Systems Biology in Prokaryote - Eukaryote Symbiosis

Monday, June 25, 2012 - 12:00pm

Allen M. Orville, Brookhaven National Laboratory

Frontier challenges for macromolecular crystallography (MX) now include determining structures of trapped reactive intermediates, large macromolecules and viruses, membrane proteins, protein-protein complexes, and protein-nucleic acid complexes. Although structure and function are intimately linked, knowledge of the former does not necessarily provide certainty of the latter. Therefore, many of these frontier questions will require more than simply atomic coordinates to answer. Indeed, the “structure-function” relationship derives from the combination of atomic, electronic and vibrational structures; but, information about the latter two are not accessible by diffraction methods. Fortunately, spectroscopic methods provide data to help resolve uncertainties inherent in the interpretation of electron density maps, especially when such data are collected from the same sample and similar conditions.

According to BioSync (biosync.sbkb.org) there are over 130 beamlines at synchrotrons worldwide devoted to structural biology. However, beamline X26-C of the National Synchrotron Light Source (NSLS) is perhaps the only one in the world devoted full-time to collecting single-crystal spectroscopic data correlated with X-ray diffraction data. We integrated optical spectroscopy instruments into the beamline in order to collect spectroscopic data during the X-ray diffraction data collection. Electronic absorption data (typically 250 – 850 nm) is collected from a 25 μm diameter area of the crystal that is coincident with the X-ray beam. We also collect off-resonance and resonance Raman spectroscopic data from the same 25 μm diameter region of the crystal with either 785 nm, 532 nm or 473 nm laser excitation.  Thus, we integrate three types of data (two spectroscopic and X-ray diffraction) and collect these complementary datasets, nearly simultaneously, from the same sample and under the same experimental conditions. Several examples will be discussed to highlight the applications of the methods.

To leverage these capabilities further, we are creating a high throughput pipeline for the structural and biophysical analysis of macromolecules involved in bacterial N2-fixation in plants. The bacteria Sinorhizobium meliloti 1021 and WSM419 are free-living or N2-fixing microbes. But, they only fix N2 under symbiotic, microaerobic conditions within root nodules of legumes such as alfalfa and its diploid model, Medicago truncatula. The genome sequences of S. meliloti and M. truncatula are known. Scientists from BNL, WA State Univ. (M. Kahn et al), Pacific Northwest National Lab (M. Lipton et al), Stanford Univ. (S. Long et al), the City Univ. of New York (H. Chen et al) and the NY Structural Genomics Research Consortium (S. Almo et al) collaborate to better understand this symbiotic relationship. The initial targets in the pipeline include S. meliloti ORFs annotated as proteins that bind either iron (~144 ORFs), heme (63 ORFs), copper (28 ORFs), or is an oxidoreductase (535 ORFs). They are being characterized by small/wide angle X-ray scattering and by X-ray (micro)crystallography that is often correlated with single-crystal spectroscopy from the same crystal. In complementary studies, whole root nodules have been analyzed by mass-tag metabalomic and microproteomic analysis, as well as by microprobe X-ray fluorescence. Together these results provide the identity and relative abundance of bacterial and plant proteins, as well as the total distribution of first row transition metals in N2-fixing root nodules.

Supported by NIH/BTRC and USA DOE/BER

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