Porphyrin Induced Multimerization of Solution-State Proteins

Abstract

Interactions between charged porphyrins and complimentary or similarly charged proteins provide important models systems for studies of electron transfer processes, artificial photosynthesis, and control of protein-protein interactions. Typically, the experimental results are analyzed and discussed assuming that the proteins exist in a monodisperse state. Here, we explored interaction of four solution-state proteins (horse heart cytochrome c, hen egg-white lysozyme, 3-heme c-type cytochrome PpcA from Geobacter sulfurreducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several cationic and anionic water-soluble derivatives of tetraphenylporphyrin. Combined small- and wide-angle X-ray scattering experiments revealed formation of multimers with a wide range of complex sizes. Thermodynamic interaction parameters and complex binding stoichiometries were established with isothermal calorimetry. Locations of porphyrin binding sites were determined with heteronuclear single quantum coherence (HSQC) and total correlation spectroscopy (TOCSY) NMRs for PpcA and cytochrome c, while covalent labeling shielding experiments followed by LC-MS analysis of tryptic digests were used to map ligand binding sites on cyt c4 and lysozyme surfaces. The obtained results demonstrate that multimerization of solution-state proteins by large water-soluble ligands appears to be a wide-spread phenomenon controlled by a delicate interplay of electrostatic and hydrophobic forces. Molecular level mapping of the binding sites allows us to build a theory explaining the size of the formed complexes and provides opportunities for targeted design and assembly of multi-subunit protein complexes.