Due to the enhanced stability and ease of production of small high-affinity binding proteins, there is a growing desire to incorporate these proteins into biosensors. Sensitivity of these devices is strongly correlated with high protein density and complete 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. Recent work has established a clear effect of protein molecular weight on protein-polymer conjugate ordering quality: the strongest ordering is observed in conjugates with protein block molecular weights around 29 kDa. Thus, exploring approaches to increase the molecular weight of the protein block in protein-polymer conjugates may yield methods to create well-ordered structures of these conjugates incorporating a low molecular weight protein.<br/><br/>In this work, we demonstrate that oligomerization of small proteins significantly improves ordering in protein-polymer conjugates. A series of four oligomers—monomer to tetramer—of a small binding protein are synthesized by connecting the proteins via a short, flexible peptide linker. Each oligomer is engineered to contain an N-terminal cysteine residue, providing a thiol group to which a maleimide-functionalized poly(<i>N</i>-ispropylacrylamide) (PNIPAM) block is conjugated using a thiol-Michael addition. 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 quality of ordering is greatly enhanced. It is further demonstrated that the oligomer conjugates order significantly better than previously-studied protein-polymer conjugates, and it is hypothesized that this improved ordering results from the greater flexibility of the oligomer protein domains.

Effect of protein oligomerization on self-assembly in protein-polymer conjugates

256th National Meeting (2018)

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