Photoinduced unfolding of beta-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.

An anionic porphyrin binds beta-lactoglobulin A at a superficial site rich in lysine residues.

Binding of small ligands to globular proteins remains a major research topic in biophysics. We have studied the binding of several photoactive dyes to beta-lactoglobulin (BLG), as a model to investigate the photoinduced effects of porphyrins on proteins. A combination of optical spectroscopies (fluorescence, circular dichroism) and molecular docking simulations were used to estimate the pH-dependence of the binding parameters and the docking location for meso-tetrakis(sulfonatophenyl)-porphyrin (TPPS). We have observed that the binding of TPPS is not modulated by the pH-mediated conformational transition of the protein (i.e., Tanford transition). Binding of TPPS appears to occur with some degree of negative cooperativity. Moreover, TPPS remains bound even upon partial denaturation of the protein. These results are consistent with a superficial binding site at a location removed from the aperture of the interior beta-barrel. Binding occurs through electrostatic interactions between the negative SO(3) (-) groups of TPPS and positively charged Lys and Arg residues. This is the first study that explores the interaction of an anionic porphyrin with BLGA in a pH range that spans across the Tanford transition. Establishing the location of the binding site will enable us to explain the photoinduced conformational effects mediated by TPPS on BLG.

Irradiation of the porphyrin causes unfolding of the protein in the protoporphyrin IX/beta-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.

Binding of porphyrins to tubulin heterodimers.

We have investigated the binding of two porphyrins, meso-tetrakis ( p-sulfonatophenyl) porphyrin (TSPP) and protoporphyrin IX (PPIX), to tubulin alpha,beta-heterodimers. TSPP had been shown to directly target microtubules in cells. A comparative study between TSPP and PPIX was carried out because the latter is used in clinical applications and is hydrophobic, in comparison with the water soluble TSPP. The results presented in this manuscript show that both porphyrins bind tubulin with nearly identical stoichiometry but with different affinity (1.76 x 10 (5) M (-1) for PPIX; 1.1 x 10 (6) M (-1) for TSPP). The combination of spectroscopic data and molecular simulations suggests that both porphyrins bind as monomers and that their binding site is in proximity of one (or more) Trp residues but do not overlap with the binding site of other well characterized ligands. Molecular simulations also show that the sites that yield the lower energy minima place the porphyrins near the surface of the protein. In the case of TSPP, binding is favored by replacing the ion-dipole interaction of monodispersed TSPP in water with ion-ion interactions provided by two basic residues (His and Lys) at the location of the binding site. Although preliminary, the data show that porphyrin binding could be used to explain some of the effects that photosensitizers may directly produce on protein targets.