Rational Design of Supramolecular Dynamic Protein Assemblies by Using a Micelle‐Assisted Activity‐Based Protein‐Labeling Technology

Abstract

The self-assembly of proteins into higher-order superstructures is ubiquitous in biological systems. Genetic methods comprising both computational and rational design strategies are emerging as powerful methods for the design of synthetic protein complexes with high accuracy and fidelity. Although useful, most of the reported protein complexes lack a dynamic behavior, which may limit their potential applications. On the contrary, protein engineering by using chemical strategies offers excellent possibilities for the design of protein complexes with stimuli-responsive functions and adaptive behavior. However, designs based on chemical strategies are not accurate and therefore, yield polydisperse samples that are difficult to characterize. Here, we describe simple design principles for the construction of protein complexes through a supramolecular chemical strategy. A micelle-assisted activity-based protein-labeling technology has been developed to synthesize libraries of facially amphiphilic synthetic proteins, which self-assemble to form protein complexes through hydrophobic interaction. The proposed methodology is amenable for the synthesis of protein complex libraries with molecular weights and dimensions comparable to naturally occurring protein cages. The designed protein complexes display a rich structural diversity, oligomeric states, sizes, and surface charges that can be engineered through the macromolecular design. The broad utility of this method is demonstrated by the design of most sophisticated stimuli-responsive systems that can be programmed to assemble/disassemble in a reversible/irreversible fashion by using the pH or light as trigger.

Rational Design of Semi‐Synthetic Protein Complexes with the Defined Oligomeric State

“…Motivated by the challenges and the opportunities, synthetic protein engineering presents, we recently introduced a new chemical strategy for the synthesis of semi‐synthetic proteins by developing a micelle‐assisted protein labelling technology . This chemical technology offers site‐specific protein modification and access to a large number of building blocks starting from small molecules to highly complex branched macromolecule.…”

“…This chemical technology offers site‐specific protein modification and access to a large number of building blocks starting from small molecules to highly complex branched macromolecule. In addition, this technology was utilized to make monodisperse photo‐responsive functional protein complex . In our previous study, we exploited the hydrophobic interaction as the sole driving force for the protein complex formation.…”

“…The next challenge in the project is developing the chemistry for site‐specific bioconjugation of a hydrophobic peptide with minimal perturbation to the protein structure. From our previous studies, we knew that the direct attachment of a hydrophobic peptide is not synthetically possible . Therefore, we thought the presence of hydrophilic linker equipped with an appropriate chemical functionality between the globular protein and hydrophobic peptide domain might facilitate site‐specific bioconjugation.…”

“…“FP” was chosen because of its ability to site‐specifically react with a catalytic serine residue of serine proteases (> 200 proteins) . For bioconjugation, we utilized the technology pioneered by our group “Micelle‐Assisted Protein Labelling” method as it is proved to be a highly reliable method even for most hydrophobic high molecular weight compounds …”

“…For the synthesis of facially amphiphilic protein‐peptide conjugates (PPCs) in the library I (Figure a), we chose to use the most hydrophobic synthetic peptide (AP‐1). The peptide was solubilized using the triton X‐100 and allowed to react with serine proteases such as trypsin, chymotrypsin, and proteinase K for 12 hours followed by purification of the reaction mixture by adopting a previous method disclosed by our group . Briefly, the neutral triton X‐100 and the unreacted amphiphilic peptide were separated using ion‐exchange chromatography followed by size‐exclusion chromatography at ionic strength of 200 mM NaCl, to separate the modified protein from the native protein.…”