, A.S., Powell, D.R., Raines, R.T.
ISSN: 03678377
CODEN: IJPPC
PubMed ID: 7822103
Document Type: Article
Source Type: Journal
Year: 1994
4(S)-Hydroxyproline (Hyp) residues constitute about 10% of most forms of collagen, the most abundant protein in vertebrates. X-Ray diffraction analysis was used to ascertain how the structure of proline residues is affected by the inductive effect elicited by the hydroxyl group of Hyp residues. N-Acetylproline methylester (1), N-acetyl-4(S)-hydroxyproline methylester (2) and N-acetyl-4(S)-fluoroproline methylester (3) were synthesized, and their crystalline structures were determined at high resolution. The amide bond of crystalline 1 was in the cis conformation, which is the minor isomer in solution, and the pyrrolidine ring of 1 had C(γ)-endo pucker. In crystalline 2 and 3 the amide bonds were in the trans conformation, and the pyrrolidine rings had C(γ)-exo pucker. The lengths of the bonds between sp3-hybridized carbon atoms in the pyrrolidine ring were significantly shorter in 2 and 3 than in 1, as was predicted by ab initio molecular orbital calculations at the RHF/3-21G level of theory. No significant change in bond length was observed in the other bonds of 1, 2 or 3. The pyramidylization of the nitrogen atom increased dramatically in the order: 1 < 2 < 3. Together, these results indicate that electron-withdrawing substituents in the 4-position of proline residues can have a significant influence on the structure of these residues. In particular, the change in pyramidylization suggests that such substituents increase the sp3-character of the prolyl nitrogen atom and could thereby alter the rate of prolyl peptide bond isomerization.
Thermodynamic Origin of Prolyl Peptide Bond Isomers
Author(s): Eberhardt, E.S., Loh, S.N., Raines, R.T.
ISSN: 00404039
CODEN: TELEA
DOI: 10.1016/S0040-4039(00)93377-X
Document Type: Article
Source Type: Journal
Year: 1993
The preference for the trans isomer of prolyl peptide bonds arises almost entirely from enthalpy in aqueous buffer and in toluene.
NMR Strategy for Determining Xaa-Pro Peptide Bond Configurations in Proteins: Mutants of Staphylococcal Nuclease with Altered Configuration at Proline-117
Author(s): Andrew P. Hinck, Eric S. Eberhardt, John L. Markley
Journal: Biochemistry – BIOCHEMISTRY-USA , vol. 32, no. 44, pp. 11810-11818, 1993
DOI: 10.1021/bi00095a009
Year: 1993
Solvent effects on the energetics of prolyl peptide bond isomerization
Author(s): Eberhardt, E.S., Loh, S.N., Hinck, A.P., Raines, R.T.
ISSN: 00027863
CODEN: JACSA
Document Type: Article
Source Type: Journal
Year: 1992