Sequence–Structure–Binding Relationships Reveal Adhesion Behavior of the Car9 Solid-Binding Peptide: An Integrated Experimental and Simulation Study

Abstract

Solid-binding peptides (SBPs) recognizing inorganic and synthetic interfaces have enabled a broad range of materials science applications and hold promise as adhesive or morphogenetic control units that can be genetically encoded within desirable or designed protein frameworks. To date, the underlying relationships governing both SBP-surface and SBP-SBP interactions and how they give rise to different adsorption mechanisms remain unclear. Here, we combine protein engineering, SPR characterization, and molecular dynamics (MD) simulations initiated from Rosetta predictions to gain insights on the interplay of amino acid composition, structure, self-association and adhesion modality in a panel of variants of the Car9 silica-binding peptide (DSARGFKKPGKR) fused to the C-terminus of superfolder green fluorescent protein (sfGFP). Analysis of kinetics, energetics and MD-predicted structures shows that the high affinity binding of Car9 to the silanol-rich surface of silica is dominated by electrostatic contributions and a spectrum of several persistent interactions that, along with a high surface population of bound molecules, promote cooperative interactions between neighboring SBPs and higher order structure formation. Transition from cooperative to Langmuir adhesion in sfGFP-Car9 variants occurs in concert with a reduction of stable surface interactions and self-association, as confirmed by AFM imaging of proteins exhibiting the two different binding behaviors. We discuss the implications of these results for the de novo design of SBP-surface binding systems.