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K H Gardner

Publications and source records attributed to K H Gardner.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of the structure and composition of gecko adhesive setae.

The ability of certain reptiles to adhere to vertical (and hang from horizontal) surfaces has been attributed to the presence of specialized adhesive setae on their feet. Structural and compositional studies of such adhesive setae will contribute significantly towards the design of biomimetic fibrillar adhesive materials. The results of electron microscopy analyses of the structure of such setae are presented, indicating their formation from aggregates of proteinaceous fibrils held together by a matrix and potentially surrounded by a limiting proteinaceous sheath. Microbeam X-ray diffraction analysis has shown conclusively that the only ordered protein constituent in these structures exhibits a diffraction pattern characteristic of beta-keratin. Raman microscopy of individual setae, however, clearly shows the presence of additional protein constituents, some of which may be identified as alpha-keratins. Electrophoretic analysis of solubilized setal proteins supports these conclusions, indicating the presence of a group of low-molecular-weight beta-keratins (14-20 kDa), together with alpha-keratins, and this interpretation is supported by immunological analyses.

Adhesiveness↗
PubMed2006

Multicriteria decision analysis: a comprehensive decision approach for management of contaminated sediments.

Contaminated sediments and other sites present a difficult challenge for environmental decisionmakers. They are typically slow to recover or attenuate naturally, may involve multiple regulatory agencies and stakeholder groups, and engender multiple toxicological and ecotoxicological risks. While environmental decision-making strategies over the last several decades have evolved into increasingly more sophisticated, information-intensive, and complex approaches, there remains considerable dissatisfaction among business, industry, and the public with existing management strategies. Consequently, contaminated sediments and materials are the subject of intense technology development, such as beneficial reuse or in situ treatment. However, current decision analysis approaches, such as comparative risk assessment, benefit-cost analysis, and life cycle assessment, do not offer a comprehensive approach for incorporating the varied types of information and multiple stakeholder and public views that must typically be brought to bear when new technologies are under consideration. Alternatively, multicriteria decision analysis (MCDA) offers a scientifically sound decision framework for management of contaminated materials or sites where stakeholder participation is of crucial concern and criteria such as economics, environmental impacts, safety, and risk cannot be easily condensed into simple monetary expressions. This article brings together a multidisciplinary review of existing decision-making approaches at regulatory agencies in the United States and Europe and synthesizes state-of-the-art research in MCDA methods applicable to the assessment of contaminated sediment management technologies. Additionally, it tests an MCDA approach for coupling expert judgment and stakeholder values in a hypothetical contaminated sediments management case study wherein MCDA is used as a tool for testing stakeholder responses to and improving expert assessment of innovative contaminated sediments technologies.

Journal Article↗
PubMed2005

Neutron vibrational spectroscopy gives new insights into the structure of poly(p-phenylene terephthalamide).

The vibrational spectra of benzanilide and poly(p-phenylene terephthalamide) have been measured using inelastic neutron scattering. These compounds have similar hydrogen-bond networks, which, for poly(p-phenylene terephthalamide), lead to two-dimensional hydrogen-bonded sheets in the crystal. Experimental spectra are compared with solid-state, quantum chemical calculations based on density functional theory (DFT). Such "parameter-free" calculations allow the structure-dynamics relation in this type of compound to be quantified, which is demonstrated here for benzanilide. In the case of poly(p-phenylene terephthalamide), vibrational spectroscopy and DFT calculations help resolve long-standing questions about the packing of hydrogen-bonded sheets in the solid state.

Journal Article↗
PubMed2001

The plug domain of FepA, a TonB-dependent transport protein from Escherichia coli, binds its siderophore in the absence of the transmembrane barrel domain.

FepA, an outer membrane iron siderophore transporter from Escherichia coli, is composed of a 22-stranded membrane-spanning beta barrel with a globular N-terminal "plug" domain of 148 residues that folds up inside the barrel and completely occludes the barrel's interior (1). We have overexpressed and purified this plug domain by itself and find that it behaves in vitro as a predominantly unfolded yet soluble protein, as determined by circular dichroism, thermal denaturation, and NMR studies. Despite its unfolded state, the isolated domain binds ferric enterobactin, the siderophore ligand of FepA, with an affinity of 5 microM, just 100-fold reduced from that of intact FepA. These findings argue against the hypothesis that the plug domain is pulled intact from the barrel during transport in vivo but may be consistent either with a model where the plug rearranges within the barrel to create a channel large enough to allow transport or with a model where the plug unfolds and comes out of the barrel.

Bacterial Outer Membrane Proteins↗
PubMed2001

PAS kinase: an evolutionarily conserved PAS domain-regulated serine/threonine kinase.

PAS domains regulate the function of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli. PAS domain-regulated histidine kinases are common in prokaryotes and control a wide range of fundamental physiological processes. Similarly regulated kinases are rare in eukaryotes and are to date completely absent in mammals. PAS kinase (PASK) is an evolutionarily conserved gene product present in yeast, flies, and mammals. The amino acid sequence of PASK specifies two PAS domains followed by a canonical serine/threonine kinase domain, indicating that it might represent the first mammalian PAS-regulated protein kinase. We present evidence that the activity of PASK is regulated by two mechanisms. Autophosphorylation at two threonine residues located within the activation loop significantly increases catalytic activity. We further demonstrate that the N-terminal PAS domain is a cis regulator of PASK catalytic activity. When the PAS domain-containing region is removed, enzyme activity is significantly increased, and supplementation of the purified PAS-A domain in trans selectively inhibits PASK catalytic activity. These studies define a eukaryotic signaling pathway suitable for studies of PAS domains in a purified in vitro setting.

Amino Acid Sequence↗
PubMed1999

A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins.

A selective protonation strategy is described that uses [3-2H] 13C alpha-ketoisovalerate to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of approximately 100 mg/L alpha-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] alpha-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-delta1 methyl)-isoleucine (> 90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.

Carbon Isotopes↗
PubMed1998

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: a role for N-terminal residues in tandem binding.

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of 2H, 15N, 13C labelled intact and (des2-7) trp repressor (delta 2-7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone 1HN, 15N, 13c alpha positions in both forms of the protein and for 13 C beta in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of delta 2-7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178-182]. Comparison of the backbone chemical shifts of DNA-bound delta 2-7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2-12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C alpha chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and delta 2-7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.

Amino Acid Sequence↗
PubMed1998

The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins.

During the past thirty years, deuterium labeling has been used to improve the resolution and sensitivity of protein NMR spectra used in a wide variety of applications. Most recently, the combination of triple resonance experiments and 2H, 13C, 15N labeled samples has been critical to the solution structure determination of several proteins with molecular weights on the order of 30 kDa. Here we review the developments in isotopic labeling strategies, NMR pulse sequences, and structure-determination protocols that have facilitated this advance and hold promise for future NMR-based structural studies of even larger systems. As well, we detail recent progress in the use of solution 2H NMR methods to probe the dynamics of protein sidechains.

Carbon Isotopes↗
PubMed1997

Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.

The development of 15N, 13C, 2H multidimensional NMR spectroscopy has facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses of over 30 kDa. The success of these methods has been achieved through the production of highly deuterated proteins; replacing carbon-bound protons with deuterons significantly improves the sensitivity of many of the experiments used in chemical shift assignment. Unfortunately, uniform deuteration also radically depletes the number of interproton distance restraints available for structure determination, degrading the quality of the resulting structures. Here we describe an approach for improving the precision and accuracy of global folds determined from highly deuterated proteins through the use of deuterated, selectively methyl-protonated samples. This labeling profile maintains the efficiency of triple-resonance NMR experiments while retaining a sufficient number of protons at locations where they can be used to establish NOE-based contacts between different elements of secondary structure. We evaluate how this deuteration scheme affects the sensitivity and resolution of experiments used to assign 15N, 13C, and 1H chemical shifts and interproton NOEs. This approach is tested experimentally on a 14 kDa SH2/phosphopeptide complex, and a global protein fold is obtained from a set of methyl-methyl, methyl-NH, and NH-NH distance restraints. We demonstrate that the inclusion of methyl-NH and methyl-methyl distance restraints greatly improves the precision and accuracy of structures relative to those generated with only NH-NH distance restraints. Finally, we examine the general applicability of this approach by determining the structures of several proteins with molecular masses of up to 40 kDa from simulated distance and dihedral angle restraint tables.

Deuterium↗
PubMed1997

Solution NMR spectroscopy beyond 25 kDa.

Improvements in NMR instrumentation, higher magnetic field strengths, novel NMR experiments and new deuterium-labeling strategies have significantly increased the scope of structural problems that can now be addressed by solution NMR methods. To date, a number of structures of proteins of 30 kDa have been solved using multidimensional 15N,13C,2H NMR techniques, and this molecular weight limit will probably be surpassed in the near future.

Binding Sites↗
PubMed1996

Selective methyl group protonation of perdeuterated proteins.

Deuteration of aliphatic sites in proteins has shown great potential to increase the range of molecules amenable to study by NMR spectroscopy. One problem inherent in high-level deuterium incorporation is the loss of 1H-1H distance information obtainable from NOESY spectra of the labeled proteins. In the limit of perdeuteration, the available NH-NH NOEs are insufficient in many cases to define the three-dimensional structure of a folded protein. We describe here a method of producing proteins that retains all the advantages of perdeuteration, while enabling observation of many NOEs absent from spectra of fully deuterated samples. Overexpression of proteins in bacteria grown in 2H2O medium containing protonated pyruvate as the sole carbon source results in complete deuteration at C alpha and > 80% deuteration at C beta positions of nearly all amino acids. In contrast, the methyl groups of Ala, Val, Leu and Ile (gamma 2 only) remain highly protonated. This labeling pattern can be readily understood from analysis of bacterial pathways for pyruvate utilization and amino acid biosynthesis. As Ala, Val, Leu and Ile are among the most highly represented residue types in protein hydrophobic cores and at protein-protein interfaces, selectively methyl-protonated samples will be useful in many areas of structural analysis of larger molecules and molecular complexes by NMR.

Amino Acids↗
PubMed1995

HeteroTOCSY-based experiments for measuring heteronuclear relaxation in nucleic acids and proteins.

While both 31P and 113Cd are present at locations of interest in many different macromolecular systems, heteronuclear-detected relaxation measurements on these nuclei have been restrained by limitations in either resolution or signal-to-noise ratio. We have developed heteroTOCSY-based methods to overcome both of these problems. Two-dimensional versions of these experiments were utilized to measure 31P T1 and T2 values in DNA oligonucleotides; the additional resolution offered by a second dimension allowed determination of these values for most of the 31P resonances in a DNA dodecamer. The results from the experiments indicated that there was little significant variation in T1 values for the different phosphates in the DNA dodecamer; however, the T2 values showed a clear pattern, with lower values in the interior of the sequence than at the ends of the helix. Furthermore, a significant correlation between 31P chemical shifts and T2 values was observed. One-dimensional, frequency-selective versions of these experiments were also developed for use on systems containing a smaller number of heteronuclear spins. These methods were applied to investigate the heteronuclear relaxation properties of 113Cd in 113Cd2LAC9(61), a Cys6Zn2 DNA-binding domain. Data from the experiments confirm biochemical evidence that more significant differences occur in the metal-protein interactions between the two metal-binding sites than has been previously identified for proteins containing this motif.

Antimetabolites, Antineoplastic↗
PubMed1995

Novel metal-binding proteins by design.

We describe the successful design of a tetrahedral His3Cys Zn(II)-binding site in a small protein of known structure: the B1 domain of Streptococcal protein G. The B1 variants containing the novel metal-binding site were characterized using a combination of optical absorption, circular dichroism and NMR spectroscopies. The results indicate that the designed proteins bind Zn(II) with high affinity and tetrahedral coordination geometry, and that the overall secondary and tertiary structure of the B1 domain is maintained.

Amino Acid Sequence↗
PubMed1995

Solution structure of the Kluyveromyces lactis LAC9 Cd2 Cys6 DNA-binding domain.

The Zn2Cys6 DNA-binding domain has been identified by sequence homology in approximately forty fungal proteins, including the K. lactis LAC9 transcriptional activator. Using 1H NMR spectroscopy, we have determined the solution structure of a cadmium-substituted form of the LAC9 DNA-binding domain. We have complemented this approach by applying a series of 113Cd-1H NMR experiments, including several novel heteroTOCSY-based techniques. The DNA-binding domain forms a core of two alpha-helix/extended strand segments around the Cd2 binuclear cluster, with a network of amide proton-cysteinyl S gamma hydrogen bonds stabilizing the cluster. Comparison with other Zn2Cys6 domain structures provides insight into the common structural elements used in metal coordination and DNA binding.

Amino Acid Sequence↗
PubMed1994

Solution structure of a DNA dodecamer containing the anti-neoplastic agent arabinosylcytosine: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement.

The effect of araC incorporation into the dodecamer duplex [d(CGCGAATT) (araC)d(GCG)]2 was examined by comparing its nuclear magnetic resonance (NMR)-determined solution structure with that of the control duplex d[(CGCGAATTCGCG)]2. 1H and 31P resonances in both duplexes were assigned using a combination of 2-D 1H NMR and a 3-D 31P-1H heteroTOCSY-NOESY experiment. Proton-proton distances (determined from NOESY data) and sugar dihedral angles (from NOESY and COSY data) were used in restrained molecular dynamics simulations starting from canonical A- or B-form DNA models. Both the control and araC sets of simulations converged to B-type structures. These structures were subjected to full relaxation matrix refinement to produce final structures which were in excellent agreement (R1/6 < 0.05) with the observed NOE intensities. A detailed comparison of the final control and araC structures revealed a global similarity (overall RMSD approximately 1.3 A), with significant differences localized at the araC site and neighboring bases. These included changes in sugar pucker, backbone torsion angles, base stacking, and other helical parameters. These findings are in general agreement with the previously published X-ray structure of a decamer duplex containing araC. One intriguing feature of the NMR solution structure not found in the crystal structure is the presence of an intramolecular hydrogen bond between the 2' hydroxyl on the araC sugar and the 3' phosphate group.

Base Composition↗
PubMed1994

113Cd-1H heteroTOCSY: a method for determining metal-protein connectivities.

113Cd-1H NMR correlation experiments have been extremely useful for determining the amino acid ligands that form metal-binding sites in proteins. To date, the majority of these methods have used heteronuclear multiple-quantum transfer as the basis for establishing correlations. In this paper, we demonstrate the feasibility of using correlation methods that employ heteronuclear cross-polarization (heteroTOCSY) as viable alternatives. Additionally, we couple heteroTOCSY with selective excitation and transfer procedures to take advantage of the small number of heteronuclei usually present in metalloprotein systems. One- and two-dimensional experiments are presented as examples of these techniques.

Cadmium↗
PubMed1991

Structure of the binuclear metal-binding site in the GAL4 transcription factor.

The GAL4 transcription factor from yeast contains within its N-terminal DNA-binding domain an amino acid sequence containing six cysteine residues, C11-X2-C14-X6-C21-X6-C28-X2-C31-X6-C38. The six Cys residues will form a binuclear metal cluster with either Zn(II) or Cd(II) in which two of the -S- donors are bridging ligands between the two metal ions. Binding of Zn(II) or Cd(II) to the GAL4 DNA-binding domain is essential to induce the conformation of GAL4 required for the protein to recognize the specific DNA sequence, UASG, to which GAL4 binds. Evidence for the presence of the binuclear cluster has come from 113Cd NMR and 2D 1H-113Cd heteronuclear NMR studies of the cloned DNA-binding domain of GAL4 consisting of the N-terminal 62 residues, GAL4(62*) [Pan and Coleman (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077]. Cd(II) binding to the GAL4 DNA is highly cooperative, thus the Cd2Cys6 cluster is always formed. On the other hand, Zn(II) forms well-defined Zn1 and Zn2 complexes with the DNA-binding domain of GAL4, both of which bind specifically to the UASG DNA sequence. The structural details of the Cd2-, Zn2-, and Zn1GAL4(62*) proteins have been determined by a variety of heteronuclear and 2D NMR techniques. When Cd(II) is exchanged for Zn(II), the cluster appears to expand to accommodate the larger Cd(II) ion as suggested by changes of 2 to 4 Hz in the 3JHN alpha coupling constants for the amino acid residues which form the polypeptide loops enclosing the cluster, residues 10-40. These changes suggest alterations in the backbone phi torsional angles of from 20 degrees to 30 degrees. A metal-ligand structure derived from the 1H-113Cd heteronuclear NMR as well as the polypeptide backbone connectivity around the cluster as determined from short-range 1H-1H NOE's is presented. The metal ions also determine the major folding of GAL4(62*), since the chemical shift dispersion in the entire NH-alpha CH fingerprint region of the 1H-1H COSY spectrum collapses on removal of the metal ion. Two short segments of the GAL4(62*) polypeptide (residues 14-19 and 30-36 in the cluster forms, 12-19 and 30-36 in the Zn1 species) show significant dNN(i,i + 1) NOE's. These short segments of polypeptide chain are the only ones that could be helical in the GAL4(62*).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗