Design and Characterization of SAKe, a Building Block for Protein Self-Assembly.

The nanofabrication of functional protein-based surfaces is challenging due to the chemical complexity of proteins and their unpredictable behavior at the solid-liquid interface. Many proteins of interest-such as antibodies or large enzymatic complexes-lack strong and dynamic protein-protein and protein-surface interactions necessary to drive self-assembly of stable arrays with high surface coverage. Additionally, adsorption-induced conformational changes at the solid-liquid interface could lead to a loss of activity and increase the risk of undesirable interfacial processes. Here we introduce SAKe, a Kelch-like designer protein, as a versatile platform to address these challenges. Ancestral sequence reconstruction led to high thermal stability, and the high symmetry allowed modularity of the protein's core. Rational engineering of the bottom side allowed SAKe to form large (up to 5 μm in length), well-defined and pH-dependent two-dimensional assemblies while maintaining structural integrity, which is key for further development of functional materials. SAKe self-assembly was investigated through in-liquid atomic force microscopy on muscovite mica. High resolution imaging confirmed the integrity of the SAKe protein upon adsorption on the solid-liquid interface. These results showcase the SAKe protein as a platform for the further engineering of functional protein-based two-dimensional materials.