Orthogonal Crosslinking: A Strategy to Generate Novel Protein Topology and Function.

Area of Science:

• Biochemistry and Molecular Biology
• Protein Engineering
• Synthetic Biology

Background:

• Disulfide bonds are common protein crosslinks, but other natural intramolecular crosslinks (orthogonal crosslinks) are less studied despite their impact on protein topology and function.
• Limited understanding and methods for creating orthogonal crosslinks restrict their application in protein engineering.

Purpose of the Study:

• To present examples of natural orthogonal crosslinks and their functional significance.
• To summarize and evaluate recent strategies for expanding the repertoire of orthogonal crosslinks.
• To highlight novel applications of orthogonal crosslinks in protein design, including domain antibodies.

Main Methods:

• Review of existing literature on natural non-disulfide crosslinks.
• Summary of methods for introducing orthogonal crosslinks: enzyme-catalyzed protein circularization and genetic code expansion for site-specific incorporation of electrophilic amino acids.
• Detailed presentation of a novel spontaneous orthogonal crosslinking method generating carbamate-based crosslinks in situ.

Main Results:

• Demonstration of natural orthogonal crosslinks influencing protein topology and function.
• Comparison of the advantages and disadvantages of enzymatic and genetic code expansion strategies for orthogonal crosslinking.
• Successful application of spontaneous carbamate crosslinking to design orthogonally crosslinked domain antibodies mimicking bacterial adhesins.

Conclusions:

• Orthogonal crosslinks offer a powerful tool for engineering novel protein topologies and functions beyond disulfide bonds.
• Spontaneous orthogonal crosslinking provides an efficient in situ method for creating unique protein architectures.
• Engineered orthogonal crosslinks have significant potential in developing advanced protein-based therapeutics and biomaterials.