The irradiation of the complex formed by meso-tetrakis (sulfonatophenyl) porphyrin (TSPP) and tubulin was investigated as well as its effects on the structure and function of the protein. We have used tubulin as a model target to investigate whether photoactive ligands docked to the protein can affect the structure and function of the protein upon exposure to visible light. We observed that laser irradiation prompts bleaching of the porphyrin which is accompanied by a sharp decrease (∼2 ns) in the average fluorescence lifetime of the protein and a change in the dichroic spectrum consistent with a decrease of helical structure. The result indicated the photoinduced partial unfolding of tubulin. We also observed that such partial conformational change inhibits the formation of microtubules in vitro. We investigated whether photosensitization of reactive oxygen species was responsible for these effects. Even upon removal of O2 the protein still undergoes conformational changes indicating that irradiation of the bound porphyrin does not require the presence of O2 to prompt conformational and functional effects opening the possibility that other mechanisms (e.g., charge transfer) are responsible for the photoinduced mechanism.

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The ability to modify the conformation of a protein by controlled partial unfolding may have practical applications such as inhibiting its function or providing non-native photosensitive properties. A water-soluble porphyrin, meso-tetrakis (p-sulfonatophenyl) porphyrin (TSPP), non-covalently bound to tubulin can be used as a photosensitizer, which upon irradiation can lead to conformational changes of the protein. To fully understand the mechanism responsible for this partial unfolding and determine the amino acid residues and atoms involved, it is essential to find the most likely binding location and the configuration of the ligand and protein. Techniques typically used to analyze atomic position details, such as nuclear magnetic resonance and X-ray crystallography, require large concentrations, which are incompatible with the dilute conditions required in experiments for photoinduced mechanisms. Instead, we develop an atomistic description of the TSPP-tubulin complex using vibrational mode analysis from density functional theory calculations correlated to resonance Raman spectra of the porphyrin paired with docking simulations. Changes in the Raman peaks of the porphyrin molecule correlate with changes in its structural vibrational modes when bound to tubulin. The data allow us to construct the relative geometry of the porphyrin when bound to protein, which are then used with docking simulations to find the most likely configuration of the TSPP-tubulin complex.

The water-soluble porphyrin meso-tetrakis(p-sulfonatophenyl)porphyrin (TSPP) can be noncovalently bound to tubulin and used as a photosensitizer, which upon irradiation triggers photochemical reactions that lead to conformational changes of the protein. These conformational changes in turn inhibit tubulin's primary function of polymerizing into microtubules. We explored the possibility of using two-photon excitation of the bound porphyrin to induce photosensitized protein unfolding. Although TSPP has a relatively low cross section (∼30 GM) our results did find that two-photon excitation of the ligand causes partial unfolding of the tubulin host and the inhibition of the in vitro formation of microtubules. Conversely, irradiating tubulin alone caused no such effects despite the large irradiance per pulse (97-190 GW/cm(2)). The conformational changes were characterized using spectroscopic studies and provide a promising protocol for the future application of non-native photosensitization of proteins.

The formation and the effects of the laser irradiation of the complex formed by protoporphyrin IX (PPIX) and tubulin was investigated. We have used tubulin as a model protein to investigate whether docked photoactive ligands can affect the structure and function of polypeptides upon exposure to visible light. We observed that laser irradiation in the Soret band prompts bleaching of the PPIX which is accompanied by a sharp decrease in the intensity and average fluorescence lifetime of the protein (dominated by the four tryptophan residues of the tubulin monomer). The kinetics indicate non-trivial effects and suggest that the photosensitization of the PPIX bound to tubulin prompts structural alterations of the protein. These modifications were also observed through changes in the energy transfer between Trp residues and PPIX. The result suggest that laser irradiation produces localized partial unfolding of tubulin and that the changes prompt modification of the formation of microtubules in vitro. Measurements of singlet oxygen formation were inconclusive in determining whether the changes are prompted by reactive oxygen species or other excited state mechanisms.

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