De novo targeting to the cytoplasmic and luminal side of bacterial microcompartments.

Bacterial microcompartments, BMCs, are proteinaceous organelles that encase a specific metabolic pathway within a semi-permeable protein shell. Short encapsulation peptides can direct cargo proteins to the lumen of the compartments. However, the fusion of such peptides to non-native proteins does not guarantee encapsulation and often causes aggregation.

Structural basis of pyrrole polymerization in human porphobilinogen deaminase.

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor.

Mask pressure effects on the nasal bridge during short-term noninvasive ventilation.

The aim of this study was to assess the influence of different masks, ventilator settings and body positions on the pressure exerted on the nasal bridge by the mask and subjective comfort during noninvasive ventilation (NIV). We measured the pressure over the nasal bridge in 20 healthy participants receiving NIV four different NIV masks (three oronasal masks, one nasal mask) at three different ventilator settings and in the seated or supine position. Objective pressure measurements were obtained with an I-Scan pressure-mapping system.

A Generic Self-Assembly Process in Microcompartments and Synthetic Protein Nanotubes.

Bacterial microcompartments enclose a biochemical pathway and reactive intermediate within a protein envelope formed by the shell proteins. Herein, the orientation of the propanediol-utilization (Pdu) microcompartment shell protein PduA in bacterial microcompartments and in synthetic nanotubes, and the orientation of PduB in synthetic nanotubes are revealed. When produced individually, PduA hexamers and PduB trimers, tessellate to form flat sheets in the crystal, or they can self-assemble to form synthetic protein nanotubes in solution.

A review of drug therapy for sporadic fatal insomnia.

Background: Sporadic fatal insomnia (sFI) is a rapid progressive neurodegenerative disease characterised by gradual to perpetual insomnia, followed by dysautonomia, coma and death. The cause of sFI was recently mapped to a mutation in a protein, the prion, found in the human brain. It is the unfolding of the prion that leads to the generation of toxic oligomers that destroy brain tissue and function.

The role of intrinsic disorder and dynamics in the assembly and function of the type II secretion system.

Many Gram-negative commensal and pathogenic bacteria use a type II secretion system (T2SS) to transport proteins out of the cell. These exported proteins or substrates play a major role in toxin delivery, maintaining biofilms, replication in the host and subversion of host immune responses to infection. We review the current structural and functional work on this system and argue that intrinsically disordered regions and protein dynamics are central for assembly, exo-protein recognition, and secretion competence of the T2SS.

Crystal structure of CobK reveals strand-swapping between Rossmann-fold domains and molecular basis of the reduced precorrin product trap.

CobK catalyzes the essential reduction of the precorrin ring in the cobalamin biosynthetic pathway. The crystal structure of CobK reveals that the enzyme, despite not having the signature sequence, comprises two Rossmann fold domains which bind coenzyme and substrate respectively. The two parallel β-sheets have swapped their last β-strands giving a novel sheet topology which is an interesting variation on the Rossmann-fold.

Structural insights into the loss of catalytic competence in pectate lyase activity at low pH.

Pectate lyase, a family 1 polysaccharide lyase, catalyses cleavage of the α-1,4 linkage of the polysaccharide homogalacturonan via an anti β-elimination reaction. In the Michaelis complex two calcium ions bind between the C6 carboxylate of the d-galacturonate residue and enzyme aspartates at the active centre (+1 subsite), they withdraw electrons acidifying the C5 proton facilitating its abstraction by the catalytic arginine. Here we show that activity is lost at low pH because protonation of aspartates results in the loss of the two catalytic calcium-ions causing a profound failure to correctly organise the Michaelis complex.

A practical approach for the validation and clinical implementation of a high-sensitivity cardiac troponin I assay across a North American city.

Objectives: Despite several publications on the analytical performance of high-sensitivity cardiac troponin (hs-cTn) assays, there has been little information on how laboratories should validate and implement these assays into clinical service. Our study provides a practical approach for the validation and implementation of a hs-cTn assay across a large North American City.

Design And Methods: Validation for the Abbott ARCHITECT hs-cTnI assay (across 5 analyzers) consisted of verification of limit of blank (LoB), precision (i.

FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR).

Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion.

Structural insights into higher order assembly and function of the bacterial microcompartment protein PduA.

Bacterial microcompartments are large proteinaceous assemblies that are found in the cytoplasm of some bacteria. These structures consist of proteins constituting a shell that houses a number of enzymes involved in specific metabolic processes. The 1,2-propanediol-utilizing microcompartment is assembled from seven different types of shell proteins, one of which is PduA.

The structure, function and properties of sirohaem decarboxylase--an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway.

Some bacteria and archaea synthesize haem by an alternative pathway, which involves the sequestration of sirohaem as a metabolic intermediate rather than as a prosthetic group. Along this pathway the two acetic acid side-chains attached to C12 and C18 are decarboxylated by sirohaem decarboxylase, a heterodimeric enzyme composed of AhbA and AhbB, to give didecarboxysirohaem. Further modifications catalysed by two related radical SAM enzymes, AhbC and AhbD, transform didecarboxysirohaem into Fe-coproporphyrin III and haem respectively.

Randomized trial of 'intelligent' autotitrating ventilation versus standard pressure support non-invasive ventilation: impact on adherence and physiological outcomes.

Background And Objective: Effective non-invasive ventilation (NIV) therapy is dependent on optimal ventilator settings to maximize clinical benefit and patient tolerance. Intelligent volume-assured pressure support (iVAPS) is a hybrid mode of servoventilation, providing constant automatic adjustment of pressure support (PS) to achieve a target ventilation determined by the patient's requirements. In a randomized crossover trial, we tested the hypothesis that iVAPS, with automated selection of ventilator settings, was non-inferior to standard PS ventilation, with settings determined by an experienced health-care professional, for controlling nocturnal hypoventilation in patients naive to NIV.

The effect of the adaptor protein Isd11 on the quaternary structure of the eukaryotic cysteine desulphurase Nfs1.

Small inorganic assemblies of alternating ferrous/ferric iron and sulphide ions, so-called iron-sulphur (Fe-S) clusters, are possibly nature's most ancient prosthetic groups. One of the early actors in Fe-S cluster biosynthesis is a protein complex composed of a cysteine desulphurase, Nfs1, and its functional binding partner, Isd11. Although the essential function of Nfs1·Isd11 in the liberation of elemental sulphur from free cysteine is well established, little is known about its structure.

Anatomy of secretin binding to the Dickeya dadantii type II secretion system pilotin.

The secretins are a family of large multimeric channels in the outer membrane of Gram-negative bacteria that are involved in protein export. In Dickeya dadantii and many other pathogenic bacteria, the lipoprotein pilotin targets the secretin subunits to the outer membrane, allowing a functional type II secretion system to be assembled. Here, the crystal structure of the C-terminal peptide of the secretin subunit bound to its cognate pilotin is reported.

Substrate channels revealed in the trimeric Lactobacillus reuteri bacterial microcompartment shell protein PduB.

Lactobacillus reuteri metabolizes two similar three-carbon molecules, 1,2-propanediol and glycerol, within closed polyhedral subcellular bacterial organelles called bacterial microcompartments (metabolosomes). The outer shell of the propanediol-utilization (Pdu) metabolosome is composed of hundreds of mainly hexagonal protein complexes made from six types of protein subunits that share similar domain structures. The structure of the bacterial microcompartment protein PduB has a tandem structural repeat within the subunit and assembles into a trimer with pseudo-hexagonal symmetry.

An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.

The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B(12)) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite.

Assessment of a four hour delay for urine samples stored without preservatives at room temperature for urinalysis.

Objectives: To determine whether urine storage at room temperature for up to 2h versus 4h changes urinalysis results.

Design And Methods: We compared the rejection rate at eight different hospital laboratories and concordance of urinalysis results (n=83; two laboratories) between urines analyzed within 2h and 4h after collection.

Results: The rejection rate at the two hour cutoff was significantly higher as compared to the four hour cutoff.

Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the type 2 secretion system.

The type II secretion system (T2SS) secretes enzymes and toxins across the outer membrane of Gram-negative bacteria. The precise assembly of T2SS, which consists of at least 12 core-components called Gsp, remains unclear. The outer membrane secretin, GspD, forms the channels, through which folded proteins are secreted, and interacts with the inner membrane component, GspC.

Structural and functional insights into the pilotin-secretin complex of the type II secretion system.

Gram-negative bacteria secrete virulence factors and assemble fibre structures on their cell surface using specialized secretion systems. Three of these, T2SS, T3SS and T4PS, are characterized by large outer membrane channels formed by proteins called secretins. Usually, a cognate lipoprotein pilot is essential for the assembly of the secretin in the outer membrane.

Solution structure of homology region (HR) domain of type II secretion system.

The type II secretion system of Gram-negative bacteria is important for bacterial pathogenesis and survival; it is composed of 12 mostly multimeric core proteins, which build a sophisticated secretion machine spanning both bacterial membranes. OutC is the core component of the inner membrane subcomplex thought to be involved in both recognition of substrate and interaction with the outer membrane secretin OutD. Here, we report the solution structure of the HR domain of OutC and explore its interaction with the secretin.