On the Origin of Hemoglobin Cooperativity under Non-equilibrium Conditions.

Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve, which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the oxygen dissociation curve of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure.

Nanoscale imaging using differential expansion microscopy.

Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures.

Human skeletal muscle cell atlas: Unraveling cellular secrets utilizing 'muscle-on-a-chip', differential expansion microscopy, mass spectrometry, nanothermometry and machine learning.

The 'Human Cell Atlas' project has been launched to obtain a comprehensive understanding of all cell types, the fundamental living units that constitute the human body. This is a global partnership and effort involving experts from many disciplines, from computer science, engineering to medicine, and is supported by several private and public organizations, among them, the Chan Zuckerberg Foundation, the National Institutes of Health, and Google, that will greatly benefit humanity. Nearly 37 trillion cells of various shapes, sizes, and composition, are precisely organized to constitute the human body.

Nodal versus Total Axonal Strain and the Role of Cholesterol in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a health threat that affects every year millions of people involved in motor vehicle and sporting accidents, and thousands of soldiers in battlefields. Diffuse axonal injury (DAI) is one of the most frequent types of TBI leading to death. In DAI, the initial traumatic event is followed by a cascade of biochemical changes that take time to develop in full, so that symptoms may not become apparent until days or weeks after the original injury.

Multidimensional mutual information methods for the analysis of covariation in multiple sequence alignments.

Background: Several methods are available for the detection of covarying positions from a multiple sequence alignment (MSA). If the MSA contains a large number of sequences, information about the proximities between residues derived from covariation maps can be sufficient to predict a protein fold. However, in many cases the structure is already known, and information on the covarying positions can be valuable to understand the protein mechanism and dynamic properties.

Biapenem inactivation by B2 metallo β-lactamases: energy landscape of the hydrolysis reaction.

Background: A general mechanism has been proposed for metallo β-lactamases (MβLs), in which deprotonation of a water molecule near the Zn ion(s) results in the formation of a hydroxide ion that attacks the carbonyl oxygen of the β-lactam ring. However, because of the absence of X-ray structures that show the exact position of the antibiotic in the reactant state (RS) it has been difficult to obtain a definitive validation of this mechanism.

Methodology/principal Findings: We have employed a strategy to identify the RS, which does not rely on substrate docking and/or molecular dynamics.

Accurate simulation and detection of coevolution signals in multiple sequence alignments.

Background: While the conserved positions of a multiple sequence alignment (MSA) are clearly of interest, non-conserved positions can also be important because, for example, destabilizing effects at one position can be compensated by stabilizing effects at another position. Different methods have been developed to recognize the evolutionary relationship between amino acid sites, and to disentangle functional/structural dependencies from historical/phylogenetic ones.

Methodology/principal Findings: We have used two complementary approaches to test the efficacy of these methods.

Biapenem inactivation by B2 metallo β-lactamases: energy landscape of the post-hydrolysis reactions.

Background: The first line of defense by bacteria against β-lactam antibiotics is the expression of β-lactamases, which cleave the amide bond of the β-lactam ring. In the reaction of biapenem inactivation by B2 metallo β-lactamases (MβLs), after the β-lactam ring is opened, the carboxyl group generated by the hydrolytic process and the hydroxyethyl group (common to all carbapenems) rotate around the C5-C6 bond, assuming a new position that allows a proton transfer from the hydroxyethyl group to C2, and a nucleophilic attack on C3 by the oxygen atom of the same side-chain. This process leads to the formation of a bicyclic compound, as originally observed in the X-ray structure of the metallo β-lactamase CphA in complex with product.

Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation.

Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation.

The contribution of coevolving residues to the stability of KDO8P synthase.

Background: The evolutionary tree of 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes a key step in the biosynthesis of bacterial endotoxin, is evenly divided between metal and non-metal forms, both having similar structures, but diverging in various degrees in amino acid sequence. Mutagenesis, crystallographic and computational studies have established that only a few residues determine whether or not KDO8PS requires a metal for function. The remaining divergence in the amino acid sequence of KDO8PSs is apparently unrelated to the underlying catalytic mechanism.

Common basis for the mechanism of metallo and non-metallo KDO8P synthases.

The three-dimensional structures of metal and non-metal enzymes that catalyze the same reaction are often quite different, a clear indication of convergent evolution. However, there are interesting cases in which the same scaffold supports both a metal and a non-metal catalyzed reaction. One of these is 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes the synthesis of KDO8P and inorganic phosphate (P(i)) from phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and water.

Defining the pathogenesis of the human Atp12p W94R mutation using a Saccharomyces cerevisiae yeast model.

Studies in yeast have shown that a deficiency in Atp12p prevents assembly of the extrinsic domain (F(1)) of complex V and renders cells unable to make ATP through oxidative phosphorylation. De Meirleir et al. (De Meirleir, L.

The energy landscape of 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P, a key precursor in the biosynthesis of the endotoxin of Gram-negative bacteria. Earlier studies have established that the condensation occurs with a syn addition of water to the si side of C2(PEP) and of C3(PEP) to the re side of C1(A5P). Two stepwise mechanisms have been proposed for this reaction.

Chaperones of F1-ATPase.

Mitochondrial F(1)-ATPase contains a hexamer of alternating alpha and beta subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to beta and alpha. In the absence of Atp11p and Atp12p, the hexamer is not formed, and alpha and beta precipitate as large insoluble aggregates.

Electronic structure of the metal center in the Cd(2+), Zn(2+), and Cu(2+) substituted forms of KDO8P synthase: implications for catalysis.

Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) is active with a variety of different divalent metal ions bound in the active site. The Cd(2+), Zn(2+), and Cu(2+) substituted enzymes display similar values of k(cat) and similar dependence of K(m)(PEP) and K(m)(A5P) on both substrate and product concentrations. However, the flux-control coefficients for some of the catalytically relevant reaction steps are different in the presence of Zn(2+) or Cu(2+), suggesting that the type of metal bound in the active site affects the behavior of the enzyme in vivo.

Structural and mechanistic changes along an engineered path from metallo to nonmetallo 3-deoxy-D-manno-octulosonate 8-phosphate synthases.

There are two classes of KDO8P synthases characterized respectively by the presence or absence of a metal in the active site. The nonmetallo KDO8PS from Escherichia coli and the metallo KDO8PS from Aquifex aeolicus are the best characterized members of each class. All amino acid residues that make important contacts with the substrates are conserved in both enzymes with the exception of Pro-10, Cys-11, Ser-235, and Gln-237 of the A.

The catalytic and conformational cycle of Aquifex aeolicus KDO8P synthase: role of the L7 loop.

KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (P(i)). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired.

Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al.

The molecular chaperone, Atp12p, from Homo sapiens. In vitro studies with purified wild type and mutant (E240K) proteins.

Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled alpha subunits of F(1) and in so doing prevents the alpha subunit from associating with itself in non-productive complexes during assembly of the F(1) moiety of the mitochondrial ATP synthase. We have developed a method to prepare recombinant Atp12p after expression of its human cDNA in bacterial cells. The molecular chaperone activity of HuAtp12p was studied using citrate synthase as a model substrate.

Structure-based design of novel inhibitors of 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) is the phosphorylated precursor of KDO, an essential sugar of the lipopolysaccharide of Gram negative bacteria. KDO8P is produced by a specific synthase (KDO8PS) by condensing arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP), with release of inorganic phosphate. As KDO8PS is present in bacteria and plants, but not in mammalian cells, and mutations that inactivate KDO8PS also block cell replication, KDO8PS is a promising target for the design of new antimicrobials that act by blocking lipopolysaccharide biosynthesis.

Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues.

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate.

Function of His185 in Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or Fe(2+) in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd(2+).

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide.