Date of Award
Doctor of Philosophy (PhD)
Genetically encoded, Lipidation, Phase behavior, Post-translational modifications, Protein polymers, Self-assembly
Post-translational modification (PTM) of protein polymers is emerging as a powerful bioinspired strategy to create protein-based hybrid materials with molecularly encoded assembly and function for applications in nanobiotechnology and drug delivery systems. Inspired by nature's use of PTMs to control the location and function of proteins, we have leveraged lipidation to synthesize genetically encoded lipidated proteins with controllable hierarchical assembly. We investigated the effect of changing molecular syntax (lipid type, lipidation site, proteins' length and sequence) of lipidated proteins on their self-assembly and phase behavior. The repertoire of canonical lipids in biology is constrained as the lipidation machinery catalyzes the transfer of only a few types of lipids to recognized substrates at one terminus of proteins, limiting the design space of available lipoproteins. To address this issue, we used the substrate promiscuity of N-myristoyl transferase (NMT) to incorporate an artificial lipid into lipoproteins using a non-canonical PTM (ncPTM). The non-natural functionality of these artificial lipoproteins results in a distinctive temperature-triggered assembly—absent from the canonical lipoproteins— and can be used to prepare hybrid bolaamphiphiles with tunable nano-assembly. The study demonstrates the promise of expanding the repertoire of PTMs into developing nanomaterials with unique assembly and function. Despite broad interest in understanding the biological implications of protein prenylation in regulating different facets of cell biology, the use of this PTM to develop new protein-based materials and therapies remains underexplored. Progress has been slow due to the lack of accessible methodologies to rapidly generate prenylated proteins with broad physicochemical diversities. This limitation, in turn, has hindered the empirical elucidation of prenylated proteins' sequence–structure–function rules. To address this gap, we genetically engineered E. coli to develop operationally simple, high-yield biosynthetic routes to produce farnesylated protein. Using scattering, calorimetry, and microscopy, we revealed determinants of their emergent material properties (nano-aggregation and phase behavior). We combined the hierarchical assembly of fatty acids with the thermoresponsive character of elastin-like polypeptides (ELPs) to form nanocarriers with temperature-dependent structural (shape or size) characteristics. We then explored the biophysical underpinnings of the thermo-responsive behavior of the lipidated proteins using spectroscopy, scattering, microscopy, and computational nanoscopy. This integrated approach revealed that temperature and molecular syntax alter the structure, contact, and hydration of lipids, lipidation site, and proteins, aligning with the changes in the nanomorphology of the lipidated proteins. We also investigated the effect of altering the architecture of the lipidated proteins. We envisioned that two orthogonal lipidation pathways with different regio-selectivity and substrate preferences can be combined inside E.coli to produce recombinant nanomaterials with distinct lipidation domains at each terminus of proteins. We demonstrated the orthogonality of N-myristoylation and C-cholesterylation pathways for recombinant production of lipidated proteins with a unique triblock architecture, a hydrophilic protein block flanked by two lipid tails, i.e., inverse bolaamphiphiles. The study indicates that lipidations' architecture and the polypeptide sequence can be used to control the hierarchical self-assembly of these materials. We then explored the effect of complex topology along with lipidation. Inspired by the findings of topological engineering for accessing rare mesophases formed by synthetic macromolecules, we explored this design principle in biomacromolecular assemblies. We used PTMs to produce lipidated proteins with precise topological and compositional asymmetry. Using an integrated experimental and computational approach, we showed that the material properties (thermoresponse and nanoscale assembly) of these hybrid amphiphiles are modulated by their amphiphilic architecture. Importantly, we demonstrate that the judicious choice of amphiphilic architecture can be used to program the assembly of proteins into adaptive nanoworms that undergo a morphological transition (sphere-to-nanoworms) in response to temperature stimuli. These findings enable us to understand better the biophysical consequence of lipidation in biology and the rational design of the biomaterials and therapeutics that rival biological systems' exquisite hierarchy and capabilities. We envision these bio-enabled approaches yield novel recombinant hybrid biomaterials with tunable nanoscale structure and morphology with applications in medicines and nanoscience.
Hossain, Md Shahadat, "LEVERAGING THE POST-TRANSLATIONAL LIPIDATIONS TO MODULATE THE ASSEMBLY AND PHASE-BEHAVIOR OF PROTEINS" (2023). Dissertations - ALL. 1697.
Available for download on Tuesday, December 12, 2023