Engineering of Thermally Stable Proteins and Photo-switchable Proteins

The aim of this project is to develop general approaches to the control of protein structures and functions. The project mainly consists of three aspects: (1) stabilizing folded protein structures, (2) reversible photo-control of protein folding and function, (3) design of a photo-switchable dominant negative protein to photo-control DNA binding. (1) Most proteins adopt specific folded structures to perform biological functions. Stabilizing the active folded forms of peptides or proteins is thus important for maintaining or enhancing the functions of these molecules. We hypothesized that a protein’s folded structure could be stabilized by introduction of a rigid cross-linker with its length matching the distance of the two attachment points in the folded structure. To test this, we synthesized a thiol-reactive alkyne-based rigid cross-linker and tested its effect. When it was introduced at i and i+11 positions of a model α-helical peptide, a significant promotion in the folded structure as well as strong resistance against thermal melting was observed. The rigid cross-linker was also applied to the Fyn SH3 domain, a protein with tertiary structure and a similar stabilization effect was obtained. This work demonstrates that a rigid cross-linker can be generally used to stabilize folded peptide/protein structures. (2) Reversible photo-switch of protein folding/unfolding offers exciting prospects for external manipulation of protein function because of its fast response, high spatial resolution and compatibility with living systems. I developed a general approach to the design of photo-switchable proteins based on the introduction of photo-switchable intramolecular cross-linkers. An azobenzene based photo-switch was used because the energy available from photoisomerization is higher than the free energy of protein folding. I chose the FynSH3 domain as a model protein. Taking the experimentally determined structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the L3C-L29C-T47AFynSH3 mutant was cross-linked with the trans cross-linker, the protein was destabilized so that folded and unfolded forms coexisted. Irradiation of the cross-linker to produce the cis isomer recovered the folded state of the protein. Photo-control of FynSH3 binding to a proline-rich peptide was also demonstrated. This work shows that structure-based introduction of switchable cross-linkers is a feasible general approach for photo-control of global folding/unfolding of globular proteins, and thereby photo-control of their activity. (3) The third aspect of my PhD research is to apply the photo-switchable proteins to photo-control of Jun/Fos DNA binding activity. Fos and Jun are important transcription factors implicated in numerous cancers. They form a hetero-dimer that binds to specific DNA sequences. Dominant negative proteins are mutants of Fos or Jun that prevent native Jun/Fos DNA binding activity. I designed photo-switchable versions of these dominant negative proteins by covalently introducing photo-switchable cross-linkers. These proteins do not have any function in the dark due to their disrupted structures induced by the trans form cross-linkers. They only function as dominant negative proteins when the cross-linker is in the cis form after photo-irradiation. Several such proteins were synthesized and their effectiveness was tested. These photo-switchable dominant negative proteins are powerful tools for temporal control of Jun/Fos regulated gene expression.

Keywords: Protein engineering
chemical biology
Advisor: Woolley, Andrew