Structural analysis and biosynthesis gene cluster of an antigenic glycopeptidolipid from Mycobacterium intracellulare

Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2'-methyl-3'-hydroxy-4'-methoxy-pentanoyl-amido-3,6-dideoxy-beta-hexose-(1-->3)-4-O-methyl-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->2)-6-deoxy-alpha-L-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.     

Structural states of the binuclear copper cluster of Cancer magister methemocyanin.

The binuclear cupric copper cluster of $\text{Cancer}\text{magister}$ methemocyanin prepared from hemocyanin and hydrogen peroxide is diamagnetic (1). Upon treatment with azide, it is transformed into magnetic dipolar coupled (paramagnetic) Cu(II) pairs and then into magnetically isolated Cu(II) complexes. This progressive uncoupling of the binuclear cupric pairs in methemocyanin is interpreted in terms of a relaxation of superexchange through one or more bridging ligands.     

Structural homology among the peroxidase enzyme family revealed by hydrophobic cluster analysis

A number of peroxidase amino acid sequences show limited homology to short regions comprising the known active site cleft of yeast cytochrome c peroxidase. Otherwise no clear homology is visible in linear alignments between this enzyme and other peroxidases. We have subjected eight peroxidase sequences to hydrophobic cluster analysis. Our results suggest that these peroxidases are evolutionary related and that they share many folding characteristics.     

Structural change in response to ligand binding

The minutiae of subtle changes that occur in response to ligand binding in multiprotein complexes are often difficult to assess without resource to high resolution X-ray analysis. Recent developments in mass spectrometry, however, are providing insight into dynamic changes within components. In this article we review recent applications of MS for selection of ligands and definition of their binding characteristics for individual protein targets through to macromolecular complexes such as ribosomes.     

A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.

A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of 7.0 nm. The cluster's visibility can be enhanced with silver development for use in EM or light microscopy for histological purposes, or to detect less than or equal to 0.2 pg of antigen in immunoblots. By using a gold compound with covalent attachment, a number of advantages over colloidal gold probes are realized, including better resolution, stability, uniformity, sensitivity, and complete absence of aggregation; its small size should also improve penetration and more quantitative labeling of antigenic sites.     

Structural change in the cell center during fibroblast polarization and movement

In polarizing and migrating 3T3/Balb mouse fibroblasts, the centrioles are located between the nucleus and the leading edge of the cell. In cells within the monolayer and in migrating cells, the centrioles have a random orientation towards the substrate. In polarized cells, that still remain in the monolayer, one centriole may be perpendicular to the substrate plane in 70% of cases. Upon polarization and migration of fibroblasts, the number of microtubules, which radiate from the centriolar region, increases. These data support a hypothesis that the number of microtubules in the cell centre characterizes the rate of their renovation in the cytoplasm. It is concluded that the cell centre is strongly involved in polarization and migration of fibroblasts.     

Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2

The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior. The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site. In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains. We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography. The structures of the metal-free forms of wild-type C. ammoniagenes R2 and the mutant E. coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible. Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E. coli apo proteins as compared to wild-type apo R2. Further structural variations are revealed by the wild-type apo C. ammoniagenes R2 structure. The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues. Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.     

Lessons from the rifamycin biosynthetic gene cluster.

There is currently intense interest in unravelling the modus operandi of type I modular polyketide synthases in order to lay the ground work for their use in the combinatorial biosynthesis of new bioactive molecules. Much of our knowledge is derived from studies on 6-deoxyerythronolide B (DEBS), the enzyme assembling the polyketide backbone of erythromycin. Work on the rifamycin polyketide synthase has revealed a number of features that differ from those seen with DEBS.     

Structural change in a B-DNA helix with hydrostatic pressure

Study of the effects of pressure on macromolecular structure improves our understanding of the forces governing structure, provides details on the relevance of cavities and packing in structure, increases our understanding of hydration and provides a basis to understand the biology of high-pressure organisms. A study of DNA, in particular, helps us to understand how pressure can affect gene activity. Here we present the first high-resolution experimental study of B-DNA structure at high pressure, using NMR data acquired at pressures up to 200 MPa (2 kbar). The structure of DNA compresses very little, but is distorted so as to widen the minor groove, and to compress hydrogen bonds, with AT pairs compressing more than GC pairs. The minor groove changes are suggested to lead to a compression of the hydration water in the minor groove.     

Structural basis of the honey bee PBP pheromone and pH-induced conformational change

The behavior of insects and their perception of their surroundings are driven, in a large part, by odorants and pheromones. This is especially true for social insects, such as the honey bee, where the queen controls the development and the caste status of the other individuals. Pheromone perception is a complex phenomenon relying on a cascade of recognition events, initiated in antennae by pheromone recognition by a pheromone-binding protein and finishing with signal transduction at the axon membrane level. With to the objective of deciphering this initial step, we have determined the structures of the bee antennal pheromone-binding protein (ASP1) in the apo form and in complex with the main component of the queen mandibular pheromonal mixture, 9-keto-2(E)-decenoic acid (9-ODA) and with nonpheromonal components. In the apo protein, the C terminus obstructs the binding site. In contrast, ASP1 complexes have different open conformations, depending on the ligand shape, leading to different volumes of the binding cavity. The binding site integrity depends on the C terminus (111-119) conformation, which involves the interplay of two factors; i.e. the presence of a ligand and a low pH. Ligand binding to ASP1 is favored by low pH, opposite to what is observed with other pheromone-binding proteins, such as those of Bombyx mori and Anopheles gambiae.     

In vivo liberation of gold ions from gold implants. Autometallographic tracing of gold in cells adjacent to metallic gold

For some years, the implantation of small pieces of gold has been used as an unauthorised remedy for osteoarthritis and pain. The aim of the present study was to evaluate whether gold ions are released from gold implants. Pieces of pure gold were placed in the connective tissue of skin, bone and brains of anaesthetised animals. Ten days to several months later the animals were anaesthetised and killed by transcardial perfusion. Tissue blocks containing the gold pieces were cut, and the sections were silver-enhanced by autometallography. It was found that gold ions are released from the implanted gold and diffuse out into the surrounding tissue. The gold-containing cells in connective tissues were macrophages, mast cells and fibroblasts. In the brain, gold accumulated in astrocytes and neurons. Proton-induced X-ray emission spectroscopy analysis of the tissue surrounding gold implants confirmed that gold ions are liberated. The findings suggest that the gold implant technique, on a local scale, mimics systemic treatment with a gold-containing drug.     

Evolution of the leucine gene cluster in Buchnera aphidicola: insights from chromosomal versions of the cluster

In Buchnera aphidicola strains associated with the aphid subfamilies Thelaxinae, Lachninae, Pterocommatinae, and Aphidinae, the four leucine genes (leuA, -B, -C, and -D) are located on a plasmid. However, these genes are located on the main chromosome in B. aphidicola strains associated with the subfamilies Pemphiginae and Chaitophorinae. The sequence of the chromosomal fragment containing the leucine cluster and flanking genes has different positions in the chromosome in B. aphidicola strains associated with three tribes of the subfamily Pemphiginae and one tribe of the subfamily Chaitophorinae. Due to the extreme gene order conservation of the B. aphidicola genomes, the variability in the position of the leucine cluster in the chromosome may be interpreted as resulting from independent insertions from an ancestral plasmid-borne leucine gene. These findings do not support a chromosomal origin for the leucine genes in the ancestral B. aphidicola and do support a back transfer evolutionary scenario from a plasmid to the main chromosome.     

Structural and thermodynamic behavior of cytochrome c assembled with glutathione-covered gold nanoparticles

The conformational changes of horse heart ferricytochrome c (cyt c) after association of gold nanoparticles have been studied by electronic absorption spectroscopy and circular dichroism (CD). Our results show that the structural stability around the heme of complexed cyt c was increased successfully. Glutathione-layered gold nanoparticles caused a significant increase of the apparent pK values of the cyt c alkaline transition. Similarly, the heme crevice became more stable to heat after assembly of cyt c with gold nanoparticles. In contrast, gold nanoparticles weaken the overall thermal stability of the cyt c by decreasing the denaturation temperature estimated from far-UV CD measurements. Similar behavior has previously been reported for cyt c complexed with physiological redox partners as well as hydrophilic polyanions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.