A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus

Oxidation of molecular hydrogen catalyzed by [NiFe] hydrogenases is a widespread mechanism of energy generation among prokaryotes. Biosynthesis of the H2-oxidizing enzymes is a complex process subject to positive control by H2 and negative control by organic energy sources. In this report we describe a novel signal transduction system regulating hydrogenase gene (hox) expression in the proteobacterium Alcaligenes eutrophus. This multicomponent system consists of the proteins HoxB, HoxC, HoxJ*, and HoxA. HoxB and HoxC share characteristic features of dimeric [NiFe] hydrogenases and form the putative H2 receptor that interacts directly or indirectly with the histidine protein kinase HoxJ*. A single amino acid substitution (HoxJ*G422S) in a conserved C-terminal glycine-rich motif of HoxJ* resulted in a loss of H2-dependent signal transduction and a concomitant block in autophosphorylating activity, suggesting that autokinase activity is essential for the response to H2. Whereas deletions in hoxB or hoxC abolished hydrogenase synthesis almost completely, the autokinase-deficient strain maintained high-level hox gene expression, indicating that the active sensor kinase exerts a negative effect on hox gene expression in the absence of H2. Substitutions of the conserved phosphoryl acceptor residue Asp55 in the response regulator HoxA (HoxAD55E and HoxAD55N) disrupted the H2 signal-transduction chain. Unlike other NtrC-like regulators, the altered HoxA proteins still allowed high-level transcriptional activation. The data presented here suggest a model in which the nonphosphorylated form of HoxA stimulates transcription in concert with a yet unknown global energy-responsive factor.

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 μs at pH 7 to 45 μs at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 μs and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

A common evolutionary origin for mitochondria and hydrogenosomes.

Trichomonads are among the earliest eukaryotes to diverge from the main line of eukaryotic descent. Keeping with their ancient nature, these facultative anaerobic protists lack two "hallmark" organelles found in most eukaryotes: mitochondria and peroxisomes. Trichomonads do, however, contain an unusual organelle involved in carbohydrate metabolism called the hydrogenosome. Like mitochondria, hydrogenosomes are double-membrane bounded organelles that produce ATP using pyruvate as the primary substrate. Hydrogenosomes are, however, markedly different from mitochondria as they lack DNA, cytochromes and the citric acid cycle. Instead, they contain enzymes typically found in anaerobic bacteria and are capable of producing molecular hydrogen. We show here that hydrogenosomes contain heat shock proteins, Hsp70, Hsp60, and Hsp10, with signature sequences that are conserved only in mitochondrial and alpha-Gram-negative purple bacterial Hsps. Biochemical analysis of hydrogenosomal Hsp60 shows that the mature protein isolated from the organelle lacks a short, N-terminal sequence, similar to that observed for most nuclear-encoded mitochondrial matrix proteins. Moreover, phylogenetic analyses of hydrogenosomal Hsp70, Hsp60, and Hsp10 show that these proteins branch within a monophyletic group composed exclusively of mitochondrial homologues. These data establish that mitochondria and hydrogenosomes have a common eubacterial ancestor and imply that the earliest-branching eukaryotes contained the endosymbiont that gave rise to mitochondria in higher eukaryotes.

A coupled proton-transfer and twisting-motion fluorescence probe for lipid bilayers

A new and sensitive molecular probe, 2-(2′-hydroxyphenyl)imidazo[1,2-a]pyridine (HPIP), for monitoring structural changes in lipid bilayers is presented. Migration of HPIP from water into vesicles involves rupture of hydrogen (H) bonds with water and formation of an internal H bond once the probe is inside the vesicle. These structural changes of the dye allow the occurrence of a photoinduced intramolecular proton-transfer reaction and a subsequent twisting/rotational process upon electronic excitation of the probe. The resulting large Stokes-shifted fluorescence band depends on the twisting motion of the zwitterionic phototautomer and is characterized in vesicles of dimyristoyl-phosphatidylcholine and in dipalmitoyl-phosphatidylcholine at the temperature range of interest and in the presence of cholesterol. Because the fluorescence of aqueous HPIP does not interfere in the emission of the probe within the vesicles, HPIP proton-transfer/twisting motion fluorescence directly allows us to monitor and quantify structural changes within bilayers. The static and dynamic fluorescence parameters are sensitive enough to such changes to suggest this photostable dye as a potential molecular probe of the physical properties of lipid bilayers.

A 12R-lipoxygenase in human skin: Mechanistic evidence, molecular cloning, and expression

A recognized feature of psoriasis and other proliferative dermatoses is accumulation in the skin of the unusual arachidonic acid metabolite, 12R-hydroxyeicosatetraenoic acid (12R-HETE). This hydroxy fatty acid is opposite in chirality to the product of the well-known 12S-lipoxygenase and heretofore in mammals is known only as a product of cytochrome P450s. Here we provide mechanistic evidence for a lipoxygenase route to 12R-HETE in human psoriatic tissue and describe a 12R-lipoxygenase that can account for the biosynthesis. Initially we demonstrated retention of the C-12 deuterium of octadeuterated arachidonic acid in its conversion to 12R-HETE in incubations of psoriatic scales, indicating the end product is not formed by isomerization from 12S-H(P)ETE via the 12-keto derivative. Secondly, analysis of product formed from [10R-3H] and [10S-3H]-labeled arachidonic acids revealed that 12R-HETE synthesis is associated with stereospecific removal of the pro-R hydrogen from the 10-carbon of arachidonate. This result is compatible with 12R-lipoxygenase-catalyzed formation of 12R-HETE and not with a P450-catalyzed route to 12R-HETE in psoriatic scales. We cloned a lipoxygenase from human keratinocytes; the cDNA and deduced amino acid sequences share ≤50% identity to other human lipoxygenases. This enzyme, when expressed in Hela cells, oxygenates arachidonic acid to 12-HPETE, >98% 12R in configuration. The 12R-lipoxygenase cDNA is detectable by PCR in psoriatic scales and as a 2.5-kilobase mRNA by Northern analysis of keratinocytes. Identification of this enzyme extends the known distribution of R-lipoxygenases to humans and presents an additional target for potential therapeutic interventions in psoriasis.

Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

Smooth muscle myosin mutants containing a single tryptophan reveal molecular interactions at the actin-binding interface

Elucidation of the molecular details of the cyclic actomyosin interaction requires the ability to examine structural changes at specific sites in the actin-binding interface of myosin. To study these changes dynamically, we have expressed two mutants of a truncated fragment of chicken gizzard smooth muscle myosin, which includes the motor domain and essential light chain (MDE). These mutants were engineered to contain a single tryptophan at (Trp-546) or near (Trp-625) the putative actin-binding interface. Both 546- and 625-MDE exhibited actin-activated ATPase and actin-binding activities similar to wild-type MDE. Fluorescence emission spectra and acrylamide quenching of 546- and 625-MDE suggest that Trp-546 is nearly fully exposed to solvent and Trp-625 is less than 50% exposed in the presence and absence of ATP, in good agreement with the available crystal structure data. The spectrum of 625-MDE bound to actin was quite similar to the unbound spectrum indicating that, although Trp-625 is located near the 50/20-kDa loop and the 50-kDa cleft of myosin, its conformation does not change upon actin binding. However, a 10-nm blue shift in the peak emission wavelength of 546-MDE observed in the presence of actin indicates that Trp-546, located in the A-site of the lower 50-kDa subdomain of myosin, exists in a more buried environment and may directly interact with actin in the rigor acto-S1 complex. This change in the spectrum of Trp-546 constitutes direct evidence for a specific molecular interaction between residues in the A-site of myosin and actin.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

NMR scalar couplings across Watson–Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy

This paper describes the NMR observation of 15N—15N and 1H—15N scalar couplings across the hydrogen bonds in Watson–Crick base pairs in a DNA duplex, hJNN and hJHN. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [15N,1H]-transverse relaxation-optimized spectroscopy (TROSY) with a 15N-labeled 14-mer DNA duplex was used to measure hJNN, which is in the range 6–7 Hz, and the two-dimensional hJNN-correlation-[15N,1H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related 15N spins and to observe, for the first time, hJHN scalar couplings, with values in the range 2–3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids.

Theoretical investigation of the [1,2]-sigmatropic hydrogen migration in the mechanism of oxidation of 2-aminobenzoyl-CoA by 2-aminobenzoyl-CoA monooxygenase/reductase

The flavin hydroperoxide at the active site of the mixed-function oxidase 2-aminobenzoyl-CoA monooxygenase/reductase (Azoarcus evansii) transfers an oxygen to the 5-position of the 2-aminobenzoyl-CoA substrate to provide the alkoxide intermediate II−. Hydrogen migration from C5 to C6 follows this monooxygenation. The nature of the monooxygenation intermediate and plausible competing reactions leading to hydrogen migration have been considered. Ab initio molecular orbital theory has been used to calculate structures and electron distributions in intermediate and transition state structures. Electrostatic potential surface calculations establish that the transition state and product, associated with the C5 to C6 hydrogen transfer, are stabilized by electron distribution to the benzoyl-CoA thioester carbonyl oxygen. This is not so for the transition state and product associated with hydrogen transfer from C5 to C4. The activation energy for the 5,6-shift is 2.5 kcal/mol lower than that for the 5,4-shift. In addition, the product of the hydrogen 5,6-shift is more stable than is the product of the hydrogen 5,4-shift, by ≈6 kcal/mol. These results explain why only the shift of hydrogen from C5 to C6 is observed experimentally. Oxygen transfer and hydrogen migration almost coincide in the gas phase (activation energy of ≈0.6 kcal/mol, equivalent to a single bond vibration). Enzymatic formation of alkoxide II− requires its stabilization; thus, the rate constant for its breakdown would be slower than in the gas phase.

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: Molecular basis for serotype specificity

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab–Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations

NMR analysis and molecular dynamics simulations of d(GGTAATTACC)2 and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar–phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving ‘lock and key’ recognition.

Symmetry in chemistry from the hydrogen atom to proteins

The last 2 decades have seen discoveries in highly excited states of atoms and molecules of phenomena that are qualitatively different from the “planetary” model of the atom, and the near-rigid model of molecules, characteristic of these systems in their low-energy states. A unified view is emerging in terms of approximate dynamical symmetry principles. Highly excited states of two-electron atoms display “molecular” behavior of a nonrigid linear structure undergoing collective rotation and vibration. Highly excited states of molecules described in the “standard molecular model” display normal mode couplings, which induce bifurcations on the route to molecular chaos. New approaches such as rigid–nonrigid correlation, vibrons, and quantum groups suggest a unified view of collective electronic motion in atoms and nuclear motion in molecules.

Glass transition in DNA from molecular dynamics simulations.

Molecular dynamics simulations of the oligonucleotide duplex d(CGCGCG)2 in aqueous solution are used to investigate the glass transition phenomenon. The simulations were performed at temperatures in the 20 K to 340 K range. The mean square atomic fluctuations showed that the behavior of the oligonucleotide duplex was harmonic at low temperatures. A glass transition temperature at 223 K to 234 K was inferred for the oligonucleotide duplex, which is in agreement with experimental observations. The largest number of hydrogen bounds between the polar atoms of the oligonucleotide duplex and the water molecules was obtained at the glass transition temperature. With increasing temperature we observed a decrease in the average lifetime of the hydrogen bonds to water molecules.

Internal molecular motions of bacteriorhodopsin: hydration-induced flexibility studied by quasielastic incoherent neutron scattering using oriented purple membranes.

Quasielastic incoherent neutron scattering from hydrogen atoms, which are distributed nearly homogeneously in biological molecules, allows the investigation of diffusive motions occurring on the pico- to nanosecond time scale. A quasielastic incoherent neutron scattering study was performed on the integral membrane protein bacteriorhodopsin (BR), which is a light-driven proton pump in Halobacterium salinarium. BR is embedded in lipids, forming patches in the cell membrane of the organism, which are the so called purple membranes (PMs). Measurements were carried out at room temperature on oriented PM-stacks hydrated at two different levels (low hydration, h = 0.03 g of D2O per g of PM; high hydration, h = 0.28 g of D2O per g of PM) using time-of-flight spectrometers. From the measured spectra, different diffusive components were identified and analyzed with respect to the influence of hydration. This study supports the idea that a decrease in hydration results in an appreciable decrease in internal molecular flexibility of the protein structure. Because it is known from studies on the function of BR that the pump activity is reduced if the hydration level of the protein is insufficient, we conclude that the observed diffusive motions are essential for the function of this protein. A detailed analysis and classification of the different kinds of diffusive motions, predominantly occurring in PMs under physiological conditions, is presented.