Biomedical subjects

Kevin H Gardner

Publications and source records attributed to Kevin H Gardner.

At least 19 recordsLinked to original sources

PubMed2006

Leaching behavior of estuarine sediments and cement-stabilized sediments in upland management environments.

Contaminated surficial sediments in major ports and harbors remain a significant economic liability during routine dredging operations. Numerous beneficial uses have been suggested in recent years that promise reduced sediment management costs concomitant with a productive material use. This manuscript describes the leaching of metals and metalloids from surficial sediments and from controlled low strength material (flowable fill) produced using the estuarine sediments as replacement for sand. Sediments from two locations within the New York/New Jersey Harbor area (Gowanus Canal and Newtown Creek) (USA), were subjected to pH-dependent leaching tests and liquid-to-solid ratio dependent leaching tests. Results indicate that, in general, maximum contaminant levels for drinking water, used here as a benchmark for metals concentrations in leachate, were exceeded only at pH values less than 5 or greater than 9. Leaching as a function of increasing liquid-to-solid ratio demonstrated that pH controlled the observed behavior: unamended sediment leached lower concentrations of all elements except for the oxyanion arsenate. The flowable fill material, despite dilution of the sediment and incorporation into a cementitious matrix, leached higher element concentrations except for arsenic due to the high pH of the material. It was also shown that a much more grossly contaminated material (Newtown Creek) had a very similar leaching behavior to the less contaminated Gowanus Canal material. Speciation calculations demonstrated that dissolved organic carbon plays a significant role in the leaching observed from these estuarine sediments and the flowable fill made with the high organic matter content sediments.

Acids↗

PubMed2006

O acetylation of the enterobacterial common antigen polysaccharide is catalyzed by the product of the yiaH gene of Escherichia coli K-12.

The carbohydrate component of the enterobacterial common antigen (ECA) of Escherichia coli K-12 occurs primarily as a water-soluble cyclic polysaccharide located in the periplasm (ECA(CYC)) and as a phosphoglyceride-linked linear polysaccharide located on the cell surface (ECA(PG)). The polysaccharides of both forms are comprised of the amino sugars N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc). These amino sugars are linked to one another to form trisaccharide repeat units with the structure -->3-alpha-D-Fuc4NAc-(1-->4)-beta-D-ManNAcA-(1-->4)-alpha-D-GlcNAc-(1-->. The hydroxyl group in the 6 position of the GlcNAc residues of both ECA(CYC) and ECA(PG) are nonstoichiometrically esterified with acetyl groups. Random transposon insertion mutagenesis of E. coli K-12 resulted in the generation of a mutant defective in the incorporation of O-acetyl groups into both ECA(CYC) and ECA(PG). This defect was found to be due to an insertion of the transposon into the yiaH locus, a putative gene of unknown function located at 80.26 min on the E. coli chromosomal map. Bioinformatic analyses of the predicted yiaH gene product indicate that it is an integral inner membrane protein that is a member of an acyltransferase family of enzymes found in a wide variety of organisms. The results of biochemical and genetic experiments presented here strongly support the conclusion that yiaH encodes the O-acetyltransferase responsible for the incorporation of O-acetyl groups into both ECA(CYC) and ECA(PG). Accordingly, we propose that this gene be designated wecH.

Acetylation↗

PubMed2006

Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation.

The covalent attachment of heme cofactors to the apo-polypeptides via thioether bonds is unique to the maturation of c-type cytochromes. A number of thiol-disulfide oxidoreductases prepare the apocytochrome for heme insertion in system I and II cytochrome c maturation. Although most thiol-disulfide oxidoreductases are nonspecific, the less common, specific thiol-disulfide oxidoreductases may be key to directing the usage of electrons. Here we demonstrate that unlike other thiol-disulfide oxidoreductases, the protein responsible for reducing oxidized apocytochrome c in Bacillus subtilis, ResA, is specific for cytochrome c550 and utilizes alternate conformations to recognize redox partners. We report solution NMR evidence that ResA undergoes a redox-dependent conformational change between oxidation states, as well as data showing that ResA utilizes a surface cavity present only in the reduced state to recognize a peptide derived from cytochrome c550. Finally, we confirm that ResA is a specific thiol-disulfide oxidoreductase by comparing its reactivity to our mimetic peptide with its reactivity to oxidized glutathione, a nonspecific substrate. This study biochemically demonstrates the specificity of this thiol-disulfide oxidoreductase and enables us to outline a structural mechanism of regulating the usage of electrons in a thiol-disulfide oxidoreductase system.

Bacillus subtilis↗

PubMed2005

Uptake of polycyclic aromatic hydrocarbons (PAHs) in salt marsh plants Spartina alterniflora grown in contaminated sediments.

Polycyclic aromatic hydrocarbon (PAH) concentrations were measured in Spartina alterniflora plants grown in pots of contaminated sediment, plants grown in native sediment at a marsh contaminated with up to 900 microg/g total PAHs, and from plants grown in uncontaminated control sediment. The roots and leaves of the plants were separated, cleaned, and analyzed for PAHs. PAH compounds were detected at up to 43 microg/g dry weight in the root tissue of plants grown in pots of contaminated soil. PAH compounds were detected at up to 0.2 microg/g in the leaves of plants grown in pots of contaminated soil. Concentrations less than 0.004 microg/g were detected in the leaves of plants grown at a reference site. Root concentration factor (RCF) values ranged from 0.009 to 0.97 in the potted plants, and from 0.004 to 0.31 at the contaminated marsh site. Stem concentration factor (SCF) values ranged from 0.00004 to 0.03 in the potted plants and 0.0002 to 0.04 at the contaminated marsh. No correlation was found between the RCF value and PAH compound or chemical properties such as logKOW. SCF values were higher for the lighter PAHs in the potted plants, but not in the plants collected from the contaminated marsh. PAH concentrations in the roots of the potted plants are strongly correlated with soil concentrations, but there is less correlation for the roots grown in natural sediments. Additional plants were grown directly in PAH-contaminated water and analyzed for alkylated PAH homologs. No difference was found in leaf PAH concentrations between plants grown in contaminated water and control plants.

Gas Chromatography-Mass Spectrometry↗

PubMed2005

Evolutionary information for specifying a protein fold.

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures. The artificial proteins show thermodynamic stabilities similar to natural WW domains, and structure determination of one artificial protein shows excellent agreement with the WW fold at atomic resolution. The relative simplicity of the information used for creating sequences suggests a marked reduction to the potential complexity of the protein-folding problem.

Algorithms↗

PubMed2005

Can you hear me now? Regulating transcriptional activators by phosphorylation.

Extracellular signals often modulate the expression of specific genetic programs by triggering the phosphorylation of relevant transcription factors (TFs). Phosphorylation in turn regulates such TFs by altering their cellular localization, DNA binding affinity, or transcriptional activity. Structural approaches have revealed how phosphorylation turns some TFs on or off; but less is known about how phosphorylation regulates other transcription factors in a graded manner that depends on signal intensity. A recent paper by Graves and colleagues reveals how a group of phosphorylation sites in Ets-1 regulates its DNA binding activity. Their studies provide new insight into the importance of multisite phosphorylation for the graded regulation of transcription and highlight the involvement of allosteric mechanisms in this process.

Allosteric Regulation↗

PubMed2005

Structural basis of ARNT PAS-B dimerization: use of a common beta-sheet interface for hetero- and homodimerization.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous bHLH-PAS (Per-ARNT-Sim) protein that forms heterodimeric transcriptional regulator complexes with several other bHLH-PAS subunits to control a variety of biological pathways, some of which are centrally involved in disease initiation and/or progression. One of these is the hypoxia response pathway, which allows eukaryotic cells to respond to low oxygen tension via the formation of a heterodimeric complex between ARNT and another bHLH-PAS protein, the hypoxia-inducible factor alpha (HIF-alpha). We have previously shown that the C-terminal PAS domains of an HIF-alpha isoform (HIF-2alpha) and ARNT interact in vitro, and that mutations in the solvent-exposed beta-sheet surface of the HIF-2alpha domain not only disrupt this interaction, but also greatly attenuate the hypoxia response in living cells. Here, we have solved the solution structure of the corresponding PAS domain of ARNT and show that it utilizes a very similar interface for the interaction with the HIF-2alpha PAS domain. We also show that this domain self-associates in a concentration-dependent manner, and that the interface used in this homodimeric complex is very similar to that used in the formation of heterodimer. In addition, using experimentally derived NMR restraints, we used the program HADDOCK to calculate a low-resolution model of the complex formed in solution by these two PAS domains, and confirm the validity of this model using site-directed spin labeling to obtain long-range distance information in solution. With this information, we propose a model for the mode of multi-PAS domain interaction in bHLH-PAS transcriptional activation complexes.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed2005

Functions of the Per/ARNT/Sim domains of the hypoxia-inducible factor.

The heterodimeric transcription factor hypoxia-inducible factor (HIF) plays an important role in the progression of a number of processes in which O2 availability is compromised and, as such, has become an increasingly attractive therapeutic target. Although tremendous progress has been made in recent years in unraveling the mechanisms underlying O2-dependent regulation of HIF through its O2-dependent degradation domain and C-terminal transactivation domain, our understanding of the contributions of other structural elements, particularly the Per/ARNT/Sim (PAS)-A and PAS-B domains, to the activity of HIF is incomplete. Using insights derived from the recently determined solution structures of the HIF PAS-B domains as a starting point, we have explored the function(s) of the HIF-2alpha PAS domains via mutational analysis. In contrast to recent models, our data reveal that both PAS domains of the HIF-alpha subunit are necessary for heterodimer formation but are not required to mediate other HIF functions in which PAS domains have been implicated. Because disruption of individual PAS domains compromise HIF function independent of the mechanism of HIF induction, these data demonstrate the potential utility of targeting these domains for therapeutic applications.

Animals↗

PubMed2005

Structure of a bacterial BLUF photoreceptor: insights into blue light-mediated signal transduction.

Light is an essential environmental factor, and many species have evolved the capability to respond to it. Blue light is perceived through three flavin-containing photoreceptor families: cryptochromes, light-oxygen-voltage, and BLUF (sensor of blue light using flavin adenine dinucleotide, FAD) domain proteins. BLUF domains are present in various proteins from Bacteria and lower Eukarya. They are fully modular and can relay signals to structurally and functionally diverse output units, most of which are implicated in nucleotide metabolism. We present the high resolution crystal structure of the dark resting state of BlrB, a short BLUF domain-containing protein from Rhodobacter sphaeroides. The structure reveals a previously uncharacterized FAD-binding fold. Along with other lines of evidence, it suggests mechanistic aspects for the photocycle that is characterized by a red-shifted absorbance of the flavin. The isoalloxazine ring of FAD binds in a cleft between two helices, whereas the adenine ring points into the solvent. We propose that the adenine ring serves as a hook mediating the interaction with its effector/output domain. The structure suggests a unique photochemical signaling switch in which the absorption of light induces a structural change in the rim surrounding the hook, thereby changing the protein interface between BLUF and the output domain.

Bacterial Proteins↗

PubMed2005

Simultaneous application of dissolution/precipitation and surface complexation/surface precipitation modeling to contaminant leaching.

This paper discusses the modeling of anion and cation leaching from complex matrixes such as weathered steel slag. The novelty of the method is its simultaneous application of the theoretical models for solubility, competitive sorption, and surface precipitation phenomena to a complex system. Selective chemical extractions, pH dependent leaching experiments, and geochemical modeling were used to investigate the thermodynamic equilibrium of 12 ions (As, Ca, Cr, Ba, SO4, Mg, Cd, Cu, Mo, Pb, V, and Zn) with aqueous complexes, soluble solids, and sorptive surfaces in the presence of 12 background analytes (Al, Cl, Co, Fe, K, Mn, Na, Ni, Hg, NO3, CO3, and Ba). Modeling results show that surface complexation and surface precipitation reactions limit the aqueous concentrations of Cd, Zn, and Pb in an environment where Ca, Mg, Si, and CO3 dissolve from soluble solids and compete for sorption sites. The leaching of SO4, Cr, As, Si, Ca, and Mg appears to be controlled by corresponding soluble solids.

Adsorption↗

PubMed2005

Identification and optimization of protein domains for NMR studies.

The success of genomic sequencing projects in recent years has presented protein scientists with a formidable challenge in characterizing the vast number of gene products that have subsequently been identified. NMR has proven to be a valuable tool in the elucidation of various properties for many of these proteins, allowing versatile studies of structure, dynamics, and interactions in the solution state. But the characteristics needed for proteins amenable to this kind of study, such as folding capability, long-term stability, and high solubility, require robust and expeditious methods for the identification and optimization of target protein domains. Here we present a variety of computational and experimental methods developed for these purposes and show that great care must often be taken in the design of constructs intended for NMR-based investigations.

Deuterium Exchange Measurement↗

PubMed2004

Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity.

Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.

Amino Acid Sequence↗

PubMed2004

Identification of small-molecule antagonists that inhibit an activator: coactivator interaction.

Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 in response to hormonal stimuli triggers cellular gene expression via the recruitment of the histone acetylase coactivator paralogs CREB binding protein (CBP) and p300 to the promoter. The NMR structure of the CREB:CBP complex, using relevant interaction domains called KID and KIX, respectively, reveals a shallow hydrophobic groove on the surface of KIX that accommodates an amphipathic helix in phospho (Ser-133) KID. Using an NMR-based screening approach on a preselected small-molecule library, we identified several compounds that bind to different surfaces on KIX. One of these, KG-501 (2-naphthol-AS-E-phosphate), targeted a surface distal to the CREB binding groove that includes Arg-600, a residue that is required for the CREB:CBP interaction. When added to live cells, KG-501 disrupted the CREB: CBP complex and attenuated target gene induction in response to cAMP agonist. These results demonstrate the ability of small molecules to interfere with second-messenger signaling cascades by inhibiting specific protein-protein interactions in the nucleus.

CREB-Binding Protein↗

PubMed2004

Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy.

Phototropins are light-activated kinases from plants that utilize light-oxygen-voltage (LOV) domains as blue light photosensors. Illumination of these domains leads to the formation of a covalent linkage between the protein and an internally bound flavin chromophore, destabilizing the surrounding protein and displacing an alpha-helix from its surface. Here we use a combination of spectroscopic tools to monitor the kinetic processes that spontaneously occur in the dark as the protein returns to the noncovalent ground state. Using time-resolved two-dimensional (2D) NMR methods, we measured the rate of this process at over 100 independent sites throughout the protein, establishing that regeneration of the dark state occurs cooperatively within a 1.6-fold range of observed rates. These data agree with other spectroscopic measurements of the kinetics of protein/FMN bond cleavage and global conformational changes, consistent with these processes experiencing a common rate-limiting step. Arrhenius analyses of the temperature dependence of these rates suggest that the transition state visited during this regeneration has higher energy than the denatured form of this protein domain despite the fact that there is no global unfolding of the domain during this process.

Avena↗

PubMed2004

Cyclic enterobacterial common antigen: potential contaminant of bacterially expressed protein preparations.

We have previously reported the identification of the cyclic enterobacterial common antigen (ECA(CYC)) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol. 185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N -acetyl signals of ECA(CYC) have been observed in (15)N-(1)H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECA(CYC) in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate.

Antigens, Bacterial↗

PubMed2003

Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor.

Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFalpha and ARNT. Although this complex involves protein-protein interactions mediated by basic helix-loop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2alpha by NMR spectroscopy. We demonstrate that HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this beta-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.

Amino Acid Sequence↗

PubMed2003

Structural basis of a phototropin light switch.

Phototropins are light-activated kinases important for plant responses to blue light. Light initiates signaling in these proteins by generating a covalent protein-flavin mononucleotide (FMN) adduct within sensory Per-ARNT-Sim (PAS) domains. We characterized the light-dependent changes of a phototropin PAS domain by solution nuclear magnetic resonance spectroscopy and found that an alpha helix located outside the canonical domain plays a key role in this activation process. Although this helix associates with the PAS core in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent bond formation to kinase activation and identifies a signaling pathway conserved among PAS domains.

Amino Acid Sequence↗

PubMed2003

Changes in fractal dimension during aggregation.

Experiments were performed to evaluate temporal changes in the fractal dimension of aggregates formed during flocculation of an initially monodisperse suspension of latex microspheres. Particle size distributions and aggregate geometrical information at different mixing times were obtained using a non-intrusive optical sampling and digital image analysis technique, under variable conditions of mixing speed, coagulant (alum) dose and particle concentration. Pixel resolution required to determine aggregate size and geometric measures including the fractal dimension is discussed and a quantitative measure of accuracy is developed. The two-dimensional fractal dimension was found to range from 1.94 to 1.48, corresponding to aggregates that are either relatively compact or loosely structured, respectively. Changes in fractal dimension are explained using a conceptual model, which describes changes in fractal dimension associated with aggregate growth and changes in aggregate structure. For aggregation of an initially monodisperse suspension, the fractal dimension was found to decrease over time in the initial stages of floc formation.

Chemical Precipitation↗