Unnatural Amino Acids for Protein Engineering

As an expanding toolbox for protein engineering, incorporation of unnatural amino acids (UAAs) has been used to enhance protein stability, explore mechanisms, tune catalytic activity, tune selectivity, design enzymes, and even design synthetic life. UAA incorporation is becoming an increasingly standard practice for protein engineers, with the components of incorporation constantly being adjusted and a growing selection of UAAs with different functional groups and uses. For example, tRNA and aaRS for a specific UAA are often integrated on a single plasmid, and UAA incorporation by genetic codon amplification usually requires only the introduction of TAG codon-specific sites on the target gene and supplementation of the protein expression medium with the corresponding UAA. UAAs have been used to achieve many functions, including bioorthogonal reactions, photoreactivity, spectroscopic probes, metal chelation, and post-translational modification mimicry.

What is Protein?

Proteins are high molecular weight compounds composed of amino acids connected by peptide bonds. They are one of the most important biological macromolecules in life, participating in the construction of cell structure and life activities. Each protein has a specific amino acid sequence, which determines its three-dimensional structure and function. Proteins perform multiple functions in organisms, including enzyme catalysis, signal transduction, material transport, immune defense, and structural support. The type, quantity, and arrangement order of amino acids determine the diversity and specificity of proteins. The synthesis of proteins in the body involves DNA transcription and mRNA translation, and is an important link in gene expression. In addition, proteins play a key role in drug discovery, helping to develop new drugs through target identification, structural analysis, and functional mechanisms. High-throughput screening and quantitative proteomics technologies enable researchers to identify disease-related proteins and design targeted drugs.

Protein Function

Proteins are among the most important biomolecules in life and they play a vital role in a variety of biological processes. Proteins are composed of amino acids linked by peptide bonds to form a variety of three-dimensional structures that define their functional properties.

• Physiological Functions

• Catalysis: Enzymes are a special class of proteins that act as catalysts to speed up chemical reactions in living organisms. For example, trypsin in the digestive tract helps break down proteins in food, and cellulase helps break down plant cellulose.
• Structure and support: Certain proteins such as collagen provide physical support to maintain the shape and structure of living organisms. Collagen is the main component of connective tissue, giving skin, bones and tendons the necessary strength and elasticity.
• Transport and storage: Hemoglobin is a protein responsible for oxygen transport, carrying oxygen in the blood from the lungs to various tissues in the body. In addition, ferritin can store and release iron and is the main storage form of iron in the body.
• Signaling: Many proteins such as receptor proteins and signaling molecules are involved in signaling between and within cells. For example, insulin is a hormone protein that helps regulate blood sugar levels.
• Mobility and movement: Myosin and actin are proteins involved in muscle contraction, which interact to propel the movement of cells and entire organisms.

• Research Functions

• Basic biological research: By studying the structure and function of proteins, scientists can better understand biological processes. For example, X-ray crystallography and nuclear magnetic resonance (NMR) techniques allow researchers to resolve the three-dimensional structure of proteins and reveal how they interact with other molecules.
• Drug development: Many drugs work by targeting specific proteins. For example, antibiotics block the function of bacterial proteins, and anticancer drugs interfere with protein signaling pathways in cancer cells. Therefore, understanding the function of proteins is crucial for drug design and screening.
• Disease research: Abnormal protein function is often associated with disease. For example, Alzheimer's disease and Parkinson's disease are associated with the misfolding or aggregation of specific proteins. By studying these pathological proteins, scientists hope to find new ways to treat these diseases.
• Biotechnology applications: Genetic engineering technology can modify proteins to play a role in industry. For example, enzymes are used in areas such as food processing, biofuel production, and wastewater treatment to improve efficiency and environmental protection.

Protein Engineering

Protein engineering refers to modern biotechnology that uses genetic engineering technology to modify natural proteins according to human needs, and even creates new protein molecules that do not exist in nature and have excellent properties based on the study of the relationship between the structure and function of protein molecules. That is, according to specific needs, the protein is molecularly designed and modified, and proteins with different functions are expressed. It is also called the second generation of genetic engineering.

Fig. 1. General protein engineering processes (Trends Biotechnol. 2020, 38(7): 729-744).

The basic idea of the genetic engineering operation procedure follows the central law, from DNA→mRNA→peptide→folding to produce protein, which basically produces proteins that already exist in nature. Protein engineering is carried out according to the opposite idea, determining the function of the protein→the high-level structure that the protein should have→the folding state that the protein should have→the amino acid sequence→the base sequence in the gene, which can create proteins that do not exist in nature. The main steps of protein engineering include:

1. Goal: Design and modify the structure of the protein according to people's specific needs for protein function.

2. Method: Modify genes or synthesize genes.

3. Process: Expected protein function→design expected protein structure→infer the expected amino acid sequence→find the corresponding amino acid sequence (gene) or synthesize new genes→obtain the required protein.

Unnatural Amino Acid Incorporation

Protein engineering typically involves replacing one amino acid residue in a protein with one of the 19 other proteinogenic amino acids. This approach is limited by the limited functional groups available in proteins or natural amino acids. For example, only a few of the 20 proteinogenic amino acids are capable of coordinating metal ions. Many of these amino acids have unique structures and properties, such as the imidazole side chain of histidine and the thiol group of cysteine. This makes the engineering of metalloprotein ligands very difficult. This limitation in the diversity of amino acid structures, functional groups, and other properties has impacted protein engineering, necessitating the need to expand the amino acid alphabet. The incorporation of unnatural amino acids (UAAs) into proteins can effectively complement current protein engineering approaches and greatly expand the chemical structures available to protein chemists. It allows protein engineering using an expanded alphabet to achieve improved or novel functions, such as enhancing protein stability, probing catalytic mechanisms, tuning enzyme activity, and designing new functions.

* List of unnatural amino acids:

• Chemical Synthesis

The introduction of UAAs can be achieved by different methods. Solid phase peptide synthesis allows the introduction of UAAs such as L-amino acids, D-amino acids, and β-amino acids with different side chains into the peptide chain. Peptides longer than 50 amino acid residues are challenging to synthesize conventionally due to the accumulation of errors in each round of synthesis. To overcome the size limitation of peptide synthesis, short peptide fragments can be chemically synthesized and ligated via Staudinger ligation or native chemical ligation methods. Native chemical ligation occurs between a peptide thioester and a peptide with an N-terminal thiol group. The two peptides undergo a chemoselective transthioesterification reaction that rearranges the bond between the thiol and thioester to a native peptide bond via spontaneous acyl transfer. Expressed protein ligation (EPL) uses an engineered intein, a self-splicing protein domain, to generate peptide thioesters from recombinantly expressed proteins. Subsequently, the peptide thioester reacts with a thiol-containing peptide in a native chemical ligation-type reaction to form the full-length protein.

• Chemical Mutagenesis

In addition to chemical synthesis, certain chemical reactions can target specific natural amino acids and convert them into UAAs. Similar to site-directed mutagenesis, this approach is called chemical mutagenesis. For example, serine can be converted to selenocysteine by phenylmethylsulfonyl fluoride activation, followed by hydroselenide treatment to replace the sulfonic acid group with -SeH. Subtilisin, a serine protease, was converted to an acyltransferase after chemical mutagenesis of Ser in the active site to selenocysteine. Tyrosine can be converted to 3-nitrotyrosine (3-NO2Y) by nitration. The surface-exposed tyrosine in azurin was chemically mutagenized to 3-NO2Y. The increased reduction potential of nitrotyrosine relative to tyrosine favors electron hopping in the protein. Chemical mutagenesis provides a facile method for the incorporation of UAA, but it usually requires the host protein to be highly stable to withstand the organic solvents in which the chemical reactions take place and the chemical reagents added during the process.

• Aminoacyl tRNA Synthetase Insertion

While chemical synthesis and mutagenesis provide structural diversity in protein building blocks, they often require complex equipment and purification steps. The natural protein synthesis machinery, which includes tRNA, aminoacyl tRNA synthetases (aaRS), mRNA, ribosomes, and various protein factors (initiation, elongation, release factors, etc.), synthesizes proteins at an astonishingly high rate (10-20 aa/s) and fidelity (10-4-10-5 aa). The natural protein synthesis machinery can be engineered to produce proteins with UAAs by changing certain components of the system. The natural promiscuity of aminoacyl tRNA synthetases enables structural analogs of natural amino acids to be incorporated into proteins in vitro and in vivo. By removing natural amino acids from the growth medium, supplementing with unnatural amino acids, and using auxotrophic strains that cannot produce the replaced natural amino acids, cells can be induced to take up UAAs with structures similar to natural amino acids. This amino acid replacement method is relatively easy to perform and does not require any redesign of the translation machinery.

• Genetic Codon Expansion

To further increase the flexibility of UAA incorporation, genetic code reprogramming has been achieved in an in vitro translation system by a ribozyme that can recognize tRNA and fill it with certain amino acids. tRNAs with UAAs are synthesized using ribozymes. Adding them to a carefully designed in vitro translation system allows for the selective incorporation of a wide range of UAAs into proteins. This approach allows for the incorporation of a wide range of UAAs that cannot be done by other biological methods, such as α-N-methyl, D-, and β-amino acids.

Fig. 2. Expansion of the genetic code based on unnatural amino acids (Curr Opin Struct Biol. 2013, 23(4): 581-7).

To achieve site-specific insertion and expand the diversity of UAAs, further engineering of the protein synthesis machinery is required. Orthogonal tRNA/aaRS pairs are introduced into the organism. 'Orthogonal' means that the tRNA and aaRS are specific for each other, do not crosstalk with their endogenous counterparts, and are compatible with the translation apparatus. The aaRS recognizes a specific UAA and then acylates the tRNA with the UAA. The tRNA recognizes a blank codon (a codon that does not encode a protein amino acid), which can be an amber stop codon (UAG), a quadruple codon, or a genome from a reassigned codon. With the help of ribosomes and protein factors, the tRNA can deliver the UAA into the elongating peptide. Through the process of genetic code expansion, a specific UAA is incorporated at the position corresponding to the blank codon.

* List of unnatural amino acids:

Protein engineering has different purposes, such as improving protein stability, modulating enzyme activity or selectivity, or even designing new protein functions. Protein engineering is based on the understanding of protein function, including its three-dimensional structure, dynamics, and reaction mechanism. UAA incorporation can be used in these areas as a complement to natural amino acid mutagenesis. For example, comprehensive fluorination of aromatic residues by introducing 4-Fluoro-DL-phenylalanine, 5-fluorotryptophan, and 3-fluoro-L-tyrosine into lipase B from Candida antarctica has been shown to extend the shelf life of the enzyme. In another study, the introduction of 3-Fluoro-DL-phenylalanine and 4-fluorophenylalanine into the Thermoanaerobacterium thermohydrogensulfuron lipase increased the lipase activity by 25%.

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