Optimization of Protein Stability
We want to understand why proteins are often so unstable and how proteins fold within the cell. To help solve these problems, we have devised folding biosensors that link protein folding to antibiotic resistance. These biosensors give us a quantitative and selectable in vivo assay for protein stability. Under antibiotic selection, only stabilized variants grow, making the biosensors an extremely powerful selection for stabilizing proteins. Our approach selects for increased protein stability without selecting for protein function, providing an opportunity to separate the contributions of these contrasting forces in the evolution of proteins.
The folding pathways of various model proteins are well studied in vitro, but little is known about folding pathways in vivo. We use our biosensors to study and optimize in vivo folding pathways. We have developed biosensors specific for disulfide isomerization and used them to evolve alternative pathways that work in the absence of the normal machinery for disulfide isomerization.
An Acid-Regulated Chaperone
We also study the mechanism of action of a very small chaperone, HdeA. This 9.7-kDa protein partially unfolds at low pH and becomes active as a protein-folding helper. HdeA helps protect proteins in bacteria from the precipitous drop in pH that occurs when they are exposed to stomach acid. The flexibility gained through partial unfolding allows it to adapt to and bind various damaged proteins, protecting them from aggregation and proteolysis much like plastic wrappers stop hard candies from sticking together. HdeA, which exploits the energy from extracellular pH changes to facilitate protein folding, releases its substrate proteins slowly. This keeps the concentration of aggregation-sensitive intermediates below the aggregation threshold, providing a straightforward and ATP-independent mechanism to facilitate protein refolding.
As of May 30, 2012