Characterization and Control Over Protein Multimerization Induced by Porphyrins

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. However, combined small- and wide-angle X-ray scattering experiments revealed the formation of multimers with a wide range of complex sizes. Binding interactions were explored using wild-type and 12 mutants of PpcA, a 3-heme c-type cytochrome from Geobacter sulfurreducens,with several anionic water-soluble derivatives of tetraphenylporphyrin. Thermodynamic interaction parameters and complex binding stoichiometries were established with isothermal calorimetry.The binding sites of porphyrin were mapped out using HSQC with varying levels of porphyrin concentration to protein. All-atom molecular dynamics simulations revealed quick complex formation with binding sites well matching the areas identified in our experimental work. The obtained results demonstrate that multimerization of solution-state proteins by large water-soluble ligands can be tuned to control shape and size of the formed complexes. 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 of multi-subunit protein complexes as well as targeted disruption of self-assembled complexes.