Helicobacter pylori senses bleach (HOCl) as a chemoattractant using a cytosolic chemoreceptor.

The gastric pathogen Helicobacter pylori requires a noncanonical cytosolic chemoreceptor transducer-like protein D (TlpD) for efficient colonization of the mammalian stomach. Here, we reconstituted a complete chemotransduction signaling complex in vitro with TlpD and the chemotaxis (Che) proteins CheW and CheA, enabling quantitative assays for potential chemotaxis ligands. We found that TlpD is selectively sensitive at micromolar concentrations to bleach (hypochlorous acid, HOCl), a potent antimicrobial produced by neutrophil myeloperoxidase during inflammation. HOCl acts as a chemoattractant by reversibly oxidizing a conserved cysteine within a 3His/1Cys Zn-binding motif in TlpD that inactivates the chemotransduction signaling complex. We found that H. pylori is resistant to killing by millimolar concentrations of HOCl and responds to HOCl in the micromolar range by increasing its smooth-swimming behavior, leading to chemoattraction to HOCl sources. We show related protein domains from Salmonella enterica and Escherichia coli possess similar reactivity toward HOCl. We propose that this family of proteins enables host-associated bacteria to sense sites of tissue inflammation, a strategy that H. pylori uses to aid in colonizing and persisting in inflamed gastric tissue.

Tolerability of graded levels of tall oil fatty acids as a nutritional additive for broiler chickens: a 45-day target animal safety study.

1. The following study was conducted to evaluate the tolerability of tall oil fatty acid (TOFA) to broiler chickens, at three graded levels as a nutritional additive in complete feed.2. 256 one-day-old female and male Cobb 500 broiler chickens were assigned to four dietary treatment groups with TOFA at 0 (control), 1.0, 3.0, or 5.0 g/kg within a complete feed for 45 d.3. Birds were weighed individually on days 0, 16, 31 and 45, and the feed intake, bird weight gain, and feed conversion ratio were calculated for the respective starter, grower and finisher phases and over the whole study. On day 45, blood samples were drawn from each bird for haematology and blood chemistry measurements. Two birds per pen were subjected to gross pathological examination and sampling of several tissues for histopathology, including weighing the liver.4. The dietary treatments did not affect zootechnical performance variables or mortality over the whole study period. Bird performance was typical for the breed.5. Haematology, clinical chemistry and histopathology did not reveal any changes associated with dietary TOFA dosing. However, the 5.0 g/kg dose level increased the relative weight of the liver, as a percentage of final body weight, compared to the control group, but there was lack of corresponding histopathology findings.6. In conclusion, the study indicated that oral administration of TOFA for 45 d in feed was well tolerated by the birds at dietary levels of up to 5.0 g/kg.

Thermal compensation of molded silicone optics.

Optical grade silicone has various properties that make it attractive for solar concentrators, such as excellent transmission across the solar spectrum and flexible moldability for freeform profiles. In this study, a glass-silicone lens structure is proposed to reduce the optothermal effect on the silicone lens. Experimental measurements and simulation modeling results demonstrate that the focal length sensitivity of the glass-silicone lens with respect to temperature can be reduced by a factor of 10 when compared to a silicone lens alone. This model has been extended to the simulation of a proposed two-stage silicone solar concentrator, consisting of an array of acylindrical lenslets and rows of waveguides that focus light onto microphotovoltaic cells. The optical efficiency of the solar concentration system showed a change of less than 10% compared to the efficiency at room temperature for temperature changes from -10∘C to 70°C.

Photoinduced proton transfer inside an engineered green fluorescent protein: a stepwise-concerted-hybrid reaction.

Photoactivated proton transfer (PT) wire is responsible for the glow of green fluorescent protein (GFP), which is crucial for bioimaging and biomedicine. In this work, a new GFP-S65T/S205V double mutant is developed from wild-type GFP in which the PT wire is significantly modified. We implement femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS) to delineate the PT process in action. The excited state proton transfer proceeds on the ∼110 ps timescale, which infers that the distance of one key link (water to T203) in the PT wire of GFP-S205V is shortened by the extra S65T mutation. The rise of an imidazolinone ring deformation mode at ∼871 cm-1 in FSRS further suggests that this PT reaction is in a concerted manner. A ∼4 ps component prior to large-scale proton dissociation through the PT wire is also retrieved, indicative of some small-scale proton motions and heavy-atom rearrangement in the vicinity of the chromophore. Our work provides deep insights into the novel hybrid PT mechanism in engineered GFP and demonstrates the power of tunable FSRS methodology in tracking ultrafast photoreactions with the desirable structural specificity in physiological environments.

Excited State Structural Evolution of a GFP Single-Site Mutant Tracked by Tunable Femtosecond-Stimulated Raman Spectroscopy.

Tracking vibrational motions during a photochemical or photophysical process has gained momentum, due to its sensitivity to the progression of reaction and change of environment. In this work, we implemented an advanced ultrafast vibrational technique, femtosecond-stimulated Raman spectroscopy (FSRS), to monitor the excited state structural evolution of an engineered green fluorescent protein (GFP) single-site mutant S205V. This mutation alters the original excited state proton transfer (ESPT) chain. By strategically tuning the Raman pump to different wavelengths (i.e., 801, 539, and 504 nm) to achieve pre-resonance with transient excited state electronic bands, the characteristic Raman modes of the excited protonated (A*) chromophore species and intermediate deprotonated (I*) species can be selectively monitored. The inhomogeneous distribution/population of A* species go through ESPT with a similar ~300 ps time constant, confirming that bridging a water molecule to protein residue T203 in the ESPT chain is the rate-limiting step. Some A* species undergo vibrational cooling through high-frequency motions on the ~190 ps time scale. At early times, a portion of the largely protonated A* species could also undergo vibrational cooling or return to the ground state with a ~80 ps time constant. On the photoproduct side, a ~1330 cm-1 delocalized motion is observed, with dispersive line shapes in both the Stokes and anti-Stokes FSRS with a pre-resonance Raman pump, which indicates strong vibronic coupling, as the mode could facilitate the I* species to reach a relatively stable state (e.g., the main fluorescent state) after conversion from A*. Our findings disentangle the contributions of various vibrational motions active during the ESPT reaction, and offer new structural dynamics insights into the fluorescence mechanisms of engineered GFPs and other analogous autofluorescent proteins.

The N-B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite.

Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO(-))-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, (11)B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp(3)-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.

Mutagenic induction of an ultra-fast water-chain proton wire.

Replacement of the hydroxyl group of a hydrophilic sidechain by an H atom in the proton wire of GFP induces formation of a water-chain proton wire. Surprisingly, this "non-native" water chain functions as a proton wire with response times within 10 ps of the wild type protein. This remarkable rate retention is understood as a natural consequence of the well-known Grotthuss mechanism of proton transfer in water.

Insight into the structure and the mechanism of the slow proton transfer in the GFP double mutant T203V/S205A.

Mutations near the fluorescing chromophore of the green fluorescent protein (GFP) have direct effects on the absorption and emission spectra. Some mutants have significant band shifts and most of the mutants exhibit a loss of fluorescence intensity. In this study we continue our investigation of the factors controlling the excited state proton transfer (PT) process of GFP, in particular to study the effects of modifications to the key side chain Ser205 in wt-GFP, proposed to participate in the proton wire. To this aim we combined mutagenesis, X-ray crystallography, steady-state spectroscopy, time-resolved emission spectroscopy and all-atom explicit molecular dynamics (MD) simulations to study the double mutant T203V/S205A. Our results show that while in the previously described GFP double mutant T203V/S205V the PT process does not occur, in the T203V/S205A mutant the PT process does occur, but with a 350 times slower rate than in wild-type GFP (wt-GFP). Furthermore, the kinetic isotope effect in the GFP double mutant T203V/S205A is twice smaller than in the wt-GFP and in the GFP single mutant S205V, which forms a novel PT pathway. On the other hand, the crystal structure of GFP T203V/S205A does not reveal a viable proton transfer pathway. To explain PT in GFP T203V/S205A, we argue on the basis of the MD simulations for an alternative, novel proton-wire pathway which involves the phenol group of the chromophore and water molecules infrequently entering from the bulk. This alternative pathway may explain the dramatically slow PT in the GFP double mutant T203V/S205A compared to wt-GFP.

BefA, a microbiota-secreted membrane disrupter, disseminates to the pancreas and increases ß cell mass.

Microbiome dysbiosis is a feature of diabetes, but how microbial products influence insulin production is poorly understood. We report the mechanism of BefA, a microbiome-derived protein that increases proliferation of insulin-producing ß cells during development in gnotobiotic zebrafish and mice. BefA disseminates systemically by multiple anatomic routes to act directly on pancreatic islets. We detail BefA's atomic structure, containing a lipid-binding SYLF domain, and demonstrate that it permeabilizes synthetic liposomes and bacterial membranes. A BefA mutant impaired in membrane disruption fails to expand ß cells, whereas the pore-forming host defense protein, Reg3, stimulates ß cell proliferation. Our work demonstrates that membrane permeabilization by microbiome-derived and host defense proteins is necessary and sufficient for ß cell expansion during pancreas development, potentially connecting microbiome composition with diabetes risk.

Host-emitted amino acid cues regulate bacterial chemokinesis to enhance colonization.

Animal microbiomes are assembled predominantly from environmental microbes, yet the mechanisms by which individual symbionts regulate their transmission into hosts remain underexplored. By tracking the experimental evolution of Aeromonas veronii in gnotobiotic zebrafish, we identify bacterial traits promoting host colonization. Multiple independently evolved isolates with increased immigration harbored mutations in a gene we named sensor of proline diguanylate cyclase enzyme (SpdE) based on structural, biochemical, and phenotypic evidence that SpdE encodes an amino-acid-sensing diguanylate cyclase. SpdE detects free proline and to a lesser extent valine and isoleucine, resulting in reduced production of intracellular c-di-GMP, a second messenger controlling bacterial motility. Indeed, SpdE binding to amino acids increased bacterial motility and host colonization. Hosts serve as sources of SpdE-detected amino acids, with levels varying based on microbial colonization status. Our work demonstrates that bacteria use chemically regulated motility, or chemokinesis, to sense host-emitted cues that trigger active immigration into hosts.

Reactive oxygen species control senescence-associated matrix metalloproteinase-1 through c-Jun-N-terminal kinase.

The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox-responsive molecular signals that drive senescence-associated (SA) matrix metalloproteinase-1 (MMP-1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady-state (H(2)O(2)) 3.5-fold (13.7-48.6 pM) relative to young cells. Restricting H(2)O(2) production through low O(2) exposure or by antioxidant treatments prevented SA increases in MMP-1 expression. The H(2)O(2)-dependent control of SA MMP-1 is attributed to sustained JNK activation and c-jun recruitment to the MMP-1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK-4) and inhibitory phosphatase (MKP-1), respectively. Enforced MKP-1 expression negates SA increases in JNK phosphorylation and MMP-1 production. Overall, these studies define redox-sensitive signaling networks regulating SA MMP-1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover.

Excited state proton transfer in the red fluorescent protein mKeima.

mKeima is an unusual monomeric red fluorescent protein (lambda(em)(max) approximately 620 nm) that is maximally excited in the blue (lambda(ex)(max) approximately 440 nm). The large Stokes shift suggests that the chromophore is normally protonated. A 1.63 A resolution structure of mKeima reveals the chromophore to be imbedded in a novel hydrogen bond network, different than in GFP, which could support proton transfer from the chromophore hydroxyl, via Ser142, to Asp157. At low temperatures the emission contains a green component (lambda(em)(max) approximately 535 nm), enhanced by deuterium substitution, presumably resulting from reduced proton transfer efficiency. Ultrafast pump/probe studies reveal a rising component in the 610 nm emission with a lifetime of approximately 4 ps, characterizing the rate of proton transfer. Mutation of Asp157 to neutral Asn changes the chromophore resting charge state to anionic (lambda(ex)(max) approximately 565 nm, lambda(em)(max) approximately 620 nm). Thus, excited state proton transfer (ESPT) explains the large Stokes shift. This work unambiguously characterizes green emission from the protonated acylimine chromophore of red fluorescent proteins.

Structure and mechanism of the photoactivatable green fluorescent protein.

Crystal structures of the photoactivatable green fluorescent protein T203H variant (PA-GFP) have been solved in the native and photoactivated states, which under 488 nm illumination are dark and brightly fluorescent, respectively. We demonstrate that photoactivation of PA-GFP is the result of a UV-induced decarboxylation of the Glu222 side chain that shifts the chromophore equilibrium to the anionic form. Coupled with the T203H mutation, which stabilizes the native PA-GFP neutral chromophore, Glu222 decarboxylation yields a 100-fold contrast enhancement relative to wild-type GFP (WT). Additionally, the structures provide insights into the spectroscopic differences between WT and PA-GFP steady-state fluorescence maxima and excited-state proton transfer dynamics.

Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells.

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens-epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host-specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.

Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase.

The phosphocarrier protein IIIGlc is an integral component of the bacterial phosphotransferase (PTS) system. Unphosphorylated IIIGlc inhibits non-PTS carbohydrate transport systems by binding to diverse target proteins. The crystal structure at 2.6 A resolution of one of the targets, glycerol kinase (GK), in complex with unphosphorylated IIIGlc, glycerol, and adenosine diphosphate was determined. GK contains a region that is topologically identical to the adenosine triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and actin. IIIGlc binds far from the catalytic site of GK, indicating that long-range conformational changes mediate the inhibition of GK by IIIGlc. GK and IIIGlc are bound by hydrophobic and electrostatic interactions, with only one hydrogen bond involving an uncharged group. The phosphorylation site of IIIGlc, His90, is buried in a hydrophobic environment formed by the active site region of IIIGlc and a 3(10) helix of GK, suggesting that phosphorylation prevents IIIGlc binding to GK by directly disrupting protein-protein interactions.

Crystal structure of the Aequorea victoria green fluorescent protein.

The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

Fluorescent proteins: maturation, photochemistry and photophysics.

It has long been appreciated that green fluorescent protein (GFP) autocatalytically forms its chromophore in a host-independent process; several of the initial steps in the reaction have recently been elucidated. Nevertheless, the end points of the process are unexpectedly diverse, as six chemically distinct chromophores, including two with three rings, have been identified. All fluorescent proteins continuously produce a low level of reactive oxygen species under illumination, which, in some cases, can lead to host cell death. In one extreme but useful example, the red fluorescent protein KillerRed can be used to selectively destroy cells upon brief illumination. Finally, when photophysical processes such as excited-state proton transfer, reversible photobleaching and photoactivation are understood, useful research tools, for example, real-time biosensors and optical highlighters, can result; however, side effects of their use may lead to significant artifacts in time-dependent microscopy experiments.

Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments.

Green fluorescent protein (GFP) indicators were previously developed that rapidly and quantitatively respond to changes in the thiol/disulfide equilibrium within subcellular compartments. In these indicators, surface-exposed cysteines residues were introduced so as to form a labile redox-active disulfide that in turn controls the emission properties of the internal chromophore. The biosensors have been shown to be effective reporters of the thiol/disulfide status within reducing compartments such as the mitochondria and cytosol for several cell types. However, due to the high thermodynamic stability of the introduced disulfide bond, the indicators are not useful for quantitative analysis within more oxidizing compartments such as the endoplasmic reticulum. Here we report the development of a new family of GFP-based redox indicators (roGFP1-iX) in which the thermodynamic stability of the disulfide is substantially lowered by insertion of a single amino acid into the main chain, adjacent to cysteine 147. The insertions result in indicators with midpoint potentials of -229 to -246 mV and are thus better suited for study of relatively oxidizing subcellular compartments. Atomic resolution crystallographic analyses suggest that two important factors act to destabilize the disulfide linkage in roGFP1-iX. In the oxidized state, an unusual non-proline cis-peptide bond adjacent to one of the cysteines introduces geometric strain into the system, while in the reduced state, a dramatic loop opening lowers the effective concentration of the reacting species.

Redox-sensitive green fluorescent protein: probes for dynamic intracellular redox responses. A review.

The quantification of transient redox events within subcellular compartments, such as those involved in certain signal transduction pathways, requires specific probes with high spatial and temporal resolution. Redox-sensitive variants of the green fluorescent protein (roGFP) have recently been developed that allow the noninvasive monitoring ofintracellular thiol-disulfide equilibria. In this chapter, the biophysical properties of these probes are discussed, including recent efforts to enhance their response times. Several recent applications of roGFPs are highlighted, including roGFP expression within Arabidopsis to monitor redox status during root elongation, expression in neurons to measure oxidative stress during ischemia, and targeting of roGFPs to endosomal compartments demonstrating unexpectedly oxidizing potentials within these compartments. Possible future directions for the optimization of roGFPs or new classes of redox-sensitive fluorescent probes are also discussed.