Our project goal is to improve gene/protein functional assignments through evaluation of various high throughput experimental techniques to screen for ligand binding to proteins. We are selecting a diversity of protein families to characterize such as ABC-type transporters, transcription factors, two component sensor systems, and enzymes.

Our applications for protein-ligand mapping focus on organisms that have potential metabolic capabilities which can be leveraged as part of the DOE missions to develop novel sources of biofuels and utilize bacteria for bioremediation.

All cells contain proteins that sense the environment and mediate transport and signaling events that lead to changes in metabolism and/or initiate changes in gene expression at the level of transcription. Mapping of ligands with their binding proteins is critical to our understanding of cell biochemistry, modeling of cellular processes, which begins with the specific functional annotation of these proteins.

Current functional descriptions of proteins primarily depend on the increasingly available genome data from sequencing projects. Unfortunately, due to the extensive diversity and versatility of many proteins, the determination of specific function through a sequence based approach alone is often insufficient.  While, sequence homology is cost-effect and can provide a prediction of protein function for 50-70% of the ORFs in newly sequenced organisms, many of these predictions are very general in nature or misleading.  Additionally, this approach cannot assign function to novel proteins, a handicap that severely limits its ability to contribute to the task of functional annotation. Experimental approaches are able to provide more specific functional characterization but are often expensive and lower throughput.  In the final analysis, a comprehensive approach will require the application of both computational and experimental methods to improve the extent of specific functional annotation for proteins.

Experimental Approach:  We employ a target-independent fluorescence-based thermal shift (FTS) assay, which uses a fluorescent dye to monitor protein unfolding, for the assessment of ligand binding. The basis of the assay is altered protein stability that often accompanies ligand binding. This assay uses a commercially available real-time PCR instrument where thermal melting curves of the protein/ligand combinations can be screened in a 96-well plate format. Read more....