Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes.

The work in this laboratory takes a combined theoretical and experimental approach to problems in structural biophysics. Computer simulations play an increasingly important role in our understanding of protein folding, stability, activity, and the specificity of protein-ligand interactions. Design methods are being developed which can be used to rationally modify the structure and function of a protein. This design methodology allows us to ask very specific question firmly based on a theoretical understanding of the system, which can then be put to an experimental test. The experimental work involves molecular biology to construct genes for the designed proteins, protein purification methods and a variety of physical techniques t o study the activity, stability and structure of the designed proteins. Each design goes through several cycles of iterative improvement involving design, analysis, redesign, etc. Empirical improvement methods such as genetic selection are also used where possible.
We have developed and experimentally validated a variety of different computer algorithms that allow us to design biologically active receptors, sensors, and enzymes. This has allowed us to build novel biosensors to detect analytes of clinical (metabolites, drugs), environmental (pollutants), military and homeland defense interest (chemical or biological threats). We have also developed synthetic signal transduction pathways and genetic circuits that enable bacteria to report xenobiotics in their immediate environment via responses triggered with computationally designed receptors ("biological sentinels"). Other applications include the design of novel enzymes, and chemically controlled molecular motors that can be used in bionanotechnology.