Optimization of Field and Laboratory Sample Processing for Characterization of Metallic Residues at Military Training Ranges.

Military ranges are unlike many waste sites because the contaminants, both energetics and metals, are heterogeneously distributed in soil during explosive detonation or ballistic impact and cannot be readily characterized using conventional grab sampling. The Incremental Sampling Methodology (ISM) has been successful for characterization of energetic contamination in soils, but no published ISM processing studies for soils with small arms range metals such as Pb, Cu, Sb, and Zn exists. This study evaluated several ISM sample-processing steps: (1) field splitting to reduce the sample mass shipped to the analytical laboratory, (2) necessity of milling, and (3) processing a larger subsample mass for digestion in lieu of milling. Cone-and-quartering and rotary sectorial splitting techniques yielded poor precision and positively skewed distributions. Hence, an increase in digestion mass from 2 to 10 g was evaluated with milled and unmilled samples. Unmilled samples yielded results with the largest variability regardless of aliquot mass.

Inadequacy of Conventional Grab Sampling for Remediation Decision-Making for Metal Contamination at Small-Arms Ranges.

Research shows grab sampling is inadequate for evaluating military ranges contaminated with energetics because of their highly heterogeneous distribution. Similar studies assessing the heterogeneous distribution of metals at small-arms ranges (SAR) are lacking. To address this we evaluated whether grab sampling provides appropriate data for performing risk analysis at metal-contaminated SARs characterized with 30-48 grab samples. We evaluated the extractable metal content of Cu, Pb, Sb, and Zn of the field data using a Monte Carlo random resampling with replacement (bootstrapping) simulation approach. Results indicate the 95% confidence interval of the mean for Pb (432 mg/kg) at one site was 200-700 mg/kg with a data range of 5-4500 mg/kg. Considering the U.S. Environmental Protection Agency screening level for lead is 400 mg/kg, the necessity of cleanup at this site is unclear. Resampling based on populations of 7 and 15 samples, a sample size more realistic for the area yielded high false negative rates.

Applying Incremental Sampling Methodology to Soils Containing Heterogeneously Distributed Metallic Residues to Improve Risk Analysis.

This study compares conventional grab sampling to incremental sampling methodology (ISM) to characterize metal contamination at a military small-arms-range. Grab sample results had large variances, positively skewed non-normal distributions, extreme outliers, and poor agreement between duplicate samples even when samples were co-located within tens of centimeters of each other. The extreme outliers strongly influenced the grab sample means for the primary contaminants lead (Pb) and antinomy (Sb). In contrast, median and mean metal concentrations were similar for the ISM samples. ISM significantly reduced measurement uncertainty of estimates of the mean, increasing data quality (e.g., for environmental risk assessments) with fewer samples (e.g., decreasing total project costs). Based on Monte Carlo resampling simulations, grab sampling resulted in highly variable means and upper confidence limits of the mean relative to ISM.

Fostering innovation in contaminated sediments management through multicriteria technology assessment and public participation.

Management of contaminated sediments is problematic and costly. Several new technologies are under development that may in some cases reduce costs and environmental or ecological impacts. However, there are significant barriers to implementing new technologies, including the increased management complexity, the potential for introducing antagonistic or incommensurate objectives that are unfamiliar to stakeholders or regulators, and the difficulty of capturing private, commercial benefits from environmental improvements that may primarily benefit the public. This article identifies several innovative contaminated sediments technologies, discusses the difficulty of proving or quantifying the benefits of new technologies, and presents an agenda for research that would foster partnerships between scientific, government, and public communities of interest for the purpose of improving innovative technology assessment and environmental decision making.

Life cycle based risk assessment of recycled materials in roadway construction.

This paper uses a life-cycle assessment (LCA) framework to characterize comparative environmental impacts from the use of virgin aggregate and recycled materials in roadway construction. To evaluate site-specific human toxicity potential (HTP) in a more robust manner, metals release data from a demonstration site were combined with an unsaturated contaminant transport model to predict long-term impacts to groundwater. The LCA determined that there were reduced energy and water consumption, air emissions, Pb, Hg and hazardous waste generation and non-cancer HTP when bottom ash was used in lieu of virgin crushed rock. Conversely, using bottom ash instead of virgin crushed rock increased the cancer HTP risk due to potential leachate generation by the bottom ash. At this scale of analysis, the trade-offs are clearly between the cancer HTP (higher for bottom ash) and all of the other impacts listed above (lower for bottom ash). The site-specific analysis predicted that the contaminants (Cd, Cr, Se and Ag for this study) transported from the bottom ash to the groundwater resulted in very low unsaturated zone contaminant concentrations over a 200 year period due to retardation in the vadose zone. The level of contaminants predicted to reach the groundwater after 200 years was significantly less than groundwater maximum contaminant levels (MCL) set by the US Environmental Protection Agency for drinking water. Results of the site-specific contaminant release estimates vary depending on numerous site and material specific factors. However, the combination of the LCA and the site specific analysis can provide an appropriate context for decision making. Trade-offs are inherent in making decisions about recycled versus virgin material use, and regulatory frameworks should recognize and explicitly acknowledge these trade-offs in decision processes.

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.

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.

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.

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.

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.

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.

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.

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.

A Sensitive Pulse Scheme for Measuring the Backbone Dihedral Angle psi Based on Cross-correlation Between (13)C (alpha)- (1)Halpha Dipolar and Carbonyl Chemical Shift Anisotropy Relaxation Interactions.

A pulse scheme for measuring cross-correlation between 13Calpha-1Halpha dipolar and carbonyl chemical shift anisotropy relaxation mechanisms is presented from which the protein backbone dihedral angle psi is measured. The method offers significant sensitivity gains relative to our recently published scheme for measuring psi based on this cross-correlation effect [Yang et al. (1997) J. Am. Chem. Soc., 119, 11938-11940]. The utility of the method is demonstrated with an application to a 42 kDa complex of 15N,13C-labeled maltose binding protein and beta-cyclodextrin.

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.

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.

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.

An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated (15)N, (13)C-labeled proteins.

A biosynthetic strategy has recently been developed for the production of (15)N, (13)C, (2)H-labeled proteins using (1)H(3)C-pyruvate as the sole carbon source and D(2)O as the solvent. The methyl groups of Ala, Val, Leu and Ile (gamma2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a (15)N, (13)C, (2)H, (1)H(3)C-labeled SH2 domain at 3 degrees C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30-40 kDa molecular weight range.

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.