Polarized XANES of Co(III)(NH3)6 molecular crystals

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27914/1/0000337.pd

Polarized XANES of iron porphyrins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27901/1/0000321.pd

Determination of the chemical environment of sulphur in petroleum asphaltenes by X-ray absorption spectroscopy

Sulphur K-edge X-ray absorption spectra are analysed for a diverse series of petroleum asphaltenes. The spectra of the asphaltenes are interpreted by fitting with a linear superposition of model compound spectra. This analysis procedure is shown to work quite well in a stringent test case. The reduced forms of sulphur, thiophenes and sulphides dominate in all of the asphaltenes. Generally, the sulphoxide group is the most dominant form of the oxidized sulphur. Comparison of the X-ray data with the elemental composition of the asphaltenes shows that the sulphur and oxygen are preferentially bonded to each other. The inverse dependence of the oxygen-sulphur correlation with the fraction of sulphur which is sulphide suggests that oxidation of sulphur occurs preferentially at the sulphide group.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30280/1/0000681.pd

Characterization of the Mn sites in manganese redox proteins: Mn catalase and the photosynthetic oxygen evolving complex.

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29205/1/0000259.pd

The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis

Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse® were well correlated with sonication. Two other methods, Bugbuster® and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability

Sulfidation of organic matter associated with gold mineralization, Pueblo viejo, Dominican republic

The Pueblo Viejo district is one of the largest producers of precious metals in the world, yielding more than 11,000 kg of Au annually. Gold mineralization at Pueblo Viejo is hosted in spilite, and coarse clastic and finely laminated, fine grained carbonaceous sedimentary rocks of the Lower Cretaceous Los Ranchos Formation. Mineralization was accompanied by sulfidation as evidenced by (1) the occurrence of siderite distal to mineralization and pyrite proximal to mineralization, (2) increased S/Fe ratios associated with Au mineralization, (3) the occurrence of native S in and adjacent to mineralization, and (4) the presence of sulfidized organic matter (organo-S compounds) in mineralized rocks. Organic matter in the carbonaceous sedimentary rocks comprises vitrinite and pyrobitumen. Rock-Eval pyrolysis data indicate that this organic matter is overmature (HI 2S in the mineralizing fluid would have destabilized Au bisulfide complexes and caused deposition of gold. The restriction of S-rich organic matter to rocks in which all Fe occurs as pyrite indicates that sulfidation of organic matter postdates sulfidation of ferrous Fe and therefore, deposition of much of the Au.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28847/1/0000682.pd

Subfamily-Specific Adaptations in the Structures of Two Penicillin-Binding Proteins from Mycobacterium tuberculosis

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis

Sulfur speciation in heavy petroleums: Information from X-ray absorption near-edge structure

The chemical speciation of sulfur in heavy petroleums, petroleum source rock extracts, and source rock pyrolysis products was studied using X-ray absorption near-edge structure (XANES) spectroscopy. The good energy resolution (ca. 0.5 eV) at the sulfur K edge and the strong dependence of XANES on the sulfur environment combine to give excellent sensitivity to changes in the electronic and structural environment of the sulfur. This has permitted identification and approximate quantitation of different classes of sulfur-containing compounds (e.g., sulfur, sulfides (including disulfides and polysulfides as a group), thiophenes, sulfoxides, sulfones, sulfinic acids, sulfonic acids, and sulfate) in a series of petroleums and petroleum source rocks. Our results indicate that the sulfur speciation of geological samples can be correlated with differences in source depositional environment, thermal maturity, and aromaticity. We report organosulfur compositions for the asphaltene, maltene, and liquid Chromatographie fractions of two sulfur-rich oils. In addition, we find that the organosulfur species in some, but not all, oils are subject to oxidation upon storage and thus may also be susceptible to oxidation in shallow reservoirs exposed to oxic waters. This work illustrates the utility of XANES as a direct spectroscopic probe for the quantitative determination of sulfur species in geological samples.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29445/1/0000527.pd

Characterization of the manganese sites in manganese redox enzymes: Catalase and superoxide dismutase.

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30701/1/0000346.pd

New Molecular Reporters for Rapid Protein Folding Assays

The GFP folding reporter assay [1] uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility [2]–[8], but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites [9]. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding [10]–[12]. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter [1] and the robustly-folding “superfolder” GFP [13]. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37°C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites