EMSL: John R. Cort's Publications

Fu N, D Su, JR Cort, B Chen, Y Xiong, W Qian, A Konopka, DJ Bigelow, and TC Squier. 2013. "Synthesis and Application of an Environmentally Insensitive Cy3-Based Arsenical Fluorescent Probe to Identify Adaptive Microbial Responses Involving Proximal Dithiol Oxidation." Journal of the American Chemical Society 135(9):3567-3575. doi:10.1021/ja3117284 Abstract Reversible disulfide oxidation between proximal cysteines in proteins represents a common regulatory control mechanism to modulate flux through metabolic pathways in response to changing environmental conditions. To enable in vivo measurements of cellular redox changes linked to disulfide bond formation, we have synthesized a cell-permeable monosubstituted cyanine dye derivatized with arsenic (i.e., TRAP_Cy3) to trap and visualize dithiols in cytosolic proteins. Alkylation of reactive thiols prior to displacement of the bound TRAP-Cy3 by ethanedithiol permits facile protein capture and mass spectrometric identification of proximal reduced dithiols to the exclusion of individual cysteines. Applying TRAP_Cy3 to evaluate cellular responses to increases in oxygen and light levels in the photosynthetic microbe Synechococcus sp. PCC 7002, we observe large decreases in the abundance of reduced dithiols in cellular proteins, which suggest redox-dependent mechanisms involving the oxidation of proximal disulfides. Under these same growth conditions that result in the oxidation of proximal thiols, there is a reduction in the abundance of post-translational oxidative modifications involving nitrotyrosine and methionine sulfoxide formation. These results suggest that the redox status of proximal cysteines respond to environmental conditions, acting to regulate metabolic flux and minimize the formation of reactive oxygen species to decrease oxidative protein damage.

Isern NG, J Xue, JV Rao, JR Cort, and BK Ahring. 2013. "Novel monosaccharide fermentation products in Caldicellulosiruptor saccharolyticus identified using NMR spectroscopy." Biotechnology for Biofuels 6(4):Article No. 47. doi:10.1186/1754-6834-6-47 Abstract Profiles of metabolites produced by the thermophilic obligately anaerobic cellulose-degrading Gram-positive bacterium Caldicellulosiruptor saccharolyticus DSM 8903 strain following growth on different monosaccharides (D-glucose, D-mannose, L-arabinose, D-arabinose, D-xylose, L-fucose, and D-fucose) as carbon sources revealed several unexpected fermentation products, suggesting novel metabolic capacities and unexplored metabolic pathways in this organism. Both 1H and 13C nuclear magnetic resonance (NMR) spectroscopy were used to determine intracellular and extracellular metabolite profiles. Metabolite profiles were determined from 1-D 1H NMR spectra by curve fitting against spectral libraries provided in Chenomx software. To reduce uncertainties due to unassigned, overlapping, or poorly-resolved peaks, metabolite identifications were confirmed with 2-D homonuclear and heteronuclear NMR experiments. In addition to expected metabolites such as acetate, lactate, glycerol, and ethanol, several novel fermentation products were identified: ethylene glycol (from growth on D-arabinose, though not L-arabinose), acetoin and 2,3-butanediol (from D-glucose and L-arabinose), and hydroxyacetone (from D-mannose and L-arabinose). Production of ethylene glycol from D-arabinose was particularly notable, with around 10% of the substrate carbon converted into this uncommon fermentation product. The novel products have not previously been reported to be produced by C. saccharolyticus, nor would they be easily predicted from the current genome annotation, and show new potentials for using this strain for production of bioproducts.

Merkley ED, ES Baker, KL Crowell, DJ Orton, T Taverner, C Ansong, YM Ibrahim, MC Burnet, JR Cort, GA Anderson, RD Smith, and JN Adkins. 2013. "Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein-Protein Interactions by Chemical Cross-Linking ." Journal of the American Society for Mass Spectrometry 24(3):444-449. doi:10.1007/s13361-012-0565-x Abstract Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides can provide insights into protein structure and protein-protein interactions. However, cross-linked peptides are by necessity of low stoichometry and have different physicochemical properties than linear peptides, routine unambiguous identification of the cross-linked peptides has remained difficult. To address this challenge, we demonstrated the use of liquid chromatography and ion mobility separations coupled with mass spectrometry in combination with a heavy-isotope labeling method. The combination of mixed-isotope cross-linking and ion mobility provided unique and easily interpretable spectral multiplet features for the intermolecular cross-linked peptides. Application of the method to two different homodimeric proteins ‒ SrfN, a virulence factor from Salmonella Typhimurium and SO_2176, a protein of unknown function from Shewanella oneidensis‒ revealed several cross-linked peptides from both proteins that were identified with a low false discovery rate (estimated using a decoy approach). A greater number of cross-linked peptides were identified using ion mobility drift time information in the analysis than when the data were summed across the drift time dimension before analysis. The identified cross-linked peptides migrated more quickly in the ion mobility drift tube than the unmodified peptides.

Cort JR, PJ Alperin, and HM Cho. 2012. "Measurement and analysis of diastereomer ratios for forensic characterization of brodifacoum." Forensic Science International 214(1-3):178-181. doi:10.1016/j.forsciint.2011.08.003 Abstract The highly toxic organic compound brodifacoum exists as two diastereomers. The diasteromer ratio in a sample depends on the methods and conditions used for synthesis and purification, and may vary over time due to differential stability of the diastereomers. The stereoisomer distribution may thus be viewed as a chemical forensic signature, containing information about the production and history of unknown samples, and providing a basis of comparison between samples. A determination of diastereomer ratios can be performed by a number of techniques, notably gas or liquid chromatography or nuclear magnetic resonance (NMR) spectroscopy. An analysis of a cross-section of U.S.-made commercial technical grade brodifacoum material shows that there are detectable manufacturer-to-manufacturer and batch-to-batch variations in diastereomer ratios. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

Snyder DA, JM Aramini, B Yu, Y Huang, R Xiao, JR Cort, R Shastry, LC Ma, J Liu, B Rost, T Acton, MA Kennedy, and G Montelione. 2012. "Solution NMR structure of the ribosomal protein RP-L35Ae from Pyrococcus furiosus." Proteins. Structure, Function, and Bioinformatics 80(7):1901-1906. Abstract The ribosome consists of small and large subunits each comprised of dozens of proteins and RNA molecules several-hundred nucleotides in length. However, the functions of many of the individual protomers within the ribosome are still unknown. Here we describe the solution NMR structure of the ribosomal protein RP-L35Ae from the archaeon Pyrococcus furiosus. RP-L35Ae is buried within the large subunit of the ribosome and belongs to Pfam protein domain family PF01247, which is highly conserved in eukaryotes, present in a few archaeal genomes, but absent in bacteria. The protein adopts a six-stranded anti-parallel beta-barrel analogous to the reductase/isomerase/elongation factor domain fold in SCOP. The structure of the P. furiosus RP-L35Ae presented herein constitutes the first structural representative from this protein domain family.

Yang Y, TA Ramelot, JR Cort, M Garcia, A Yee, CH Arrowsmith, and MA Kennedy. 2012. "Solution NMR Structure of Hypothetical Protein CV_2116 Encoded by a Viral Prophage Element in Chromobacterium violaceum." International Journal of Molecular Sciences 13(6):7354-7364. doi:10.3390/ijms13067354 Abstract Protein domain family YabP (PF07873) is a family of small protein domains that are conserved in a wide range of bacteria and involved in spore coat assembly during the process of sporulation. The 62-residue fragment of Dsy0195 from Desulfitobacterium hafniense, which belongs to the YabP family, exists as a homodimer in solution under the conditions used for structure determination using NMR spectroscopy. The structure of the Dsy0195 homodimer contains two identical 62-residue monomeric subunits, each consisting of five anti-parallel beta strands (b1, 23-29; b2, 31-38; b3, 41-46; b4, 49-59; b5, 69-80). The tertiary structure of the Dsy0195 monomer adopts a cylindrical fold composed of two beta sheets. The two monomer subunits fold into a homodimer about a single C2 symmetry axis, with the interface composed of two anti-parallel beta strands, b1-b1' and b5b-b5b', where b5b refers to the C-terminal half of the bent b5 strand, without any domain swapping. Potential functional regions of the Dsy0195 structure were predicted based on conserved sequence analysis. The Dsy0195 structure reported here is the first representative structure from the YabP family.

Yang Y, TA Ramelot, JR Cort, M Garcia, A Yee, CH Arrowsmith, and MA Kennedy. 2012. "Solution NMR Structure of Hypothetical Protein CV_2116 Encoded by a Viral Prophage Element in Chromobacterium violaceum." International Journal of Molecular Sciences 13(6):7354-7364. doi:10.3390/ijms13067354 Abstract CV_2116 from Chromobacterium violaceum is a small hypothetical protein of 82 amino acids. A PSI-BLAST search using the CV_2116 protein sequence as a query identified only two hits, both with amino acid sequence identities of less than 40%. After the CV_2116 gene was cloned into the p15TvLic expression plasmid and transformed into E. coli, the desired CV_2116 protein was expressed and purified. A high quality solution structure of CV_2116 was determined by NMR spectroscopy. The tertiary structure of CV_2116 adopts a novel alpha + beta fold containing two anti-parallel beta sheets and one alpha helix in the C-terminal end. CV_2116 does not belong to any known protein sequence families and no similar structures exist in the protein data bank. To date, no function of CV_2116 can be derived from either sequence or structural similarity searches.

Barb AW, JR Cort, J Seetharaman, S Lew, HW Lee, T Acton, R Xiao, MA Kennedy, L Tong, G Montelione, and JH Prestegard. 2011. "Structures of Domains I and IV from YbbR are representative of a widely distributed protein family." Protein Science 20(2):396-405. doi:10.1002/pro.571 Abstract YbbR domains are widespread throughout eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Though the precise role of these domains remains undefined, the location of the tetratandem YbbR domain coding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin-type phosphopantetheinyl transferase suggests a role in cell growth, division and virulence. To further characterize the YbbR domains, structures of two of the four domains from the YbbR protein of Desulfitobacterium hafniense DCB-2, were solved by solution nuclear magnetic resonance and x-ray crystallography. The structures show the domains to have nearly identical topologies despite a minimal amino acid identity (23%). The topology, which has just a single representative in the structural database, is dominated by -strands, roughly following a “figure 8” pattern with some strands coiling around the domain perimeter and others crossing the center. The homologous domain in the structural database is the C-terminal domain of a stress-responsive bacterial CTC protein. Based on these models a structurally-guided amino acid alignment identifies features of the YbbR domains that are not evident from naïve amino acid alignments. A structurally conserved cisPro residue was identified in both domains, though the immediate vicinities surrounding this residue differed between the two models. The conservation and location of this cisPro plus anchoring Val residues suggests this motif may be significant to protein function.

Cort JR, M Swenson, and TS Magnuson. 2011. "1H, 13C, and 15N backbone, side-chain, and heme chemical shift assignments for oxidized and reduced forms of the monoheme c-type cytochrome ApcA isolated from the acidophilic metal-reducing bacterium Acidiphilium cryptum." Biomolecular NMR Assignments 5(1):89-92. doi:10.1007/s12104-010-9274-1 Abstract We report the 1H, 13C, and 15N chemical shift assignments of both oxidized and reduced forms of an abundant periplasmic c-type cytochrome, designated ApcA, from the acidophilic gram-negative facultatively anaerobic metal-reducing alpha-proteobacterium Acidiphilium cryptum. These resonance assignments prove that ApcA is a monoheme cytochrome c2 and the product of the Acry_2099 gene. An absence of resonance peaks in the NMR spectra for the 21 N-terminal residues suggests that a predicted N-terminal signal sequence is cleaved. We also describe the preparation and purification of the protein in labeled form from laboratory cultures of A. cryptum growing on 13C- and 15N- labeled substrates.

Yin C, JM Aramini, LC Ma, JR Cort, GVT Swapna, RM Krug, and G Montelione. 2011. "Backbone and Ile-δ1, Leu, Val Methyl 1H, 13C and 15N NMR chemical shift assignments for human interferon-stimulated gene 15 protein." Biomolecular NMR Assignments 5(2):215-219. doi:10.1007/s12104-011-9303-8 Abstract Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins in a variety of ways. Here we report nearly complete backbone 1HN, 15N, 13CO, and 13Ca, as well as side chain 13Cb, methyl (Ile-d1, Leu, Val), amide (Asn, Gln), and indole NH (Trp) NMR resonance assignments for the 157-residue human ISG15 protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically important human ISG15 protein.