Encrypting Chemical Reactivity in Protein Sequences toward Information‐Coded Reactions†

Controlling chemical reactivity has been the central theme in chemistry. Herein, we review the recent progress on the development of genetically encoded protein coupling reactions and their potential applications. The chemical reactivity is encoded in the protein sequences. The information is read out by folding and molecular recognition between two reactive components and subsequently translated into chemical bonding via autocatalysis. It has emerged as a unique way to tune the chemical reactivity and is regarded as one type of information‐coded reactions. Not only has it received many applications such as protein topology engineering, bioconjugation, biomaterials and synthetic biology, but also its principle may be extended beyond protein chemistry to enable new modes of supramolecular interactions that promote chemical bonding and that are simultaneously reinforced by covalent bonds.     

Overcoming Symmetry Mismatch in Vaccine Nanoassembly through Spontaneous Amidation

Matching of symmetry at interfaces is a fundamental obstacle in molecular assembly. Virus‐like particles (VLPs) are important vaccine platforms against pathogenic threats, including Covid‐19. However, symmetry mismatch can prohibit vaccine nanoassembly. We established an approach for coupling VLPs to diverse antigen symmetries. SpyCatcher003 enabled efficient VLP conjugation and extreme thermal resilience. Many people had pre‐existing antibodies to SpyTag:SpyCatcher but less to the 003 variants. We coupled the computer‐designed VLP not only to monomers (SARS‐CoV‐2) but also to cyclic dimers (Newcastle disease, Lyme disease), trimers (influenza hemagglutinins), and tetramers (influenza neuraminidases). Even an antigen with dihedral symmetry could be displayed. For the global challenge of influenza, SpyTag‐mediated display of trimer and tetramer antigens strongly induced neutralizing antibodies. SpyCatcher003 conjugation enables nanodisplay of diverse symmetries towards generation of potent vaccines.     

Native conjugation between proteins and [60]fullerene derivatives using SpyTag as a reactive handle

Herein,we report the facile conjugation between proteins and water-soluble [60]fullerene derivatives(DC₆₀) under native conditions using SpyTag as a reactive handle.Water-soluble [60] fullerene derivatives were first prepared via sequential Bingel-Hirsch reaction and "clicked" with SpyTag to give DC₆₀-SpyTag for native conjugation with proteins by the highly efficient SpyTag-SpyCatcher chemistry.The bioconjugation was confirmed by MALDI-TOF MS spectra and SDS-PAGE analysis.The TEM and UVvis spectroscopic study further revealed that the DC₆₀ could alter the optical performance and induce aggregation of the target proteins.It thus provides a general and robust method for modifying proteins with C₆₀ derivatives and could potentially be adapted for native conjugation between proteins and other nonbiological motifs as well.     

Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics

SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram‐positive bacterial adhesin. In this work, we established a phage‐display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus. SpyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli . SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.     

Unleashing chemical power from protein sequence space toward genetically encoded “click” chemistry

We propose the concept of genetically encoded “click” chemistry (GECC) to describe the “perfect” peptide-protein reactive partners and use SpyTag/SpyCatcher chemistry as a prototype to illustrate their structural plasticity, robust interaction, and versatile applications.     

Genetically Encoded Click Chemistry†

Genetically encoded click chemistry (GECC) refers to a category of peptide/protein reactions that can spontaneously form covalent linkages with high efficiency and selectivity under mild physiological conditions. The emergence of powerful SpyTag/SpyCatcher chemistry, a prototype of GECC, has opened the door to new fields such as in cellulo protein topology engineering and design of synthetic organelles. The continuing developments of GECC will surely provide great opportunities for future materials science and synthetic biology and open up a new avenue to information‐coded chemical reactions.