An ability to manipulate the activity of a specific protein inside living cells offers exciting prospects for the study of protein function in vivo. Azobenzene derivatives introduced as intramolecular bridges have been demonstrated to reversibly photoregulate secondary structures and functions of peptides and proteins in vitro. My overall goal is to create a generally-applicable process for the reversible photocontrol of protein-protein interactions within the complex environment of a living cell. Results of studies toward this aim are presented.
A blue-green absorbing (~480 nm) azobenzene derivative cross-linker was designed that reversibly controlled the helical content of attached peptides with a half-life of the cis state of ~50 ms. This rapid photoswitch may prove useful as a tool for probing dynamic processes in biochemical systems using light.
The effect of cross-linker position (N-terminus, middle, C-terminus) on a photo-switchable 32-residue helical peptide was studied. Although the activation energies for thermal cis – trans relaxations were not the same, linker position did not affect the change in helix content. This work provides useful information for the effective photoregulation of much longer helices such as occur in coiled-coils.
Fluorescently labeled, cross-linked, modified Fos/Jun peptides with and without cell-penetrating peptide (CPP) tags were prepared for the purpose of photocontrolling peptide-peptide interactions in vivo. One of the peptides showed a degree of photocontrol of helicity. Cell uptake of CPP-tagged peptides was demonstrated. However, overall peptide behavior was dominated by undesired aggregation.
A simple reporter, a cross-linked peptide bearing an environmentally sensitive fluorophore at a key site, was designed for detecting photoswitching in vivo. Photoisomerization of the cross-linker caused changes in the local chemical environment and changes in fluorescence intensity of the environmentally sensitive dyes in vitro. However, no change in fluorescence was observed in the living systems we investigated.
Conclusions and suggestions for further work aimed at achieving the overall goal stated above are discussed.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/26160 |
| Date | 15 February 2011 |
| Creators | Chi, Lei |
| Contributors | Woolley, Andrew |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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