Carbohydrate-based Clostridium difficile vaccines.

Clostridium difficile is responsible for thousands of deaths each year and a vaccine would be welcomed, especially one that would disrupt bacterial maintenance, colonization and persistence in carriers and convalescent patients. Structural explorations at the University of Guelph (ON, Canada) discovered that C. difficile may express three phosphorylated polysaccharides, named PSI, PSII and PSIII; this review captures our recent efforts to create vaccines based on these glycans, especially PSII, the common antigen that has precipitated immediate attention. The authors describe the design and immunogenicity of vaccines composed of raw polysaccharides and conjugates thereof. So far, it has been observed that anti-PSII antibodies can be raised in farm animals, mice and hamster models; humans and horses carry anti-PSII IgA and IgG antibodies from natural exposure to C. difficile, respectively; phosphate is an indispensable immunogenic epitope and vaccine-induced PSII antibodies recognize PSII on C. difficile outer surface.

A vaccine and diagnostic target for Clostridium bolteae, an autism-associated bacterium.

Constipation and diarrhea are common in autistic patients. Treatment with antibiotics against bacteria appears to partially alleviate autistic-related symptoms. Clostridium bolteae is a bacterium that has been shown to be overabundant in the intestinal tract of autistic children suffering from gastric intestinal ailments, and as such is an organism that could potentially aggravate gastrointestinal symptoms. We set out to investigate the cell-wall polysaccharides of C. bolteae in order to evaluate their structure and immunogenicity. Our explorations revealed that C. bolteae produces a conserved specific capsular polysaccharide comprised of rhamnose and mannose units: [→3)-α-D-Manp-(1→4)-ß-d-Rhap-(1→], which is immunogenic in rabbits. These findings are the first description of a C. bolteae immunogen and indicate the prospect of using this polysaccharide as a vaccine to reduce or prevent C. bolteae colonization of the intestinal tract in autistic patients, and as a diagnostic marker for the rapid detection of C. bolteae in a clinical setting.

Crystallographic insights into the pore structures and mechanisms of the EutL and EutM shell proteins of the ethanolamine-utilizing microcompartment of Escherichia coli.

The ethanolamine-utilizing bacterial microcompartment (Eut-BMC) of Escherichia coli is a polyhedral organelle that harbors specific enzymes for the catabolic degradation of ethanolamine. The compartment is composed of a proteinaceous shell structure that maintains a highly specialized environment for the biochemical reactions inside. Recent structural investigations have revealed hexagonal assemblies of shell proteins that form a tightly packed two-dimensional lattice that is likely to function as a selectively permeable protein membrane, wherein small channels are thought to permit controlled exchange of specific solutes. Here, we show with two nonisomorphous crystal structures that EutM also forms a two-dimensional protein membrane. As its architecture is highly similar to the membrane structure of EutL, it is likely that the structure represents a physiologically relevant form. Thus far, of all Eut proteins, only EutM and EutL have been shown to form such proteinaceous membranes. Despite their similar architectures, however, both proteins exhibit dramatically different pore structures. In contrast to EutL, the pore of EutM appears to be positively charged, indicating specificity for different solutes. Furthermore, we also show that the central pore structure of the EutL shell protein can be triggered to open specifically upon exposure to zinc ions, suggesting a specific gating mechanism.

Structural characterization of the organic solvent-stable cholesterol oxidase from Chromobacterium sp. DS-1.

Cholesterol oxidase is of significant commercial interest as it is widely used as a biosensor for the detection of cholesterol in clinical samples, blood serum and food. Increased stability of this enzyme with regards to temperature and different solvent conditions are of great importance to the reliability and versatility of its applications. We here report the crystal structure of the cholesterol oxidase of Chromobacterium sp. DS-1 (CHOLOX). In contrast to other previously characterized cholesterol oxidases, this enzyme retains high activity in organic solvents and detergents at temperatures above 85 degrees C despite its mesophilic origin. With the availability of one other homologous oxidase of known three-dimensional structure, a detailed comparison of its sequence and structure was performed to elucidate the mechanisms of stabilization. In contrast to factors that typically contribute to the stability of thermophilic proteins, the structure of CHOLOX exhibits a larger overall cavity volume, less charged residues and less salt bridge interactions. Moreover, the vast majority of residue substitutions were found on or near the protein's solvent exposed surface. We propose that the engineering of enhanced stability may also be accomplished through selective engineering of the protein periphery rather than by redesigning its entire core.

Real-time in vivo imaging of mercury uptake in Caenorhabditis elegans through the foodchain.

Mercury is a strong poison that poses significant and immediate hazards to human health. Due to its bioaccumulative properties, even small amounts of the metal are usually very poisonous or lethal when absorbed over long periods of time. Even though the possible dangers of mercury interactions with proteins are well understood, little is known about its uptake and dynamics within an organism. In particular, the concentration and distribution of the metal within a cell or a tissue are only poorly understood. In this study, we describe the application of a recently developed biosensor [Chapleau R.R., Blomberg R., Ford P.C., Sagermann M., 2008. Design of a highly specific and non-invasive biosensor suitable for real-time in vivo imaging of mercury(II) uptake. Protein Sci. 17(4), 614-622] that facilitates unprecedented non-invasive real-time imaging of ionic mercury uptake by an organism under in vivo conditions. Specifically, we here show that mercury ions can be taken up from the environment within minutes by prokaryotic as well as eukaryotic organisms. This rapid uptake can still be detected if the sensor expressing cells are shielded by layers of surrounding tissues suggesting that neither individual cell walls nor tissues provide a serious barrier for the metal. Furthermore, we show that this biosensor is suitable for the direct imaging of mercury uptake through the food chain. Our results suggest that ionic mercury remains available for extended periods of time and can rapidly contaminate surface as well as embedded tissue cells.

Crystal structure of the EutL shell protein of the ethanolamine ammonia lyase microcompartment.

Bacterial microcompartments (BMCs) are specialized organelles that use proteinaceous membranes to confine chemical reaction spaces. The ethanolamine ammonialyase microcompartment of Escherichia coli represents such a class of cytosolic organelles that enables bacteria to survive on small organic molecules such as ethanolamine as the sole source for carbon and nitrogen. We present here the crystal structure of the shell protein EutL at 2.2-A resolution. With 219 residues, it is the largest representative of this BMC's shell proteins. In the crystal, EutL forms a trimer that exhibits a hexagonally shaped tile structure. The tiles arrange into a tightly packed 2D array that is likely to resemble the proteinaceous membrane of the intact BMC. In contrast to other BMC shell proteins, which have only 1 pore per tile, EutL exhibits 3 pores per tile, thereby significantly increasing the overall porosity of this protein membrane. Each of the individual pores is lined with negatively charged residues and aromatic residues that are proposed to facilitate passive transport of specific solutes. The characteristic shape of the hexagonal tile, which is also found in the microcompartments of carbon-fixating bacteria, may present an inherent and fundamental building unit that may provide a general explanation for the formation of differently sized microcompartments.

Preliminary structural investigations of the Eut-L shell protein of the ethanolamine ammonia-lyase metabolosome of Escherichia coli.

The ethanolamine ammonia-lyase microcompartment is composed of five different shell proteins that have been proposed to assemble into symmetrically shaped polyhedral particles of varying sizes. Here, preliminary X-ray analysis of crystals of the bacterial microcompartment shell protein Eut-L from Escherichia coli is reported. Cloning, overexpression and purification resulted in highly pure protein that crystallized readily under many different conditions. In all cases the protein forms thin hexagonal plate-shaped crystals belonging to space group P3 that are of unusually high stability against different solvent conditions. The crystals diffracted to a resolution of 2.0 A using synchrotron radiation but proved to be radiation-sensitive. Preparations of heavy-atom-derivatized crystals for use in determining the three-dimensional structure are under way.

Using affinity chromatography to engineer and characterize pH-dependent protein switches.

Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione-S-transferase to convert this protein into a pH-dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH-dependent GSH-binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector-specific allostery into a protein structure.

Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase.

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53-62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K(m) and V(max) values of the cholesterol oxidase were estimated from Lineweaver-Burk plots. The V(max)/K(m) ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.

Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance.

A new screening method for 6beta-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0-7.5, showing optimal activity at pH 7.0 and 65 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.

Design of a highly specific and noninvasive biosensor suitable for real-time in vivo imaging of mercury (II) uptake.

Mercury is a ubiquitous pollutant that when absorbed is extremely toxic to a wide variety of biochemical processes. Mercury (II) is a strong, "invisible" poison that is rapidly absorbed by tissues of the intestinal tract, kidneys, and liver upon ingestion. In this study, a novel fluorescence-based biosensor is presented that allows for the direct monitoring of the uptake and distribution of the metal under noninvasive in vivo conditions. With the introduction of a cysteine residue at position 205, located in close proximity to the chromophore, the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensor for this metal ion. The mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH. Absorbance and fluorescence properties with respect to the metal concentration exhibit sigmoidal binding behavior with a detection limit in the low nanomolar range. Time-resolved binding studies indicate rapid subsecond binding of the metal to the protein. The crystal structures obtained of mutant eGFP205C indicate a possible access route of the metal into the core of the protein. To our knowledge, this engineered protein is a first example of a biosensor that allows for noninvasive and real-time imaging of mercury uptake in a living cell. A major advantage is that its expression can be genetically controlled in many organisms to enable unprecedented studies of tissue specific mercury uptake.

Sequential reorganization of beta-sheet topology by insertion of a single strand.

Insertions, duplications, and deletions of sequence segments are thought to be major evolutionary mechanisms that increase the structural and functional diversity of proteins. Alternative splicing, for example, is an intracellular editing mechanism that is thought to generate isoforms for 30%-50% of all human genes. Whereas the inserted sequences usually display only minor structural rearrangements at the insertion site, recent observations indicate that they may also cause more dramatic structural displacements of adjacent structures. In the present study we test how artificially inserted sequences change the structure of the beta-sheet region in T4 lysozyme. Copies of two different beta-strands were inserted into two different loops of the beta-sheet, and the structures were determined. Not surprisingly, one insert "loops out" at its insertion site and forms a new small beta-hairpin structure. Unexpectedly, however, the second insertion leads to displacement of adjacent strands and a sequential reorganization of the beta-sheet topology. Even though the insertions were performed at two different sites, looping out occurred at the C-terminal end of the same beta-strand. Reasons as to why a non-native sequence would be recruited to replace that which occurs in the native protein are discussed. Our results illustrate how sequence insertions can facilitate protein evolution through both local and nonlocal changes in structure.

Relocation or duplication of the helix A sequence of T4 lysozyme causes only modest changes in structure but can increase or decrease the rate of folding.

In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associated with the C-terminal domain in the folded structure. To investigate the implications of this arrangement for the folding of the protein, we first created a circularly permuted variant with a new amino terminus at residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminus of the molecule. The protein crystallized nonisomorphously with the wild type but has a very similar structure, showing that the unit consisting of helix A and the C-terminal domain can be reconstituted from a contiguous polypeptide chain. The protein is less stable than the wild type but folds slightly faster. We then produced a second variant in which the helix A sequence was appended at the C-terminus (as in the first variant), but was also restored at the N-terminus (as in the wild type). This variant has two helix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete for the same site in the folded protein. The crystal structure shows that it is the N-terminal sequence that folds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loop out. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantly slower. The reduction in rate is attributed to the presence of the two identical sequence segments which compete for a single, mutually exclusive, site.

Long-distance conformational changes in a protein engineered by modulated sequence duplication.

There are few, if any, known instances in which a biological signal is transmitted via a large conformational change through the body of a protein. We describe here a mutant of T4 lysozyme that was engineered to permit structural change at a distance. The design uses a tandem sequence repeat that makes it possible to transmit large-scale structural changes from one end of an alpha-helix to the other over a distance of 17-25 A. The method should be of general applicability and may make it possible to introduce a mutation at one site in a protein that will induce large-scale changes in the structure at a spatially remote site.

Crystal structures of a T4-lysozyme duplication-extension mutant demonstrate that the highly conserved beta-sheet region has low intrinsic folding propensity.

Residues 24 to 35 of T4 lysozyme correspond to the second and third strands of a region of beta-sheet that is highly conserved in all known lysozyme and chitinase structures. To evaluate the intrinsic propensity of these amino acid residues to form a defined structure they were added at the C terminus of the native protein, together with a dipeptide linker. Two crystal structures of this active, mutant protein were obtained, to 1.9A and 2.3A resolution, respectively. Even though the crystal conditions are similar, the appended sequence adopts very different secondary structures. In one case it is weakly structured and appears to extend through the active-site cleft, perhaps in part adding an extra strand to the original beta-sheet. In the other crystal form the extension is largely alpha-helical. The formation of these alternative structures shows that the sequence does not have a strong intrinsic propensity to form a unique fold (either beta-sheet or otherwise). The results also suggest that structural conservation during evolution does not necessarily depend on sequence conservation or the conservation of folding propensity.

A test of proposed rules for helix capping: implications for protein design.

alpha-helices within proteins are often terminated (capped) by distinctive configurations of the polypeptide chain. Two common arrangements are the Schellman motif and the alternative alpha(L) motif. Rose and coworkers developed stereochemical rules to identify the locations of such motifs in proteins of unknown structure based only on their amino acid sequences. To check the effectiveness of these rules, they made specific predictions regarding the structural and thermodynamic consequences of certain mutations in T4 lysozyme. We have constructed these mutants and show here that they have neither the structure nor the stability that was predicted. The results show the complexity of the protein-folding problem. Comparison of known protein structures may show that a characteristic sequence of amino acids (a sequence motif) corresponds to a conserved structural motif. In any particular protein, however, changes in other parts of the sequence may result in a different conformation. The structure is determined by sequence as a whole, not by parts considered in isolation.