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.

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.

Photocontrol, the use of light-sensitive proteins to control events within living tissue, allows complex processes in higher organisms to be studied. The Halorhodospira halophila photoactive yellow protein (PYP) can be used to regulate transcription factor activity with blue light. Before any PYP-based system can probe complex processes in higher organisms, proof of functional expression in vivo is required. We linked d25 PYP to the C-terminus of blue fluorescent protein (BFP) and expressed variants of the fusion protein (BFPd25PYP) in E. coli and human embryonic kidney (HEK293) cells. Expression of BFPd25PYP in E. coli verified in vitro photoswitching. The fusion protein was successfully expressed in HEK293. Fluorescence studies of intact cells indicated chromophore uptake and incorporation into PYP in HEK293, while photoswitching of PYP was measured in protein isolated from HEK293. These findings are promising for the development of applications using PYP for in vivo mammalian photocontrol of biological events.

Photocontrol, the use of light-sensitive proteins to control events within living tissue, allows complex processes in higher organisms to be studied. The Halorhodospira halophila photoactive yellow protein (PYP) can be used to regulate transcription factor activity with blue light. Before any PYP-based system can probe complex processes in higher organisms, proof of functional expression in vivo is required. We linked d25 PYP to the C-terminus of blue fluorescent protein (BFP) and expressed variants of the fusion protein (BFPd25PYP) in E. coli and human embryonic kidney (HEK293) cells. Expression of BFPd25PYP in E. coli verified in vitro photoswitching. The fusion protein was successfully expressed in HEK293. Fluorescence studies of intact cells indicated chromophore uptake and incorporation into PYP in HEK293, while photoswitching of PYP was measured in protein isolated from HEK293. These findings are promising for the development of applications using PYP for in vivo mammalian photocontrol of biological events.

Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.

Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.