We propose to develop sulfur K-edge X-ray absorption near-edge spectroscopy so that it can be used to monitor and to improve our understanding of sulfur metabolism in living systems, and especially in mammalian cells. There are currently no effective spectroscopic probes of sulfur in biological systems. X-ray absorption spectroscopy will detect all chemical forms of sulfur in a sample, in solid, in aqueous solution or in any other form, and different forms of sulfur display strikingly different spectra. Under favorable conditions we can therefore quantitatively discriminate between different forms of sulfur in biological samples. No pre-treatment is needed and the technique is at least potentially non-destructive. The ultimate goal of the work we propose is to provide an in vivo probe of metabolic status in healthy and diseased tissue. The proposed work can be divided into two major categories: 1. Bulk measurements, where a large X-ray beam is used to interrogate samples to provide the average sulfur speciation. We propose to study the sulfur biochemistry of living mammalian cell cultures, and specifically the responses of polarized epithelial cultures to osmotic shock, changes during cell differentiation, apoptosis, and responses associated with mechanical stimulation of the cells.2. Chemically-specific imaging, where we propose to develop sulfur K-edge X-ray absorption spectroscopy imaging as a tool for investigating the localization of different chemical forms of sulfur in intact living cells. In this technique X-ray micro-focusing optics are used to produce a very small beam, and the sample is raster scanned using carefully chosen incident X-ray energies to discriminate specific categories of compounds.Our initial studies will focus on plant cells, because of their large size and high sulfur content. As the resolution of the technique and the experimental methodology improve, we will apply the method to more challenging systems, with the ultimate goal of a 1 pm resolution probe that can be applied to mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM057375-04A1
Application #
6579300
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Carter, Anthony D
Project Start
1998-08-01
Project End
2005-11-30
Budget Start
2002-12-15
Budget End
2003-11-30
Support Year
4
Fiscal Year
2003
Total Cost
$275,616
Indirect Cost
Name
Stanford University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Pickering, Ingrid J; Sneeden, Eileen Yu; Prince, Roger C et al. (2009) Localizing the chemical forms of sulfur in vivo using X-ray fluorescence spectroscopic imaging: application to onion (Allium cepa) tissues. Biochemistry 48:6846-53
George, Simon J; Drury, Owen B; Fu, Juxia et al. (2009) Molybdenum X-ray absorption edges from 200 to 20,000eV: the benefits of soft X-ray spectroscopy for chemical speciation. J Inorg Biochem 103:157-67
George, Graham N; Gnida, Manuel; Bazylinski, Dennis A et al. (2008) X-ray absorption spectroscopy as a probe of microbial sulfur biochemistry: the nature of bacterial sulfur globules revisited. J Bacteriol 190:6376-83
Rigby, Kevin; Zhang, Limei; Cobine, Paul A et al. (2007) characterization of the cytochrome c oxidase assembly factor Cox19 of Saccharomyces cerevisiae. J Biol Chem 282:10233-42
Gnida, Manuel; Sneeden, Eileen Yu; Whitin, John C et al. (2007) Sulfur X-ray absorption spectroscopy of living mammalian cells: an enabling tool for sulfur metabolomics. In situ observation of uptake of taurine into MDCK cells. Biochemistry 46:14735-41
George, Graham N; Doonan, Christian J; Rothery, Richard A et al. (2007) X-ray absorption spectroscopic characterization of the molybdenum site of Escherichia coli dimethyl sulfoxide reductase. Inorg Chem 46:2-4
Liu, Tong; Ramesh, Arati; Ma, Zhen et al. (2007) CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator. Nat Chem Biol 3:60-8
Popescu, Bogdan F Gh; Pickering, Ingrid J; George, Graham N et al. (2007) The chemical form of mitochondrial iron in Friedreich's ataxia. J Inorg Biochem 101:957-66
Coyne 3rd, H Jerome; Ciofi-Baffoni, Simone; Banci, Lucia et al. (2007) The characterization and role of zinc binding in yeast Cox4. J Biol Chem 282:8926-34
Doonan, Christian J; Kappler, Ulrike; George, Graham N (2006) Structure of the active site of sulfite dehydrogenase from Starkeya novella. Inorg Chem 45:7488-92

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