Coiled-Coil Domains for Self-Assembly and Sensitivity Enhancement of Protein–Polymer Conjugate Biosensors

While protein self-assembly has been used to improve the performance of biosensors and biocatalysts, few general techniques have been demonstrated to achieve well-ordered protein arrays under the highly concentrated conditions required to optimize device performance. The strong association strength of coiled-coils, supercoiled bundles of alpha helices wrapped around each other, makes these structures promising scaffolds from which proteins can be linked into ordered assemblies in concentrated solution. Herein, the two component alpha helices of a heterodimeric coiled-coil are fused to a weakly ordering protein, 2x rcSso7d.SA (2x), to improve its self-assembly as a protein–poly(N-isopropylacrylamide) (PNIPAM) conjugate. Three fusion protein conjugates are developed, from which two distinct heterodimeric coiled-coil conjugates are formed, but only one of these heterodimer conjugates significantly improves the ordering quality of 2x. Additionally, the alpha helical segment in one of the three synthesized fusion protein conjugates remains almost entirely unfolded, and this conjugate is found to achieve both the strongest ordering quality and over an order of magnitude enhancement in biosensor performance compared to unmodified 2x. These findings suggest that certain arrangements of coiled-coil fusions produce well-ordered protein–polymer conjugates, and unfolded coiled-coil sequences may also serve as effective order-inducing protein sequences.