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Dmitriy A Vinarov

Publications and source records attributed to Dmitriy A Vinarov.

7 recordsLinked to original sources
PubMed2006

Solution structure of a single-domain thiosulfate sulfurtransferase from Arabidopsis thaliana.

We describe the three-dimensional structure of the product of Arabidopsis thaliana gene At5g66040.1 as determined by NMR spectroscopy. This protein is categorized as single-domain sulfurtransferase and is annotated as a senescence-associated protein (sen1-like protein) and ketoconazole resistance protein (http://arabidopsis.org/info/genefamily/STR_genefamily.html). The sequence of At5g66040.1 is virtually identical to that of a protein from Arabidopsis found by others to confer ketoconazole resistance in yeast. Comparison of the three-dimensional structure with those in the Protein Data Bank revealed that At5g66040.1 contains an additional mobile beta-hairpin not found in other rhodaneses that may function in binding specific substrates. This represents the first structure of a single-domain plant sulfurtransferase. The enzymatically active cysteine-containing domain belongs to the CDC25 class of phosphatases, sulfide dehydrogenases, and stress proteins such as senescence specific protein 1 in plants, PspE and GlpE in bacteria, and cyanide and arsenate resistance proteins. Versions of this domain that lack the active site cysteine are found in other proteins, such as phosphatases, ubiquitin hydrolases, and sulfuryltransferases.

Amino Acid Sequence↗
PubMed2006

Wheat germ cell-free platform for eukaryotic protein production.

We describe a platform that utilizes wheat germ cell-free technology to produce protein samples for NMR structure determinations. In the first stage, cloned DNA molecules coding for proteins of interest are transcribed and translated on a small scale (25 microL) to determine levels of protein expression and solubility. The amount of protein produced (typically 2-10 microg) is sufficient to be visualized by polyacrylamide gel electrophoresis. The fraction of soluble protein is estimated by comparing gel scans of total protein and soluble protein. Targets that pass this first screen by exhibiting high protein production and solubility move to the second stage. In the second stage, the DNA is transcribed on a larger scale, and labeled proteins are produced by incorporation of [(15)N]-labeled amino acids in a 4 mL translation reaction that typically produces 1-3 mg of protein. The [(15)N]-labeled proteins are screened by (1)H-(15)N correlated NMR spectroscopy to determine whether the protein is a good candidate for solution structure determination. Targets that pass this second screen are then translated in a medium containing amino acids doubly labeled with (15)N and (13)C. We describe the automation of these steps and their application to targets chosen from a variety of eukaryotic genomes: Arabidopsis thaliana, human, mouse, rat, and zebrafish. We present protein yields and costs and compare the wheat germ cell-free approach with alternative methods. Finally, we discuss remaining bottlenecks and approaches to their solution.

Animals↗
PubMed2006

Solution structure of Arabidopsis thaliana protein At5g39720.1, a member of the AIG2-like protein family.

The three-dimensional structure of Arabidopsis thaliana protein At5g39720.1 was determined by NMR spectroscopy. It is the first representative structure of Pfam family PF06094, which contains protein sequences similar to that of AIG2, an A. thaliana protein of unknown function induced upon infection by the bacterial pathogen Pseudomonas syringae. The At5g39720.1 structure consists of a five-stranded beta-barrel surrounded by two alpha-helices and a small beta-sheet. A long flexible alpha-helix protrudes from the structure at the C-terminal end. A structural homology search revealed similarity to three members of Pfam family UPF0131. Conservation of residues in a hydrophilic cavity able to bind small ligands in UPF0131 proteins suggests that this may also serve as an active site in AIG2-like proteins.

Arabidopsis Proteins↗
PubMed2005

Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies.

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.

Arabidopsis↗
PubMed2005

High-throughput automated platform for nuclear magnetic resonance-based structural proteomics.

The development of new systems and strategies capable of synthesizing any desired soluble, labeled protein or protein fragment on a preparative scale is one of the most important tasks in biotechnology today. The Center for Eukaryotic Structural Genomics (WI, USA), in co-operation with Ehime University (Matsuyama, Japan) and CellFree Sciences Co., Ltd, has developed an automated platform for nuclear magnetic resonance-based structural proteomics that employs wheat germ extracts for cell-free production of labeled protein. The platform utilizes a single construct for all targets without any redesign of the DNA or RNA. Therefore, it offers advantages over commercial cell-free methods utilizing Escherichia coli extracts that require multiple constructs or redesign of the open reading frame. The protein production and labeling protocol is no more costly than E. coli cell-based approaches, is robust and scalable for high-throughput applications. This protocol has been used in the authors center to screen eukaryotic open reading frames from the Arabidopsis thaliana and human genomes and for the determination of nuclear magnetic resonance structures. With the recent addition of the GeneDecoder 1000 (CellFree Sciences Co., Ltd) robotic system, the Center for Eukaryotic Structural Genomics is able to carry out as many as 384 small-scale (50 microl) screening reactions per week. Furthermore, the Protemist (CellFree Sciences Co., Ltd) robotic system enables the Center for Eukaryotic Structural Genomics to carry out 16 production-scale (4 ml) reactions per week. Utilization of this automated platform technology to screen targets for expression and solubility and to produce stable isotope-labeled samples for nuclear magnetic resonance structure determinations is discussed.

Automation↗
PubMed2004

Cell-free protein production and labeling protocol for NMR-based structural proteomics.

Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods. To illustrate this platform, we describe its application to a specific target (At3g01050.1) from Arabidopsis thaliana. After cloning the target gene into a specialized plasmid, we carry out a small-scale (50 mul) in vitro sequential transcription and translation trial to ascertain the level of protein production and solubility. Next, we prepare mRNA for use in a 4-ml semicontinuous cell-free translation reaction to incorporate (15)N-labeled amino acids into a protein sample that we purify and test for suitability for NMR structural analysis. We then repeat the cell-free approach with (13)C,(15)N-labeled amino acids to prepare a doubly labeled sample. The three-dimensional (3D) structure of At3g01050.1 shows that this protein is an unusual member of the beta-grasp protein family.

Arabidopsis Proteins↗