Stabilization of flavobacterium meningosepticum glycerol kinase by introduction of a hydrogen bond

The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp [Protein Eng., 14, 663-667 (2001)]. Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme. If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329. We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn. As we expected, S414N showed comparable thermostability to that of S329D. On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed.     

Quantitative comparison of the hydrogen bond network of A-state and native ubiquitin by hydrogen bond scalar couplings

The backbone hydrogen bond (H-bond) network of the partially folded A-state of ubiquitin (60% methanol, 40% water, pH 2) has been characterized quantitatively by (h3)J(NC)(') H-bond scalar couplings between the (15)N nuclei of amino acid H-bond donors and the (13)C carbonyl nuclei of the acceptors. Results on (h3)J(NC)(') couplings and the amide proton ((1)H(N)) chemical shifts for the A-state are compared quantitatively to the native state. The (h3)J(NC)(') correlations of the A-state show intact, nativelike H-bonds of the first beta-hairpin beta1/beta2 and the alpha-helix, albeit at lower strength, whereas the H-bonds in the C-terminal part change from a pure beta-structure to an all alpha-helical H(N)(i)-->O(i-4) connectivity pattern. A residue-specific analysis reveals that the conformations within the conserved secondary structure segments are much more homogeneous in the A-state than in the native state. Thus, the strong asymmetry of (h3)J(NC)(') couplings and (1)H(N) chemical shifts between the interior and exterior sides of the native state alpha-helix vanishes in the A-state. This indicates that the bend of this helix around the native state hydrophobic core is released in the homogeneous solvent environment of the A-state. Similarly, an irregularity in the behavior of H-bond I3-->L15 in hairpin beta1/beta2, which results from strong contacts to strand beta5 in the native state, is absent in the A-state. These findings rationalize the behavior of the (1)H(N) chemical shifts in both states and indicate that the A-state is in many aspects similar to the onset of thermal denaturation of the native state.     

A helix initiation motif, XLLRA, is stabilized by hydrogen bond, hydrophobic and van der Waals interactions

Five partially overlapping synthetic peptides containing the N-terminal portion of the leucine zipper (LZ)-like domain of human immunodeficiency virus envelope glycoprotein gp41 were used to deduce the helix initiation site. Circular dichroism (CD) data suggested a strong helix-inducing motif, LLRA. The coupling constant and nuclear Overhauser effect (NOE) results obtained from nuclear magnetic resonance experiments in 20% trifluoroethanol aqueous solution at 280 K for the four decapeptides under study suggested that the motif XLLRA, where X is a group or an amino acid residue capable of forming hydrogen bond to arginine, constitutes a helix nucleation core. A similar conclusion was reached for a pentadecapeptide in water, suggesting that the result was not dependent on both chain length and the helix promoting medium. Detailed analysis of NOE and CD data from the four decapeptides indicated that the acetyl group and asparagine had a strong tendency to be helix N-capping, in confirmation of previous studies. Molecular modeling using restraints derived from NOE data showed that van der Waals, hydrophobic interactions and hydrogen bonds contribute synergetically to the stability of the core structure. The concept of nucleation core consisting of a few amino acids may be generally applied in proton design and folding studies.     

A hyperthermophilic plant-type [2Fe-2S] ferredoxin from Aquifex aeolicus is stabilized by a disulfide bond

A [2Fe-2S] ferredoxin (Fd1) from the hyperthermophilic bacterium Aquifex aeolicus has been obtained by heterologous expression of the encoding gene in Escherichia coli. Sequence comparisons show that this protein belongs to the extended family of plant- and mammalian-type [2Fe-2S] ferredoxins but also indicate that it is not closely similar to either the plant-type or mammalian-type subfamilies. Instead, it appears to bear some similarity to novel members of this family, in particular the Isc-type ferredoxins involved in the assembly of iron-sulfur clusters in vivo. The two redox levels of the [2Fe-2S](2+/+) metal site of A. aeolicus ferredoxin have been studied by UV-visible, resonance Raman, EPR, variable temperature magnetic circular dichroism, and M?ssbauer spectroscopies. A full-spin Hamiltonian analysis is given for the M?ssbauer spectra. In aggregate, the spectroscopic data reveal differences with both the plant-type and mammalian-type ferredoxins, in keeping with the sequence comparisons. The midpoint potential of the [2Fe-2S](2+/+) couple, at -375 mV versus the normal hydrogen electrode, is more negative than those of mammalian-type ferredoxins and at the upper end of the range covered by plant-type ferredoxins. A. aeolicus ferredoxin contains two cysteines in addition to the four that are committed as ligands of the [2Fe-2S] cluster. These two residues have been shown by chemical modification and site-directed mutagenesis to form a disulfide bridge in the native protein. While that cystine unit plays a significant role in the exceptional thermostability of A. aeolicus ferredoxin (T(m) = 121 degrees C at pH 7 versus T(m) = 113 degrees C in a molecular variant where the disulfide bridge has been removed), it does not bear on the properties of the [2Fe-2S](2+/+) chromophore. This observation is consistent with the large distance (ca. 20 A) that is predicted to separate the iron-sulfur chromophore from the disulfide bridge.     

Nickel(II) complexes stabilized by bis[N-(6-pivalamido-2-pyridylmethyl)]benzylamine: Synthesis and characterization of complexes stabilized by a hydrogen bonding network

Intramolecular hydrogen bonds in metalloproteins are key in directing reactivity yet these effects have been difficult to achieved in synthetic systems. We have been developing a synthetic system that uses hydrogen-bonding interactions to modulate the secondary coordination around a transition metal ion. This was accomplished with the ligand bis[N-(6-pivalamido-2-pyridylmethyl)]benzylamine (H₂pmb), which contains two carboxyamido units appended from pyridine rings. Several nickel complexes were prepared and structurally characterized. In particular, we found that the appended carboxyamido groups either provide intramolecular H-bond donors or can be converted to bind directly to a metal center. We established that the complex Ni^IIH₂pmb(Cl)₂ can be sequentially deprotonated with potassium tert-butoxide, causing coordination of the carboxyamido oxygen atoms and concomitant loss of the chloro ligands. The chloro ligands were also removed with silver(I) salts in the presence of acetate ions and the complex Ni^IIH₂pmb(κ²-OAc)(κ¹-OAc) was isolated, in which an intramolecular H-bonding network occurs between the H₂pmb ligand and the coordinate acetato ligands.     

Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic

Fertility is a highly complex and regulated phenomenon essential for the survival of any species. To identify Drosophila fertility-specific neural networks, we used a GAL4/UAS enhancer trap genetic screen that selectively inactivates groups of neurons. We identified a GAL4 line (bwktqs) that has a female sterile phenotype only when it expresses the tetanus toxin light chain (TeTxLC). These flies lack oviduct contraction, lay almost no eggs, sperm accumulate in the oviducts, and fewer than normal are seen in the storage organs. In insects, two neuroactive substances are important for oviduct contraction: octopamine (OA), a monoamine that inhibits oviduct contraction, and glutamate (Glu), a neurotransmitter that induces contraction. It is known that octopaminergic neurons of the thoracic abdominal ganglion (TAG) modulate oviduct contraction, however, the glutamatergic neurons that innervate the oviduct have not been identified yet and the interaction between these two neuroactive substances is not well understood. Immunostaining experiments revealed that the bwktqs line trapped an octopaminergic neural network that innervates the genital tract. We show that wt like oviduct contraction in TeTxLC-inactivated flies can only be rescued by simultaneous application of Glu and OA suggesting that the abdominal bwktqs neurons are both octopaminergic and glutamatergic, the use of an agonist and an antagonist for Glu receptors as well as their direct visualization confirmed its participation in this phenomenon. Our work provides the first evidence that adult abdominal type II visceral innervations co-express Glu and OA and allows us to re-evaluate the previous model of neuronal network controlling insect oviduct contraction.     

The novel 1D chain and 3D network of mercury carborane-diphenylphosphine complexes constructed by covalent bond or hydrogen bond interactions

The closo- and nido-carborane-diphenylphosphine complexes [Hg₂{1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀}₂(μ-Cl₂)₂(μ-HgCl₂)₃]·2CH₂Cl₂ (1) and [HgCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] (2) have been synthesized and characterized by elemental analysis, FT-IR and X-ray structure determination. The X-ray structure analysis for these two complexes showed that the carborane cage ligand was coordinated bidentately to the Hg(II) center through its two phosphorus atoms. The coordination geometry of the mercury atom complexed by P₂Cl₂ unit in complex 1 or P₃Cl unit in complex 2 was a distorted tetrahedron, while the mercury atom in complex 2 coordinated to six Cl atoms was a slightly distorted octahedron. X-ray analysis reveals that the complex 1 forms a 1D chain coordination polymer via bridged Hg-Cl bonds. For complex 2, it displays a 3D network constructed by the C-H···Cl hydrogen bonds and C-H···H-B dihydrogen bonds.     

SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the gamma-tubulin-mediated addition of centriolar microtubules

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating gamma-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires gamma-tubulin and microtubule assembly. These results indicate that gamma-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.     

Sequence and temperature effect on hydrogen bond disruption in DNA determined by a statistical analysis

We use the modified self-consistent phonon approximation theory to calculate temperature dependent interbase hydrogen bond disruption profiles for a number of six base pair repeating sequence infinite B-DNA polymers with various guanine-cytosine/adenine-thymine ratios. For comparison we also include results we have obtained in our earlier work on several B-DNA homopolymers, copolymers and a four-base-pair repeating sequence polymer. Our theory gives a statistical estimate of thermal fluctuational disruption probability of individual hydrogen bonds in individual base pairs in DNA as a function of temperature. The calculated probabilities show no sequence dependence at premelting temperatures, in agreement with proton exchange measurements. These probabilities however become very sensitive to base sequence at temperatures close to the observed melting temperatures. Multi-phasic critical transitions are found in which a portion of base pairs are disrupted at temperatures below the final disruption temperature. These transitions include localized as well as non-localized base pair opening. The localized transitions involve disruption of a few base-pairs at every other location without large scale base unstacking, and they may not appear in the observed UV curves with current resolution. On the other hand the overall disruption behavior is consistent with observations. The midpoint transition temperatures are close to the observed melting temperatures and these temperatures show the observed linear dependence on guanine-cytosine content. Our calculations indicate that our theory can be used effectively to calculate H-bond disruption behavior of different DNA sequences. Received: 20 February 1996 / Accepted: 2 May 1996     

Counterfactual choice and learning in a neural network centered on human lateral frontopolar cortex

Decision making and learning in a real-world context require organisms to track not only the choices they make and the outcomes that follow but also other untaken, or counterfactual, choices and their outcomes. Although the neural system responsible for tracking the value of choices actually taken is increasingly well understood, whether a neural system tracks counterfactual information is currently unclear. Using a three-alternative decision-making task, a Bayesian reinforcement-learning algorithm, and fMRI, we investigated the coding of counterfactual choices and prediction errors in the human brain. Rather than representing evidence favoring multiple counterfactual choices, lateral frontal polar cortex (lFPC), dorsomedial frontal cortex (DMFC), and posteromedial cortex (PMC) encode the reward-based evidence favoring the best counterfactual option at future decisions. In addition to encoding counterfactual reward expectations, the network carries a signal for learning about counterfactual options when feedback is available-a counterfactual prediction error. Unlike other brain regions that have been associated with the processing of counterfactual outcomes, counterfactual prediction errors within the identified network cannot be related to regret theory. Furthermore, individual variation in counterfactual choice-related activity and prediction error-related activity, respectively, predicts variation in the propensity to switch to profitable choices in the future and the ability to learn from hypothetical feedback. Taken together, these data provide both neural and behavioral evidence to support the existence of a previously unidentified neural system responsible for tracking both counterfactual choice options and their outcomes.     

Computational design of a new hydrogen bond network and at least a 300-fold specificity switch at a protein-protein interface

The redesign of protein-protein interactions is a stringent test of our understanding of molecular recognition and specificity. Previously we engineered a modest specificity switch into the colicin E7 DNase-Im7 immunity protein complex by identifying mutations that are disruptive in the native complex, but can be compensated by mutations on the interacting partner. Here we extend the approach by systematically sampling alternate rigid body orientations to optimize the interactions in a binding mode specific manner. Using this protocol we designed a de novo hydrogen bond network at the DNase-immunity protein interface and confirmed the design with X-ray crystallographic analysis. Subsequent design of the second shell of interactions guided by insights from the crystal structure on tightly bound water molecules, conformational strain, and packing defects yielded new binding partners that exhibited specificities of at least 300-fold between the cognate and the non-cognate complexes. This multi-step approach should be applicable to the design of polar protein-protein interactions and contribute to the re-engineering of regulatory networks mediated by protein-protein interactions.     

Glycoprotein hyposialylation gives rise to a nephrotic-like syndrome that is prevented by sialic acid administration in GNE V572L point-mutant mice

Mutations in the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase, result in distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusion body myopathy (HIBM) in humans. Sialic acid is an acidic monosaccharide that modifies non-reducing terminal carbohydrate chains on glycoproteins and glycolipids, and it plays an important role in cellular adhesions and interactions. In this study, we generated mice with a V572L point mutation in the GNE kinase domain. Unexpectedly, these mutant mice had no apparent myopathies or motor dysfunctions. However, they had a short lifespan and exhibited renal impairment with massive albuminuria. Histological analysis showed enlarged glomeruli with mesangial matrix deposition, leading to glomerulosclerosis and abnormal podocyte foot process morphologies in the kidneys. Glycan analysis using several lectins revealed glomerular epithelial cell hyposialylation, particularly the hyposialylation of podocalyxin, which is one of important molecules for the glomerular filtration barrier. Administering Neu5Ac to the mutant mice from embryonic stages significantly suppressed the albuminuria and renal pathology, and partially recovered the glomerular glycoprotein sialylation. These findings suggest that the nephrotic-like syndrome observed in these mutant mice resulted from impaired glomerular filtration due to the hyposialylation of podocyte glycoproteins, including podocalyxin. Furthermore, it was possible to prevent the nephrotic-like disease in these mice by beginning Neu5Ac treatment during gestation.     

Sleep apnea is induced by a high-fat diet and reversed and prevented by metformin in non-obese rats

Objective: We assessed the relationship between a high‐fat (HF) diet and central apnea during rapid eye movement and non‐rapid eye movement sleep stages by recording ventilatory parameters in 28 non‐obese rats in which insulin resistance had been induced by an HF diet. We also studied whether metformin (an anti‐hyperglycemic drug frequently used to treat insulin resistance) could reverse sleep apnea or prevent its occurrence in this experimental paradigm. Research Methods and Procedures: Rats were fed with a standard diet (10 rats), an HF diet (8 rats), or an HF diet concomitantly with metformin treatment (10 rats). Each animal was instrumented for electroencephalographic and electromyographic recording. After 3 weeks, ventilatory parameters during sleep were recorded with a body plethysmograph. All rats were treated with metformin for 1 week, after which time the ventilatory measurements were measured again. Results: Our results showed that the three groups of animals did not differ in terms of body growth over the entire experimental period. The HF diet did not modify sleep structure or minute ventilation in the different sleep stages. A great increase (+266 ± 48%) in central apnea frequency was observed in insulin‐resistant rats. This was explained by an increase in both post‐sigh (+195 ± 35%) and spontaneous apnea (+437 ± 65%) in the different sleep stages. These increases were suppressed by metformin treatment. Discussion: Insulin resistance induced by the HF diet could be the promoter of sleep apnea in non‐obese rats. Metformin is an efficient curative and preventive treatment for sleep apnea, suggesting that insulin resistance modifies the ventilatory drive independently of obesity.     

Evidence that phosphate release is the rate-limiting step on the overall ATPase of psoas myofibrils prevented from shortening by chemical cross-linking

It has been suggested that the mechanical condition determines the rate-limiting step of the ATPase of the myosin heads in fibers: when fibers are isometrically contracting, the ADP release kinetics are rate-limiting, but as the strain is reduced and the fibers are allowed to shorten, the ADP release kinetics accelerate and P(i) release becomes rate-limiting. We have put this idea to the test with myofibrils as a model because with these both mechanical and chemical kinetic measurements are possible. With relaxed or rapidly shortening myofibrils, P(i) release is rate-limiting and (A)M.ADP.P(i) states accumulate in the steady state [Lionne, C., et al. (1995) FEBS Lett. 364, 59]. We have now studied the kinetics of P(i) release with chemically cross-linked myofibrils that, when adequately cross-linked, appear to be a good model for isometric contraction. By using a method that is specific for free P(i) and rapid quench flow that measures the amount of (A)M.ADP.P(i) states and free P(i), we show that (A)M.ADP.P(i) states predominate which suggests that the overall ATPase is limited by P(i) release kinetics. Therefore, under our experimental conditions with myofibrils prevented from shortening, the concentration of (A)M.ADP states is low, as with rapidly shortening and relaxed myofibrils. This result is difficult to reconcile with the sensitivity of force development in fibers and myofibrils to P(i) which implies interaction of P(i) with an (A)M.ADP state. We discuss two models for accommodating the mechanical and chemical kinetics with reference to the duty cycle in skeletal muscle.     

Improvement of tumor targeting and antitumor activity by a disulphide bond stabilized diabody expressed in Escherichia coli

We have generated an anti-Pgp/anti-CD3 diabody which can effectively inhibit the growth of multidrug-resistant human tumors. However, the two chains of the diabody are associated non-covalently and are therefore capable of dissociation. Cysteine residues were introduced into the V-domains to promote disulphide cross-linking of the dimer as secreted by Escherichia coli. Compared with the parent diabody, the ds-Diabody obtained was more stable in human serum at 37°C, without loss of affinity or cytotoxicity activity in vitro. Furthermore, the ds-Diabody showed improved tumor localization and a twofold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts. Our data demonstrate that ds-Diabody may be more useful in therapeutic applications than the parent diabody.     

Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic "honeycomb" surface coat

Three-dimensional "deep-etch" electron microscopy (DEEM) resolves a longstanding controversy concerning poxvirus morphogenesis. By avoiding fixative-induced membrane distortions that confounded earlier studies, DEEM shows that the primary poxvirus envelope is a single membrane bilayer coated on its external surface by a continuous honeycomb lattice. Freeze fracture of quick-frozen poxvirus-infected cells further shows that there is only one fracture plane through this primary envelope, confirming that it consists of a single lipid bilayer. DEEM also illustrates that the honeycomb coating on this envelope is completely replaced by a different paracrystalline coat as the poxvirus matures. Correlative thin section images of infected cells freeze substituted after quick-freezing, plus DEEM imaging of Tokuyasu-type cryo-thin sections of infected cells (a new application introduced here) all indicate that the honeycomb network on immature poxvirus virions is sufficiently continuous and organized, and tightly associated with the envelope throughout development, to explain how its single lipid bilayer could remain stable in the cytoplasm even before it closes into a complete sphere.     

Theoretical calculations on an acetic acid, 5-methylimidazole, methanol hydrogen bond network: a model charge relay system

The CNDO/2 MO SCF method is used to calculate canonical form stabilities, proton potential energy curves, charge redistributions, and hydrogen bond strengths in a model charge relay system. The model system consists of methanol, 5-methylimidazole and acetic acid. The carboxylate group is observed to (i) increase the negative charge on the methanol oxygen, (ii) increase the methanol-imidazole hydrogen bond strength and (iii) reduce the proton transfer energies. Consideration of an extended model system indicates that solvation of the model system further reduces the proton transfer energies. These results are consistant with the proposed role of the buried aspartate group in activating the reactive serine oxygen in the serine proteinase enzymes.     

Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy.

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (delta delta G(folding) = 0.46 +/- 0.15 kcal mol-1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in lambda repressor.

In the N-terminal domain of lambda repressor, the Asp 14 side chain forms an intrahelical, hydrogen bond/salt bridge with the Arg 17 side chain and a tertiary hydrogen bond with the Ser 77 side chain. By measuring the stabilities to urea denaturation of the wild-type N-terminal domain and variants containing single, double, and triple alanine substitutions at positions 14, 17, and 77, the side-chain interaction energies, the coupling energy between interactions, and the intrinsic effects of each wild-type side chain on protein stability have been estimated. These studies indicate that the Asp 14-Arg 17 and Asp 14-Ser 77 interactions are stabilizing by roughly 0.8 and 1.5 kcal/mol, respectively, but that Asp 14, by itself, is destabilizing by roughly 0.9 kcal/mol. We also show that a peptide model of alpha-helix 1, which contains Asp 14 and Arg 17, forms a reasonably stable, monomeric helix in solution and responds to alanine mutations at positions 14 and 17 in the fashion expected from the intact protein studies. These studies suggest that it is possible to view the stability effects of mutations in intact proteins in a hierarchical fashion, with the stability of units of secondary structure being distinguishable from the stability of tertiary structure.