Daniel D. Clark

The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response

In plants, salicylic acid (SA) plays an important role in signaling both local and systemic defense responses. Previous efforts to identify SA effector proteins in tobacco have led to the isolation of two soluble cytoplasmic SA-binding proteins (SABPs): catalase, SABP, and an ≈25-kDa protein, SABP2. Here we describe the identification of an SA-binding protein, SABP3, in the stroma of tobacco chloroplasts. SABP3 bound SA with an apparent dissociation constant (Kd) of 3.7 μM and exhibited much greater affinity for biologically active than inactive analogs. Purification and partial sequencing of SABP3 indicated that it is the chloroplast carbonic anhydrase (CA). Confirming this finding, recombinant tobacco chloroplast CA exhibited both CA enzymatic and SA-binding activities. Expression of this protein in yeast also demonstrated that CA/SABP3 has antioxidant activity. A second gene encoding CA was also cloned, and its encoded protein was shown to behave similarly to that purified as SABP3. Finally, silencing of CA gene expression in leaves suppressed the Pto:avrPto-mediated hypersensitive response in disease resistance. These results demonstrate that SA may act through multiple effector proteins in plants and shed further light on the function of CA in chloroplasts.

Shotgun proteomics of Xanthobacter autotrophicus Py2 reveals proteins specific to growth on propylene

Coenzyme M (CoM, 2-mercaptoethanesulfonate), once thought to be exclusively produced by methanogens, is now known to be the central cofactor in the metabolism of short-chain alkenes by a variety of aerobic bacteria. There is little evidence to suggest how, and under what conditions, CoM is biosynthesized by these organisms. A shotgun proteomics approach was used to investigate CoM-dependent propylene metabolism in the Gram-negative bacterium Xanthobacter autotrophicus Py2. Cells were grown on either glucose or propylene, and the soluble proteomes were analyzed. An average of 395 proteins was identified from glucose-grown replicates, with an average of 419 identified from propylene-grown replicates. A number of linear megaplasmid (pXAUT01)-encoded proteins were found to be specifically produced by growth on propylene. These included all known to be crucial to propylene metabolism, in addition to an aldehyde dehydrogenase, a DNA-binding protein, and five putative CoM biosynthetic enzymes. This work has provided fresh insight into bacterial alkene metabolism and has generated new targets for future studies in X. autotrophicus Py2 and related CoM-dependent alkene-oxidizing bacteria.

Crystallization and preliminary X-ray analysis of an R-2-hydroxypropyl-coenzyme M dehydrogenase

The R-2-hydroxypropyl-coenzyme M (2-mercaptoethanesulfonate) dehydrogenase is a key enzyme in the microbial conversion of propylene to the central metabolite acetoacetate. This enzyme is an interesting member of the NAD(P)H-dependent short-chain dehydrogenase/reductase (SDR) family of enzymes, being one of a pair of homologous dehydrogenases that act in concert in a single pathway to convert the R- and S-enantiomers of hydroxypropyl-coenzyme M to the achiral ketopropyl-coenzyme M product. Crystallization trials have revealed that the highest diffraction quality crystals (better than 2.0 A resolution) could be achieved when the reaction substrates were added to the enzyme in a stoichiometric excess prior to crystallization.

Mechanism of inhibition of aliphatic epoxide carboxylation by the coenzyme M analog 2-bromoethanesulfonate.

The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys82) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys87 was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys82. These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.

Crystallization and preliminary X-ray analysis of a NADPH 2-ketopropyl-coenzyme M oxidoreductase/carboxylase

NADPH 2-ketopropyl-coenzyme M (2-mercaptoethanesulfonate) oxidoreductase/carboxylase is the terminal enzyme in a metabolic pathway that results in the conversion of propylene to the central metabolite acetoacetate. This enzyme is an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase family of enzymes and catalyzes the cleavage and carboxylation of 2-ketopropyl-coenzyme M to form acetoacetate and coenzyme M. Crystallization trials have revealed that the highest diffraction quality crystals (better that 2.0 A resolution) could be achieved when the substrate or product of the reaction was added to the enzyme in a stoichiometric excess.

Preliminary investigation of deoxyoligonucleotide binding to ribonuclease A using mass spectrometry: An attempt to develop a lab experience for undergraduates

Deoxyoligonucleotide binding to bovine pancreatic ribonuclease A (RNase A) was investigated using electrospray ionization ion-trap mass spectrometry (ESI-IT-MS). Deoxyoligonucleotides included CCCCC (dC 5) and CCACC (dC 2AC 2).xa0 This work was an attempt to develop a biochemistry lab experience that would introduce undergraduates to the use of mass spectrometry for the analysis of protein-ligand interactions.xa0 Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations.xa0 Samples at equilibrium were infused directly into the mass spectrometer under native conditions.xa0 For each deoxyoligonucleotide, mass spectra showed one-to-one binding stoichiometry, with marked increases in the total ion abundance of ligand-bound RNase A complexes as a function of concentration, but the accurate determination of dC 5 and dC 2AC 2 dissociation constants was problematic.

Virtual protein purification: A simple exercise to introduce ph as a parameter that effects ion exchange chromatography

This article describes a simple exercise using a free, easy‐to‐use, established online program. The exercise helps to reinforce protein purification concepts and introduces undergraduates to pH as a parameter that affects anion‐exchange chromatography. The exercise was tested with biochemistry majors at California State University‐Chico. Given the versatility of the program, this work is also a model for instructors that wish to develop their own exercise to help teach other protein purification techniques.

Fcp-Indi/C-Pac: Cpac Version 1.0.1B Beta

The updates to CPAC in this new version include:nnCPAC now offers De-Spiking as an option in nuisance regression, which regresses out the impact of motion-induced artifacts from the functional timeseries from volumes exhibiting motion greater than a specified threshold, without removing those volumes.nnUsers can now select which Framewise Displacement (FD) calculation to use (Jenkinsons or Powers) when applying the motion threshold for either Scrubbing or De-Spiking.nnCPAC can now automatically select a Framewise Displacement (FD) threshold based on a percentage value provided in the pipeline configuration. For example, if provided 5%, CPAC will select a cut-off derived from the top 5% of highest-motion volumes. See the User Guide for more information.nnScrubbing has been moved to the Nuisance Regression tab in the GUIs pipeline configuration editor. The pipeline configuration YAML keys have changed for scrubbing settings. See the User Guide Nuisance Regression page and the sample pipeline configuration file for more details.nnRe-introduced the ability to stop pipeline runs easily from the GUI.nnFixed a bug in the data configuration (subject list) builder that would cause non-NIfTI files to be included if the user did not explicitly define the file extension in the file template.nnFixed a bug in the data configuration (subject list) builder where some fields would not get populated when re-loading the settings in the GUI.nnAdded better error-catching and messages in nuisance regression which warn the user if nuisance parameters are too stringent for the regression to complete properly.nnnUpdated user documentation for this release can be found here:nhttp://fcp-indi.github.io/docs/user/index.htmlnAnd as always, you can contact us here for user support and discussion:nhttps://groups.google.com/forum/#!forum/cpax_forumnRegards,nThe CPAC development team.