(165j) Synthesis of Active Fusion Protein Nanosheets

Two dimensional nanosheets with thickness of less than 100 nm have been synthesized from inorganic and organic materials including graphene oxide, biomimetic peptoids, and membrane proteins. In this work we report for the first time the self-assembly of protein nanosheets from active, globular proteins. These protein nanosheets were synthesized from mixtures containing two fusion proteins. The hydrophobic fusion protein contains an elastin-like polypeptide (ELP) flanked by two arginine-rich leucine zippers (Z_R) and the hydrophilic fusion protein contains a globular, functional protein (i.e. model fluorescent protein, mCherry) that is fused to a glutamate-rich leucine zipper (Z_E). The leucine zippers link one globular, hydrophilic domain to each end of the hydrophobic ELP creating amphiphiles that self-assemble into nanosheets upon end-over-end rotation. The assembly of the amphiphiles into nanosheets is dependent on the expansion and contraction of the air-water interface that occurs due to the end-over-end rotation of the solution. To investigate parameters that effect nanosheet assembly, the rotation speed, Z_E/Z_R ratio, and globular protein identity were varied. The results demonstrate that the formation of nanosheets is dependent on speed of rotation and the fill fraction of the container because these parameters both effect the degree of expansion and contraction of the air-water interface. Importantly, they also demonstrate that nanosheets can be loaded with a Additionally, we show for the first time that bioactivity can be easily incorporated into the nanosheets by synthesizing nanosheets from globular enzymes without the use of covalent chemistry. To demonstrate the self-assembly of nanosheets from active enzymes, nanosheets were formed from the enzymes human carbonic anhydrase II and E. coli malate synthase G. The enzymes retained approximately 80% of their respective activities. The high loading capacity of nanosheets along with their ability to incorporate bioactivity makes them a valuable material for use as biosensors or biocatalytic supports.