We want to understand how proteins fold within the cell. To help solve this problem, 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.
This approach also provides a powerful platform for the discovery of chaperones, the proteins that assist in the folding of other proteins.
Chaperones have traditionally been identified through upregulation in response to stress, such as heat shock. Although this approach has been successful, it does not monitor protein folding directly. Because of this limitation, our understanding of factors governing protein stability in vivo is almost certainly incomplete. Our genetic selection that directly links increased protein stability to increased antibiotic resistance provides an alternate route to chaperone discovery.
Using this this "fold-or-die" selection, we have generated designer bacteria and yeast strains selected for their ability to stabilize proteins. Analysis of these strains has led to the identification of multiple new chaperones.
We have also used this selection to isolate variants of one of our new chaperones, called Spy, that show substantially improved chaperone activity against a variety of substrates. Our super Spy variants generally show tighter binding to client proteins and are more flexible than wild-type Spy. These results provide convincing evidence for the importance of disorder and flexibility in chaperone function.
We are using a variety of biochemical, biophysical, and genetic techniques to exploit these new chaperones to address two fundamental problems in biology: how chaperones affect the folding of their client proteins and how proteins in general can effectively interact with multiple partners.
Grants from the National Institutes of Health provided partial support for these projects.
As of March 23, 2016