Glyoxalase 2 is a ␤-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and 1 H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 ؎ 0.15 eq of iron and 1.31 ؎ 0.05 eq of Zn(II) and to exhibit k cat and K m values of 129 ؎ 10 s ؊1and 391 ؎ 48 M, respectively, when using S-D-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Zn(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese.1 H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-Å resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).The glyoxalase system consists of two enzymes, lactoylglutathione lyase (glyoxalase I, GLX1) 2 and hydroxyacylglutathione hydrolase (glyoxalase II, GLX2), that act coordinately to convert a variety of ␣-ketoaldehydes into hydroxy acids in the presence of glutathione (1). Aromatic and aliphatic ␣-ketoaldehydes react spontaneously with glutathione to form thiohemiacetals, which are converted to S-(2-hydroxyacyl)glutathione derivatives by GLX1. GLX2 hydrolyzes S-(2-hydroxyacyl)glutathione derivatives to regenerate glutathione and produce hydroxy acids. Glyoxalase I, a Zn(II) or Ni(II) metalloprotein, can utilize a number of ␣-ketoaldehydes (2). However, the primary physiological substrate of the enzyme is thought to be methylglyoxal (MG), a cytotoxic and mutagenic compound that is formed primarily as a by-product of carbohydrate and lipid metabolism (3, 4). MG can react with DNA to form modified guanylate residues (5) and interstrand cross-links (6). It also reacts with proteins to form glycosylamine derivatives of arginine and lysine and hemithioacetals with cysteines (7).Cells with high glycolytic rates exhibit high rates of methylglyoxal formation and increased levels of GLX1 activity. For instance, increased levels of GLX1 and GLX2 RNA and protein have been detected in tumor cells, including breast carcinoma cells (8). Because selec...

Anfinsen’s principle1 asserts that all information required to specify the structure of a protein is encoded in its amino acid sequence. However, during protein synthesis by the ribosome the N-terminus of the nascent chain can begin to fold before the C-terminus is available. We tested whether this co-translational folding can alter the folded structure of an encoded protein in vivo, versus the structure formed when refolded in vitro. We designed a fluorescent protein consisting of three half-domains, where the N- and C-terminal half-domains compete with each other to interact with the central half-domain. The outcome of this competition determines the fluorescence properties of the resulting folded structure. Upon refolding after chemical denaturation, this protein produced equimolar amounts of the N- and C-terminal folded structures, respectively. In contrast, translation in E. coli resulted in a two-fold enhancement in the formation of the N-terminal folded structure. Rare synonymous codon substitutions at the 5′ end of the C-terminal half-domain further increased selection for the N-terminal folded structure. These results demonstrate that the rate at which a nascent protein emerges from the ribosome can specify the folded structure of a protein.

Newly synthesized proteins must form their native structures in the crowded environment of the cell, while avoiding non-native conformations that can lead to aggregation. Yet remarkably little is known about the progressive folding of polypeptide chains during chain synthesis by the ribosome, or of the influence of this folding environment on productive folding in vivo. P22 tailspike is a homotrimeric protein that is prone to aggregation via misfolding of its central β-helix domain in vitro. We have produced stalled ribosome:tailspike nascent chain complexes of four fixed lengths in vivo, in order to assess co-translational folding of newly synthesized tailspike chains as a function of chain length. Partially synthesized, ribosome-bound nascent tailspike chains populate stable conformations with some native-state structural features even prior to the appearance of the entire β-helix domain, regardless of the presence of the chaperone trigger factor, yet these conformations are distinct from the conformations of released, refolded tailspike truncations. These results suggest that organization of the aggregation-prone β-helix domain occurs co-translationally, prior to chain release, to a conformation that is distinct from the accessible energy minimum conformation for the truncated free chain in solution.As a protein is synthesized by the ribosome, it begins to fold into a three-dimensional shape, and must simultaneously avoid aggregation with other proteins in the crowded cell. In this seemingly hostile environment, where total protein concentrations can exceed 200-300 mg/ mL, de novo protein folding during and after translation is, for many proteins, more efficient than in vitro refolding1. In other words, many proteins that can fold productively in the cell will aggregate severely under in vitro refolding reactions, presumably due to differences between the dominant folding pathway used. For example, experiments with both bacterial and firefly luciferase have demonstrated that these nascent chains adopt conformations cotranslationally that are not populated during refolding from denaturant2,3, and these cotranslational conformations fold to the native state much more efficiently than the conformations populated during refolding from denaturant. However, there continues to be debate as to what extent large, multi-domain proteins can fold co-translationally in prokaryotes4-7.While molecular chaperones certainly play a role in efficient protein folding in the cell, less than 20% of E. coli cytoplasmic proteins require an interaction with one of the three major chaperone systems (trigger factor (TF), DnaK/DnaJ, or GroEL/ES) in order to fold correctly under normal growth conditions8. And remarkably, both chaperone systems responsible for Correspondence should be addressed to P.L.C. (pclark1@nd.edu). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript wil...

Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein b-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

Radiopacity is a critical property of materials that are used for a range of radiological applications, including the development of phantom devices that emulate the radiodensity of native tissues and the production of protective equipment for personnel handling radioactive materials. Three-dimensional (3D) printing is a fabrication platform that is well suited to creating complex anatomical replicas or custom labware to accomplish these radiological purposes. We created and tested multiple ABS (Acrylonitrile butadiene styrene) filaments infused with varied concentrations of bismuth (1.2–2.7 g/cm3), a radiopaque metal that is compatible with plastic infusion, to address the poor gamma radiation attenuation of many mainstream 3D printing materials. X-ray computed tomography (CT) experiments of these filaments indicated that a density of 1.2 g/cm3 of bismuth-infused ABS emulates bone radiopacity during X-ray CT imaging on preclinical and clinical scanners. ABS-bismuth filaments along with ABS were 3D printed to create an embedded human nasocranial anatomical phantom that mimicked radiological properties of native bone and soft tissue. Increasing the bismuth content in the filaments to 2.7 g/cm3 created a stable material that could attenuate 50% of 99mTechnetium gamma emission when printed with a 2.0 mm wall thickness. A shielded test tube rack was printed to attenuate source radiation as a protective measure for lab personnel. We demonstrated the utility of novel filaments to serve multiple radiological purposes, including the creation of anthropomorphic phantoms and safety labware, by tuning the level of radiation attenuation through material customization.