Xanthomonas campestris Structural Genomics
Collaboration with Prof. Shan-Ho Chou,
Institute of Biochemistry, National Chung Hsing University.
The flood of sequence information available from the various genome projects coupled with the recent advances in molecular and structural biology has led to the concept of structural genomics on a genome-wide scale. Determination of the three-dimensional structures of proteins is critical for understanding the biological basis of the protein molecules. In this context, ¡¥structural genomics¡¦ is expected to pave way for understanding the intricate interactions among proteins in the whole organisms.
Xanthomonas campestris is a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. However, it also produces an exopolysaccharide called xanthan that is of great industrial importance. Owing to the immense economic impact and the simplicity of the genetic constitution (lacking introns) of this organism, we envisage, in the present proposal, to identify and characterize novel protein structures and functions in Xanthomonas campestris. Our genome sequencing team will first use shotgun approach to sequence the whole genome of Xanthomonas campestris that contains approximately five million base pairs to allow bioinformatics team to identify and annotate the genome sequence. Such annotation is necessary to serve as the ground for protein expression team (using PCR to amplify the genes or using the clones provided by genome sequencing team) to engineer efficient vectors for expressing gene products in high yield and to purify and analyze their functions. After samples are ready, the structure team will then use high resolution NMR methodology or X-ray diffraction methodology to determine the solution structures of novel target proteins. To circumvent the problem of short T2 relaxation and broad linewidth in large proteins, we plan to employ a variety of Transverse Relaxation-Optimized Spectroscopy (TROSY) technique to optimize the resolution. We will further use cryoprobe to accelerate NMR data collection so that we can collect enough data in a short time to solve approximately ten protein structures in a year. We will also use autoassign program to help assign the multidimensional spectra that is the most time-consuming part in protein structural determination. Autostructure determination program will also be used to accelerate the whole process. In addition, we intend to obtain high precision structures of the target proteins using the now popular dipolar coupling restraints. Attempts would also be made to identify potential binding substrate(s) for the target proteins using the SAR by NMR approach. The final goal of this coordinated project is to obtain a structural database of Xanthomonas campestris containing all structural and function annotation. We believe that successful execution of this project will not only promote academic excellence (to find novel proteins with novel folds), but also serve industrial and agricultural need (to prevent black rot disease and to provide xanthan gum). It will promote the academic status of Taiwan and provide personnel Taiwan desperately need in the biotechnology sector to advance into the 21-century.