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Current Research Biological X-ray Absorption Spectroscopy
Specific XAS applications of interest can be gleaned from a glance at the group publication list. In addition to using XAS to understand structure-function relationships in metalloenzymes (containing, Fe, Co, Ni, Cu, Zn, Mo, etc.), we are particularly interested in the structural characterization of metal (Co, Ni, Zn, Cd, Hg, etc.) and metalloid (As, Sb) binding sites on metallosensors and metalloregulatory proteins. We also have a particular interest in selenobiology, both in studying the local structure around Se in naturally occurring selenoenzymes and in using Se substitution for S in cofactors and substrates as a spectroscopic probe of enzymatic mechanisms. Recent work in technology development includes work on high-throughput X-ray absorption spectroscopy as a tool to characterize the metalloproteome. This term refers to the collection of metalloproteins expressed by a given organism (organ, tissue, cell type) under a given set of environmental conditions. We also have a collaboration to combine structural data from EXAFS with computational chemistry codes to provide additional structural information about biological metal sites. (more…) Discovery and Prediction of Regulatory Transcription Factors
scott@chem.uga.edu |
We use synchrotron
radiation to measure fluorescence-detected X-ray absorption spectroscopy
(XAS) of metals and metalloids in biological macromolecules. With this
technique, we can determine both molecular and electronic structural
details of metal sites in metalloproteins and other biological systems.
As a spectroscopy, XAS determines speciation of metals and metalloids,
giving us information about valence state and molecular symmetry. XAS
is also a structural biology technique (also known as EXAFS or XAFS),
yielding local structural information about the atomic neighborhood
of the metal
or
metalloid
environment, using samples in frozen solution (not single crystals).
It answers the question "How
many of what type of atoms
are at what distance from the metal(loid)?"
This provides us with a local view of the molecular environment surrounding
a selected element and allows us to examine whether a given biochemical
treatment (redox change, addition of substrate or inhibitor, catalytic
turnover) results in a change of the valence or structure at that element.
In a multi-investigator
project, we
are developing an integrative methodology to discover and predict on
a
genomic scale
regulatory transcription factors and their DNA binding sites (operators).
Currently, we are applying this methodology to prokaryotic organisms
(Pyrococcus furiosus, a hyperthermophilic marine archaeon,
and Shewanella
oneidensis, a metal-reducing bacterium) to provide a global view
of how these microorganisms respond to environmental stress. Our collaborators
provide DNA microarray expression profile results detailing the response
of the organism to a particular stress (e.g., cold-shock, redox stress,
heavy metal stress, etc.). Together, we choose highly regulated genes
and use DNA upstream of those genes to pull down sequence-specific
DNA-binding proteins from cell extract obtained from the microarray
conditions. DNA-binding proteins that are differentially present in
cell extracts from stressed vs. normal conditions are candidate regulatory
transcription factors and are identified by mass spectrometry proteomic
techniques. We then clone, express, and purify these prospective regulators
and characterize their DNA-binding sites. Bioinformatics is used to
identify other similar binding sequences as components of regulons.
Functional validation using cell-free transcription will allow us
to construct transcriptional regulatory networks for the organisms.