Ejan M. Tyler

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 μl) 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 15N-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 13C,15N-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 β-grasp protein family.

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. Proteins 2005.

Solution structure of thioredoxin h1 from Arabidopsis thaliana

Present in virtually every species, thioredoxins catalyze disulfide/dithiol exchange with various substrate proteins. While the human genome contains a single thioredoxin gene, plant thioredoxins are a complex protein family. A total of 19 different thioredoxin genes in six subfamilies has emerged from analysis of the Arabidopsis thaliana genome. Some function specifically in mitochondrial and chloroplast redox signaling processes, but target substrates for a group of eight thioredoxin proteins comprising the h subfamily are largely uncharacterized. In the course of a structural genomics effort directed at the recently completed A. thaliana genome, we determined the structure of thioredoxin h1 (At3g51030.1) in the oxidized state. The structure, defined by 1637 NMR‐derived distance and torsion angle constraints, displays the conserved thioredoxin fold, consisting of a five‐stranded β‐sheet flanked by four helices. Redox‐dependent chemical shift perturbations mapped primarily to the conserved WCGPC active‐site sequence and other nearby residues, but distant regions of the C‐terminal helix were also affected by reduction of the active‐site disulfide. Comparisons of the oxidized A. thaliana thioredoxin h1 structure with an h‐type thioredoxin from poplar in the reduced state revealed structural differences in the C‐terminal helix but no major changes in the active site conformation.

Wheat Germ Cell‐Free Expression System for Protein Production

The Center for Eukaryotic Structural Genomics, in cooperation with Ehime University and CellFree Sciences, has developed a novel wheat germ cell‐free technology for the production of eukaryotic proteins. Protein production and purification are robust and scalable for high‐throughput applications. The protocols have been used to express and purify proteins from Arabidopsis thaliana, human, mouse, rat and zebra fish. This unit describes expression and purification protocols for both small‐scale testing (microgram) and large‐scale production (milligram) of N‐His6‐ and N‐GST‐tagged proteins. The methods described in this unit can be used to produce both unlabeled and labeled proteins required for structure‐based determinations by NMR spectroscopy or X‐ray crystallography.

Letter to the Editor: Hypothetical protein At2g24940.1 from Arabidopsis thaliana has a cytochrome b5 like fold ∗

Progesterone is believed to exert rapid non-genomic actions through its interaction with membrane associated progesterone receptors (MAPRs) (Bramley, 2003; Li and O’Malley, 2003). BLAST sequence searches (Altieri et al., 1995) for mammalian MAPRs and putative MAPRs from plants have identified that these proteins all contain a cytochrome b5-like ligandbinding domain (Mifsud and Bateman, 2002). Interestingly, unlike cytochrome b5 itself, these MAPRs domains appear not to bind heme and not to be involved in redox reactions. Their distinct biological functions suggest that these steroid receptors adopt the cytochrome b5 domain as a template in order to build their own ligand-binding pockets (Mifsud and Bateman, 2002). The Center for Eukaryotic Structural Genomics is engaged in determining the three-dimensional structures of novel proteins from eukaryotic gene families. Its target selection algorithm selected Arabidopsis thaliana putative protein At2g24940.1 for structure determination. The biochemical function of At2g24940.1 currently is unknown. Its ∼40% sequence identity with mammalian MAPR suggests that At2g24940.1 may act as a steroid binding protein. In addition, its sequence is distantly similar to that of cytochrome b5 (Figure 1). Here we describe the threedimensional structure of At2g24940.1 as determined by NMR spectroscopy. At present, no structure of a MAPR is available from the Protein Data Bank. Thus, the structure of At2g24940.1 may provide clues to the function of a class of steroid binding proteins in plants.

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 β‐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.

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.

X‐ray structure of Arabidopsis At1g77680, 12‐oxophytodienoate reductase isoform 1

Brian G. Fox,* Thomas E. Malone, Kenneth A. Johnson, Stacey E. Madson, David Aceti, Craig A. Bingman, Paul G. Blommel, Blake Buchan, Brendan Burns, John Cao, Claudia Cornilescu, Jurgen Doreleijers, Jason Ellefson, Ronnie Frederick, Holokere Geetha, David Hruby, Won Bae Jeon, Todd Kimball, John Kunert, John L. Markley, Craig Newman, Andrew Olson, Francis C. Peterson, George N. Phillips Jr., John Primm, Bryan Ramirez, Nathan S. Rosenberg, Mike Runnels, Kory Seder, Jeff Shaw, David W. Smith, Hassan Sreenath, Jikui Song, Michael R. Sussman, Sandy Thao, Donna Troestler, Ejan Tyler, Robert Tyler, Eldon Ulrich, Dimitriy Vinarov, Frank Vojtik, Brian F. Volkman, Gary Wesenberg, Russell L. Wrobel, Jie Zhang, Qin Zhao, and Zolt Zolnai University of Wisconsin Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison, Madison, Wisconsin Molecular and Environmental Toxicology Program, University of Wisconsin–Madison, Madison, Wisconsin Biophysics Doctoral Program, University of Wisconsin–Madison, Madison, Wisconsin