Developing new protein-based materials with a programmable assembly is of great scientific interest and technological importance. Inspired by nature's use of posttranslational modifications (PTMs) to control the function and location of proteins, we have leveraged lipidationthe PTM of proteins with lipidsto synthesize genetically encoded lipidated proteins with controllable hierarchical assembly. Specifically, we envisioned the combination of two orthogonal lipidation pathways with different regioselectivity and substrate preferences inside Escherichia coli to produce recombinant nanomaterials with distinct lipidation domains at each terminus of proteins. In this study, we demonstrate the orthogonality of N-myristoylation and C-cholesterylation pathways for recombinant production of lipidated proteins with a unique triblock architecture, which is a hydrophilic protein block flanked by two lipid tails, i.e., inverse bolaamphiphiles. Our study indicates that the architecture of lipidated protein and the sequence of the polypeptide can be used to control the hierarchical self-assembly of these materials. We envision this bio-enabled approach yielding unexplored recombinant hybrid biomaterials with tunable nanoscale structure and morphology with applications in nanobiotechnology.

Post-translational modification of proteins is widely used in biological systems and plays an important role in modifying the function of polypeptides. The eukaryotic N-myristoyl transferase (NMT) genetically incorporates myristic acid at the N-terminus of proteins. The C-terminal domain of a hedgehog protein (HhC) can attach a cholesterol moiety to the C-terminal of the polypeptide by cleaving itself. • Elastin-like-polypeptides (ELP) are a class of proteins with rapidly increasing importance due to their unique properties. By having a lower critical solution temperature (LCST), they exhibit temperature triggered hierarchical self-assembly.