UC San Diego

Proteins are the premier building blocks that Nature uses to create sophisticated systems. From carrying out the biological processes essential for life (e.g., photosynthesis, DNA replication, nitrogen fixation, etc.), to serving as mechanical supports for cells (e.g., cytoskeleton, membrane proteins, etc.), proteins play diverse, functional roles. Due to these capabilities, the synthesis of protein-based materials has become a burgeoning field that aims to introduce new-to-nature functionalities. Notably, researchers have recently begun to view protein crystals as viable candidates to serve as molecular templates. While historically used for structural analysis, protein crystals have more recently garnered attention as hosts for various extrinsic cargos including small molecules, nanoparticles, and other guest proteins. The Tezcan Lab, in particular, has successfully integrated synthetic polymers into crystals composed of Human Heavy-chain Ferritin (HuHF), resulting in a material termed Polymer-Integrated Protein Crystals (PIX). These hydrogel-crystals exhibit unprecedent characteristics such as reversible expansion/contraction and self-healing, all while maintaining crystallinity. Herein, we have focused on exploring the potential applications and limitations of PIX. In this dissertation, we elucidate how the different components of PIX can be modified to generate a multifunctional material. Starting with the building block, we created a mutant HuHF variant that exhibits a cysteine residue on the protein surface. By labeling HuHF with a specific small molecule, control over its crystallization behavior was achieved, leading to the formation of two distinct lattice arrangements. The spatial organization of HuHF molecules subsequently influences polymer distribution during PIX formation. Through these experiments, we discovered anisotropic polymer distribution can grant PIX with directional actuation during expansion/ contraction (Chapter 2). In another study, we examined how the dynamicity of PIX can be leveraged to encapsulate macromolecular cargo. By screening different proteins and encapsulation conditions, we revealed PIX can uptake guest proteins with high loading efficiencies (up to 46% w/w). Moreover, we explored the pH-dependency of cargo uptake and release using two model proteins, cytochrome c and lysozyme (Chapter 3). Lastly, we developed core-shell protein crystals using two distinct HuHF variants. This unique spatial organization of the HuHF molecules facilitated selective modification of the crystal surface. Due to PIX’s ability to encapsulate protein, we further functionalized the interstitial space and created a cell-like system (Chapter 4).