Multimerization of Solution-State Proteins by Anionic Porphyrins

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Protein-protein interactions and formation of multi-subunit protein complexes remain among the most challenging areas of modern Biophysics. Recently, surface binding and interactions of anionic porphyins with cationic proteins gained a lot of attention as relevant models for protein surface molecular recognition and photoinitiated electron transfer. However, interpretation of data in nearly all reports explicitly or implicitly assumed interaction of porphyrin with monodisperse proteins in solutions. Here, using small-angle X-ray scattering with solution phase samples, we demonstrate that horse heart cytochrome (cyt) c, triheme cytochrome c7 PpcA from Geobacter sulfurreducens, and hen egg lysozyme multimerize in the presence of several water-soluble porphyrins. Multimerization of cyt c induced by tetrakis(4-sulfonatophenyl)porphyrin showed a pH dependence with a stronger apparent binding affinity under alkaline conditions and was weakened in the presence of a high salt concentration. Ferric-cyt c formed complexes larger than those formed by ferro-cyt c. Free base TPPS and FeTPPS facilitated formation of complexes larger than those of ZnTPPS. A number of carboxylated porphyrins induced multimer formation as well. No increase in protein aggregation state for cationic proteins was observed in the presence of cationic porphyrins or sulfonated anthraquinone. All-atom molecular dynamics simulations of cyt c and PpcA with free base TPPS corroborated X-ray scattering results and revealed a mechanism by which the tetrasubstituted charged porphyrins serve as bridging ligands nucleating multimerization of the complementarily charged protein. The final aggregation products suggest that multimerization involves a combination of electrostatic and hydrophobic interactions. The results demonstrate an overlooked complexity in the design of multifunctional ligands for protein surface recognition.