(721d) Sequential Self-Assembly of Engineered Protein Building Blocks in 3D Matrices

Self-assembly is an attractive tool to manipulate physical properties of bio-macromolecules by organizing them into supramolecular structures at physiologically relevant conditions. Naturally derived protein motifs present useful self-assembling properties, and combination of multiple motifs in a self-assembly system can provide new opportunities to control the self-assembly process. We use two protein building blocks with different characteristic self-assembling motifs. The first assembly component is a diblock copolypeptide that consists of an elastin-like polypeptide (ELP) block, which undergoes inverse phase transition and aggregation, and a leucine zipper that forms coiled-coils. Its self-assembly partner is fluorescent protein mCherry, acting as a globular model protein, fused with a leucine zipper motif. The lower critical solution temperature (LCST) of the ELP leucine zipper fusion protein ranged from 25°C to 40°C at micromolar concentrations. Consistently, upon a temperature increase from 25°C to 37°C, the diblock copolypeptide rapidly aggregated into nano- and micro-particulates. The leucine zipper domain fused with ELP allows for further assembly with mCherry leucine zipper fusion protein by formation of high-affinity, heterodimeric coiled-coils. The second step of assembly significantly influenced the phase transition, colloidal stability, and dynamic behavior of aggregated particles in solution, primarily controlled by the ratio of leucine zipper building blocks. These sequential multi-step assembly processes spontaneously occur in physiological environments, where no further stimulus is necessary, and can be applied as a new system for protein delivery. In a new strategy, non-equilibrium in situ self-assembly of the building blocks resulted in self-assembled nano- and micro-structures in 3D matrices, which promote substantial control of molecular transport of the model protein. Dynamic behavior of the self-assembled structures in 3D matrices was characterized by fluorescence microscopy to quantify the parameters that govern protein retention in tissue. This simple, all protein, self-assembling system can be applied to control delivery and extend retention of therapeutic proteins in natural tissue or artificial matrices.