Crystal structure of a Trypanosoma brucei metacaspase.

Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a metacaspase, an inactive mutant of metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.

New insights into the structure of the reaction centre from Blastochloris viridis: evolution in the laboratory.

Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs.

High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins.

An important factor in the crystallization, and subsequent structural determination, of integral membrane proteins is the ability to produce a stable and monodisperse solution of the protein. Obtaining the correct purification detergent to achieve this can be laborious and is often serendipitous. In this study, high-throughput methods are used to analyze the suitability of eight different detergents on the stability of 12 inner transmembrane proteins from Escherichia coli.

Outer membrane protein A of bovine and ovine isolates of Mannheimia haemolytica is surface exposed and contains host species-specific epitopes.

Mannheimia haemolytica is the etiological agent of pneumonic pasteurellosis of cattle and sheep; two different OmpA subclasses, OmpA1 and OmpA2, are associated with bovine and ovine isolates, respectively. These proteins differ at the distal ends of four external loops, are involved in adherence, and are likely to play important roles in host adaptation. M.

Mapping lipid and detergent molecules at the surface of membrane proteins.

Electron-density maps for the crystal structures of membrane proteins often show features suggesting binding of lipids and/or detergent molecules on the hydrophobic surface, but usually it is difficult to identify the bound molecules. In our studies, heavy-atom-labelled phospholipids and detergents have been used to unequivocally identify these binding sites at the surfaces of test membrane proteins, the reaction centres from Rhodobacter sphaeroides and Blastochloris viridis. The generality of this method is discussed in the present article.

Autotransporter passenger domain secretion requires a hydrophobic cavity at the extracellular entrance of the β-domain pore.

Whooping cough (pertussis) is a highly contagious acute respiratory illness of humans caused by the Gram-negative bacterial pathogen Bordetella pertussis. The AT (autotransporter) BrkA (Bordetella serum-resistance killing protein A) is an important B. pertussis virulence factor that confers serum resistance and mediates adherence.

Expression, purification, crystallization and initial X-ray diffraction analysis of thiol peroxidase from Yersinia pseudotuberculosis.

Thiol peroxidase is an atypical 2-Cys peroxiredoxin that reduces alkyl hydroperoxides. Wild-type and C61S mutant protein have been recombinantly expressed in Escherichia coli and purified using nickel-affinity chromatography. Initial crystallization trials yielded three crystal forms in three different space groups (P2(1), P6(4) and P2(1)2(1)2(1)) both in the presence and the absence of DTT.

Crystal structure of Leishmania major oligopeptidase B gives insight into the enzymatic properties of a trypanosomatid virulence factor.

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution.

Expression, purification, crystallization and preliminary X-ray crystallographic data from TktA, a transketolase from the lactic acid bacterium Lactobacillus salivarius.

The enzyme transketolase from the lactic acid bacterium Lactobacillus salivarius (subsp. salivarius UCC118) has been recombinantly expressed and purified using an Escherichia coli expression system. Purified transketolase from L.

Crystallization and initial X-ray diffraction analysis of a mannose-binding lectin from champedak.

Mannose-binding lectin from champedak (Artocarpus integer) is a homotetramer with a single-monomer molecular weight of 16 800 Da. Previous work has shown it to bind IgE and IgM, as well as being a mitogen of T cells in humans. Champedak mannose-binding lectin has successfully been used to detect altered glycosylation states of serum proteins.

Crystal structures of all-alpha type membrane proteins.

Integral membrane proteins are involved in a wide range of essential biological functions and the determination of their three-dimensional structures plays a central role in understanding their function. This review focuses on the structures of one class of integral membrane proteins: the functionally diverse all-alpha type membrane proteins. It gives an overview of all the structures determined by X-ray crystallography, describing each system and structure in turn.

Crystallization and preliminary structural studies of champedak galactose-binding lectin.

Galactose-binding lectin from champedak (Artocarpus integer) consists of two chains: alpha and beta (133 and 21 amino acids, respectively). It has been shown to recognize and bind to carbohydrates involved in IgA and C1 inhibitor molecules. The protein was purified and crystallized at 293 K.

A protocol for high throughput methods for the expression and purification of inner membrane proteins.

The production of diffraction quality crystals for the structural determination of inner membrane proteins relies on obtaining large amounts of stable protein. Achieving this, by finding the correct parameters to successfully express and purify these proteins is often time-consuming and frustrating. The methods described here examine the most important parameters, in both expression and purification, quickly and simply.

Raman optical activity and circular dichroism reveal dramatic differences in the influence of divalent copper and manganese ions on prion protein folding.

The binding of divalent copper ions to the full-length recombinant murine prion protein PrP23-231 at neutral pH was studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UV CD). The effect of the Cu2+ ions on PrP structure depends on whether they are added after refolding of the protein in water or are present during the refolding process. In the first case ROA reveals that the hydrated alpha-helix is lost, with UV CD revealing a drop from approximately 25% to approximately 18% in the total alpha-helix content.

Multiple roles for the C-terminal tail of the chemokine scavenger D6.

D6 is a heptahelical receptor that suppresses inflammation and tumorigenesis by scavenging extracellular pro-inflammatory CC chemokines. Previous studies suggested this is dependent on constitutive trafficking of stable D6 protein to and from the cell surface via recycling endosomes. By internalizing chemokine each time it transits the cell surface, D6 can, over time, remove large quantities of these inflammatory mediators.

Mitochondrial peroxiredoxins.

Mitochondria are the major intracellular sites of oxygen consumption producing reactive oxygen species (ROS) as toxic by-products of oxidative phosphorylation, primarily via electron leakage from the respiratory chain. The resultant types of chemical damage to lipids, DNA and proteins are described as well as the broader implications for the involvement of ROS in disease onset and progression. The relative contributions of mitochondrial, enzyme-linked, antioxidant defence systems to tissue protection are also reviewed as is the emerging importance of the peroxiredoxin family in general to H2O2-mediated signalling The constituent enzymes of the mitochondrial PrxIII pathway are discussed in detail including the roles of PrxIII and PrxV in their capacities as typical 2-cys and atypical 2-cys thioredoxin-dependent hydroperoxide reductases, respectively.

Residual structure in disordered peptides and unfolded proteins from multivariate analysis and ab initio simulation of Raman optical activity data.

Vibrational Raman optical activity (ROA), measured as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the aqueous solution structure of proteins. The large number of structure-sensitive bands in protein ROA spectra makes multivariate analysis techniques such as nonlinear mapping (NLM) especially favorable for determining structural relationships between different proteins. We have previously used NLM to map a large dataset of peptide, protein, and virus ROA spectra into a readily visualizable two-dimensional space in which points close to or distant from each other, respectively, represent similar or dissimilar structures.

Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis.

P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a beta-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K.

Brominated lipids identify lipid binding sites on the surface of the reaction center from Rhodobacter sphaeroides.

This study describes the use of brominated phospholipids to distinguish between lipid and detergent binding sites on the surface of a typical alpha-helical membrane protein. Reaction centers isolated from Rhodobacter sphaeroides were cocrystallized with added brominated phospholipids. X-ray structural analysis of these crystals has revealed the presence of two lipid binding sites from the characteristic strong X-ray scattering from the bromine atoms.

Delineation of protein structure classes from multivariate analysis of protein Raman optical activity data.

Vibrational Raman optical activity (ROA), measured as a small difference in the intensity of Raman scattering from chiral molecules in right and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the aqueous solution structure of proteins. On account of the large number of structure-sensitive bands in protein ROA spectra, multivariate analysis techniques such as non-linear mapping (NLM) are especially favourable for determining structural relationships between different proteins. Here NLM is used to map a dataset of 80 polypeptide, protein and virus ROA spectra, considered as points in a multidimensional space with axes representing the digitized wavenumbers, into readily visualizable two and three-dimensional spaces in which points close to or distant from each other, respectively, represent similar or dissimilar structures.

Purification, crystallization and preliminary X-ray diffraction analysis of RafE, a sugar-binding lipoprotein from Streptococcus pneumoniae.

Streptococcus pneumoniae contains a large number of sugar-transport systems and the system responsible for raffinose uptake has recently been identified. The substrate-binding protein component of this system shares strong sequence homology with the multiple sugar metabolism substrate-binding protein MsmE from S. mutans and contains a lipoprotein-attachment site at cysteine residue 23.

Overexpression, purification and crystallization of a choline-binding protein CbpI from Streptococcus pneumoniae.

The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni-NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column.

Raman optical activity of proteins, carbohydrates and glycoproteins.

On account of its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the structure of biomolecules. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration and side-chain conformations, and on structural elements present in unfolded states. Carbohydrate ROA spectra provide information on the central features of carbohydrate stereochemistry, especially that of the glycosidic link.

Bovine mitochondrial peroxiredoxin III forms a two-ring catenane.

A crystal structure is reported for the C168S mutant of a typical 2-Cys peroxiredoxin III (Prx III) from bovine mitochondria at a resolution of 3.3 A. Prx III is present as a two-ring catenane comprising two interlocking dodecameric toroids that are assembled from basic dimeric units.

A310 helical turn is essential for the proliferation-inhibiting properties of macrophage inflammatory protein-1 alpha (CCL3).

Despite possessing marked structural similarities, the chemokines macrophage inflammatory protein-1alpha (MIP-1alpha; CCL3) and RANTES (CCL5) display differential activity in hematopoietic progenitor-cell-inhibitory assays, with MIP-1alpha being active and RANTES inactive in this context. We have sought to identify the key structural determinants of this property of MIP-1alpha. This has involved constructing MIP-1alpha/RANTES chimeras by swapping structural domains between the 2 proteins.