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.
The function of the stacking tryptophan, W290, a second-coordination sphere residue in galactose oxidase, has been investigated via steady-state kinetics measurements, absorption, CD and EPR ...spectroscopy, and X-ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly slower in the W290 variants. The K m for d-galactose for W290H is similar to that of the wild type, whereas the K m is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding and/or positioning via the NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type−azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radicals in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has properties similar to those of the wild-type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in the W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semireduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X−azide complexes are also closely similar to the wild-type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function.
Roadblock/LC7 is a member of a class of dynein light chains involved in regulating the function of the dynein complex. We have determined the three-dimensional structure of isoform 1 of the mouse ...Roadblock/LC7 cytoplasmic dynein light chain (robl1_mouse) by NMR spectroscopy. In contrast to a previously reported NMR structure of the human homolog with 96% sequence identity (PDB 1TGQ), which showed the protein as a monomer, our results indicate clearly that robl1 exists as a symmetric homodimer. The two β
3-strands pair with each other and form a continuous ten-stranded β-sheet. The 25-residue α
2-helix from one subunit packs antiparallel to that of the other subunit on the face of the β-sheet. Zipper-like hydrophobic contacts between the two helices serve to stabilize the dimer. Through an NMR titration experiment, we localized the site on robl1_mouse that interacts with the 40 residue peptide spanning residues 243 through 282 of IC74-1_rat. These results provide physical evidence for a symmetrical interaction between dimeric robl1 and the two molecules of IC74-1 in the dynein complex.
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.
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.
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.
Abstract
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
h
1 (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
h
1 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.
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