Amino acids are the fundamental building blocks of proteins, complex molecules that carry out a vast array of functions within living organisms. Proteins are composed of long chains of these amino acid units, linked together in specific sequences. An “unnatural amino acid” (UAA) refers to any amino acid not among the 20 standard ones typically encoded by the genetic code found in nature. These UAAs can differ from natural amino acids in their side chains, backbones, or functional groups, often being chemically synthesized. The primary aim of incorporating these unnatural amino acids is to expand protein functional capabilities beyond natural limitations, opening new avenues for scientific exploration and practical applications.
Why Modify Proteins with New Building Blocks?
The 20 natural amino acids, while versatile, present limitations for advanced scientific and industrial applications due to their restricted range of chemical functionalities. Scientists introduce unnatural amino acids into proteins to overcome these inherent constraints and introduce novel properties. This expansion of the amino acid repertoire allows for the precise introduction of new chemical handles or functionalities, such as sites for specific chemical reactions or fluorescent labeling, which are not present in natural proteins.
Proteins engineered with UAAs can exhibit enhanced properties, including increased stability, new catalytic activities, or modified binding affinities. For example, replacing a natural tyrosine in phosphotriesterase with specific unnatural amino acids can significantly increase the enzyme’s catalytic activity for certain reactions. Incorporating UAAs also enables new ways to study protein structure and function, allowing researchers to precisely modify or tag proteins to observe their behavior in living systems. This provides a higher “atom level” resolution and a wider range of modifications to side chain chemistry, expanding the possibilities for protein engineering.
How Scientists Add New Amino Acids
Genetic code expansion (GCE) is a primary method for site-specific unnatural amino acid incorporation into proteins within living cells. This technique repurposes a “stop codon” (typically the amber codon, UAG) to encode an unnatural amino acid instead of signaling protein synthesis termination. This is achieved through engineered molecular tools: an orthogonal transfer RNA (tRNA) and aminoacyl-tRNA synthetase (aaRS) pair.
The engineered aaRS is designed to recognize and attach the specific unnatural amino acid to its corresponding engineered tRNA. This engineered tRNA, in turn, has an anticodon complementary to the repurposed stop codon. When the cell’s protein synthesis machinery, the ribosome, encounters this repurposed stop codon in the messenger RNA (mRNA), the engineered tRNA delivers the unnatural amino acid, which is then incorporated into the growing protein chain. This engineered pair functions independently of the cell’s natural protein synthesis machinery, preventing misincorporation. Other methods, such as in vitro protein synthesis or chemical ligation, also exist, but GCE offers the advantage of site-specific incorporation within living cells.
Real-World Impact of Engineered Proteins
Incorporating unnatural amino acids into proteins has led to tangible advancements and holds significant promise across various fields. In drug discovery and development, engineered proteins create novel therapeutics. This includes developing more stable or potent drugs, as well as precision-targeted therapies such as antibody-drug conjugates (ADCs). With ADCs, a drug is precisely attached to an antibody at a specific site, allowing for selective delivery of cytotoxic agents to cancer cells while minimizing harm to healthy tissues.
Engineered proteins also contribute to the design of advanced biomaterials with new functions. For instance, they can create self-assembling materials with tailored properties or biosensors for detecting specific molecules. Furthermore, these modified proteins serve as powerful tools in basic biological research, enabling scientists to understand fundamental life processes by precisely modifying or tagging proteins to study their interactions and functions. These capabilities push the boundaries of what is achievable in biology, medicine, and materials science, offering new solutions to complex challenges.