Lorenzo Brancaleon

Photo-induced unfolding of β-lactoglobulin mediated by a water soluble porphyrin

We investigated the effects that the irradiation of a tetra-anionic porphyrin (mesotetrakis(sulfonatophenyl)porphyrin) noncovalently bound to beta-lactoglobulin (BLG) produces on the conformation of the protein. Although BLG is not a potential target for the biomedical applications of porphyrins, it is a useful model for investigating the effects of photoactive ligands on small globular proteins. We show in this paper that irradiation causes a large unfolding of the protein and that the conformational change is not mediated by the formation of reactive oxygen species. Instead, our data are consistent with an electron-transfer mechanism that is capable of triggering structural changes in the protein and causes the Trp19 residue to undergo chemical modifications to form a derivative of kynurenine. This demonstrates that protein unfolding is prompted by a type-III photosensitizing mechanisms. Type-III mechanisms have been suggested previously, but they have been largely neglected as useful mediators of biomolecular damage. Our study demonstrates that porphyrins can be used as mediators of localized protein conformational changes and that the biomedical applications as well as the mechanistic details of electron transfer between exogenous ligands and proteins merit further investigation.

Irradiation of the Porphyrin Causes Unfolding of the Protein in the Protoporphyrin IX/β-Lactoglobulin Noncovalent Complex

Porphyrins such as protoporphyrin IX (PPIX) are known to occasionally cause conformational changes in proteins for which they are specific ligands. It has also been established that irradiation of porphyrins noncovalently intercalated between bases or bound to one of the grooves can cause conformational effects on DNA. Conversely, there is no evidence reported in the literature of conformational changes caused by noncovalently bound PPIX to globular proteins for which the porphyrin is not a specific ligand. This study shows that the irradiation of the porphyrin in the PPIX/lactoglobulin noncovalent complex indeed causes a local and limited (approximately 7%) unfolding of the protein near the location of Trp19. This event causes the intrinsic fluorescence spectrum of the protein to shift to the red by 2 nm and the average decay lifetime to lengthen by approximately 0.5 ns. The unfolding of lactoglobulin occurs only at pH >7 because of the increased instability of the protein at alkaline pH. The photoinduced unfolding does not depend on the presence of O2 in solution; therefore, it is not mediated by formation of singlet oxygen and is likely the result of electron transfer between the porphyrin and amino acid residues.

Combination of resonance Raman spectroscopy and docking simulations to study the nonspecific binding of a free-base porphyrin to a globular protein.

Understanding the conformational changes induced by small ligands noncovalently bound to proteins is a central problem in biophysics. We focus on the binding location of the water-soluble porphyrin, meso-tetrakis (p-sulfonatophenyl) porphyrin, to a globular protein, β-lactoglobulin, which has been observed to partially unfold when irradiated by laser light. Identifying the binding location is necessary to determine the mechanism of action as well as the atoms and residues involved in the photoinduced partial unfolding. Such atomic details are typically investigated by nuclear magnetic resonance or X-ray crystallography. However, for biomolecules in solution at the low concentrations (μM) required to deliver uniform laser irradiation, these traditional techniques do not currently provide sufficient information, and one must rely upon less direct spectroscopic methods. We describe a method that uses resonance Raman spectroscopy and density functional theory (DFT) to select the most likely binding configuration among a set of solutions yielded by computational docking algorithms. This methodology may be generalized to use with other ligand-protein complexes where the ligand structure is amenable to DFT simulations.

Partial Unfolding of Tubulin Heterodimers Induced by Two-Photon Excitation of Bound meso-Tetrakis(sulfonatophenyl)porphyrin

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 tubulins 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.