Structural Genomics of Eukaryotic Model Organisms
EMSL Project ID
2327
Abstract
We have recently received a NIH Center Grant in Structural Genomics establishing the Northeast Structural Genomics Consortium for evaluating the feasibility, costs, economies of scale, and value of structural genomics. The primary goal of our pilot project is to develop integrated technologies for high-throughput (htp) protein production and 3D structure determination. These will form key components of the scientific infrastructure required for the next stage of genome research beyond DNA sequencing. The primary genome targets for this methodology development are eukaryotic model organisms which are subjects of extensive functional genomics research, including S. cerevisiae, C. elegans, and D. melanogaster, as well as homologues from the human genome. Within these genomes, the pilot project focuses on proposed open reading frames (ORFs) encoding phylogenetically conserved polypeptide chains of < 340 amino acids with no predicted 3D structures. While these targets will be identified in genomes of these eukaryotic model organisms, in some cases it will be more practical (or more interesting) to express and study the corresponding prokaryotic or human homologues. Our hypothesis is that these smaller proteins are more suitable for a pilot structural genomics project because: i) they will tend to be single-domain proteins that may be easier to express in bacterial expression systems than are large multidomain proteins; ii) they can be rapidly screened for foldedness using 2D 15N-1H heteronuclear single-quantum correlation spectroscopy (HSQC); iii) they may be easier to crystallize than large multidomain proteins; and iv) their smaller size makes them better suited for the development of automated methods of structure analysis. All of the expressed and purified targets are screened by NMR and other biophysical methods to determine feasibility for structure analysis, and put through initial htp crystallization trials. Criteria have been established for assigning particular targets for crystallographic or NMR structure determination. Proteins with high feasiblity and biological interest scores are prioritized for NMR data collection and structural analysis. Some 100 proteins have been cloned and expressed to date from the genome of C elegans. Conditions have been identified for 40 of these that provide high level expression and solubility. Instrument time is requested to collect some of the triple resonance data required for determining resonance assignments and NOESY data required for determining 3D structures for five of these proteins over the next six months. In recent months we have collected triple resonance and NOE data at PNNL on the proteins WR4, WR41, WR90Ec and WR64Tt that has contributed to their structure determinations. In addition to collecting additional NMR data to refine and extend these structure determinations, we would like to begin data collection efforts on additional proteins WR33 and WR73. Sequence data and HSQC spectra for these six proteins are available from the SPINE database (http://bioinfo5.mbb.yale.edu/spine/sum.php3) of the Northeast Structural Genomics Consortium.
Project Details
Project type
Capability Research
Start Date
2001-10-01
End Date
2002-11-18
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
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Wang L, X Wang, J Luo, BN Wanjala, CM Wang, N Chernova, MH Engelhard, Y Liu, IT Bae, and CJ Zhong. 2010. "Core-Shell Structured Magnetic Ternary Nanocubes." Journal of the American Chemical Society 132(50):17686-17689. doi:10.1021/ja1091084
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