Improved Ordering in Low Molecular Weight Protein-Polymer Conjugates Through Oligomerization of the Protein Block

Due to the enhanced stability of small affinity binders such as nanobodies and engineered binding proteins, there is a growing desire to incorporate these materials into biosensors. Performance in these biosensors is strongly linked to protein density and accessibility of binding sites. While our group has previously demonstrated the ability to create arrays of densely-packed, well-oriented proteins by conjugating these proteins to polymers, the ordering with low molecular weight protein blocks is poor. Here, we demonstrate that oligomerization of small proteins significantly improves ordering in protein-polymer conjugates due to an increase in the overall molar mass of the conjugate. A series of four oligomers—monomer to tetramer—of a small binding protein are synthesized by connecting proteins via a short, flexible peptide linker. Small-angle X-ray scattering (SAXS) measurements reveal that while conjugates of the monomer exclusively exist in disordered states, oligomer conjugates self-assemble into long-range ordered lamellae. As the protein degree of oligomerization is increased, the concentration range over which strong ordering is observed is broadened. Binding assays indicate that activity is retained with oligomerization.