Correlating Solid-Binding Peptide Structure with Biomimetic Function

Abstract

While much progress has been made in elucidating the mode of action of biomineralizing proteins and in emulating their function using combinatorially selected solid-binding peptides (SBPs), we remain far from duplicating the sophisticated architectures produced by biological systems. This is in part due to a poor understanding of the relationship between SBP conformation and inorganic adhesion and precipitation. This dissertation focuses on correlating the structure of Car9, a SiO2/TiO2-binding dodecapeptide originally isolated for its ability to recognize the edges of graphitic nanostructures, with silica binding affinity and titania precipitation activity. We start by quantifying the binding kinetics of a fusion protein between superfolder green fluorescent (sfGFP) and the Car9 SBP to silica using surface plasmon resonance (SPR) measurements, develop a two-step kinetic model that accurately captures the unique cooperative adhesion behavior of the fusion protein, and demonstrate that elimination of multiple basic residues converts the binding to a Langmuir process. Using a panel of mutants in the Car9 segment, we combine SPR characterization and molecular dynamics simulations initiated from Rosetta structural predictions to gain insights on the interplay of amino acid composition, structure, and adhesion modality. We show that the high affinity binding of Car9 to silica stems from persistent electrostatic interaction with the surface that, along with high surface coverage, promotes cooperative interactions between neighboring SBPs. Additionally, we ascribe the transition to Langmuir adhesion to the loss of two or more such contacts due to mutagenesis. Finally, we show mutations in the Car9 extension biases the crystallinity of titania bioprecipitated by sfGFP-Car9 fusion proteins between anatase and TiO2 (B) phases and harness our knowledge of SiO2 adhesion to interpret these results. Collectively, our results provide a roadmap for developing a fundamental understanding of SBPs’ inorganic adhesion and offers insight in how to intuitively design peptide-protein scaffolds to produce unique composite materials for applications ranging from biomedical engineering to catalysis.