Phage display identifies Affimer proteins that direct calcium carbonate polymorph formation

A key factor in biomineralization is the use of organic molecules to direct the formation of inorganic materials. However, identification of molecules that can selectively produce the calcium carbonate polymorphs calcite or aragonite has proven extremely challenging. Here, we use a phage display approach to identify proteins - rather than the short peptides typically identified using this method - that can direct calcium carbonate formation. A 1.3 × 1010 library of Affimer proteins was displayed on modified M13 phage, where an Affimer is a ≈13 kDa protein scaffold that displays two variable regions of 9-13 residues. The phage displaying the Affimer library were then screened in binding assays against calcite and aragonite at pH 7.4, and four different strongly-binding proteins were identified. The two aragonite-binding proteins generated aragonite when calcium and magnesium ions were present at a 1 : 1 ratio, while the calcite-binding proteins produce magnesium-calcite under the same conditions. Calcite alone formed in the presence of all four proteins in the absence of magnesium ions. In combination with molecular dynamics simulations to evaluate the conformations of the proteins in solution, this work demonstrates the importance of conformation in polymorph control, and highlights the importance of magnesium ions, which are abundant in seawater, to reduce the energetic barriers associated with aragonite formation.