Unnatural Amino Acids for Protein-Protein Interaction Research

Unnatural amino acids (UAAs) provide an expanded toolkit to dissect complex protein-protein interactions with unprecedented precision. UAAs are amino acids that differ from the 20 standard amino acids encoded by the genetic code. They possess unique chemical properties that can be exploited to gain insight into the dynamics, structure, and function of protein assemblies. Incorporation of UAAs into proteins, enabled by advances in genetic encoding and synthetic chemistry, has revolutionized the study of proteins, particularly protein-protein interactions, which are critical in many biological processes.

Protein Definition

Proteins are a class of macromolecules with complex structures and perform multiple biological functions. They are widely present in organisms. From a chemical point of view, proteins are peptide chains composed of 20 different amino acids connected by peptide bonds. Specifically, a protein molecule may contain dozens to thousands of amino acids. Functionally, proteins play an irreplaceable role in the life activities of organisms. They participate in almost all functions of cells, including catalyzing chemical reactions (enzymes), transmitting signals (receptors and signal molecules), providing structural support (structural proteins, such as collagen), regulating gene expression (transcription factors), and transporting molecules (such as hemoglobin). The diversity and complexity of proteins make them an extremely important field in biological science and medical research.

Protein Structure

The function of a protein is directly dependent on its structure, and the complexity and diversity of protein structure make its functions equally rich and colorful. Protein structure can generally be divided into four levels: primary structure, secondary structure, tertiary structure and quaternary structure.

• The primary structure of a protein refers to the linear arrangement sequence of its amino acids. Each amino acid is linked together by covalent peptide bonds to form one or more polypeptide chains. The primary structure is the basis for determining the function of a protein because it determines how the protein will fold and form more complex structures.
• The secondary structure refers to the specific folding and twisting formed in the local area of the primary structure of the protein, which is mainly maintained by hydrogen bonds. Common secondary structures include alpha-helix and beta-sheet. These structures form relatively stable three-dimensional configurations in local parts of the protein through the stabilization of hydrogen bonds.
• The tertiary structure refers to the global folding of the entire peptide chain in three-dimensional space to form a complex, functional protein monomer structure. Various types of non-covalent interactions, including hydrogen bonds, hydrophobic interactions, van der Waals forces and ionic bonds, jointly stabilize the tertiary structure. Additionally, disulfide bonds (a type of covalent bond formed by the sulfhydryl groups of two cysteine residues) can further stabilize the structure.
• Quaternary structure exists in proteins that consist of multiple peptide chains (subunits) held together by non-covalent interactions or disulfide bonds. Quaternary structure describes how these peptide chains are arranged with respect to each other to form a functional protein complex. Classic examples include hemoglobin, which consists of four peptide chains (two alpha chains and two beta chains), each with a subunit that can bind an oxygen molecule.

Protein-Protein Interaction

Protein-Protein Interaction (PPI) refers to the process in which two or more protein molecules bind to each other through a specific physical contact mode and exert their biological functions. This phenomenon is widely present in organisms and plays a vital role in the life activities, signal transduction, metabolic regulation, protein synthesis and degradation of cells. Studying PPI has important scientific and application value. By identifying and analyzing these interactions, scientists can understand the molecular mechanisms of diseases and develop new drug targets. For example, in cancer research, some cancer proteins cause unlimited cell proliferation through abnormal PPI. Clarifying the interaction network of these abnormal proteins helps to develop targeted drugs.

First, PPI plays a key role in cell signal transduction. Cells recognize external signals through receptors, thereby initiating a series of signal transduction pathways. In these pathways, protein interaction is the basic way of signal transmission and amplification. For example, after receiving the signal, receptor tyrosine kinases (RTKs) initiate a series of cascade reactions by binding to downstream adapter proteins, and finally transmit the signal to the cell nucleus to regulate gene expression.

Secondly, PPI also plays an important role in maintaining cell structure and function. The cytoskeleton is composed of a series of interacting proteins, which form the basic structure of the cell through interaction, maintaining the cell's morphology and the stability of the internal and external environment. The interaction between actin and myosin is the basis for cell movement, while the interaction between tubulin and microtubule-associated proteins is the core of cell division and material transport.

In addition, protein interactions also play an important role in the regulation of gene expression. For example, transcription factors regulate the expression of specific genes by binding to other regulatory proteins to form a variety of complexes. For another example, in the immune response of an organism, antibodies specifically bind to antigens through their variable regions, thereby initiating an immune response. This process depends on precise protein-protein interactions.

Amino Acids in Protein Interaction

UAAs can capture directly interacting proteins in living cells in situ and identify the interaction interface by identifying UAAs-mediated cross-linked peptides. In addition to affinity purification and immunoprecipitation, chemical cross-linking mass spectrometry (CXMS) is increasingly used to study protein assembly and complex protein interaction networks. Existing CXMS chemical cross-linkers only target Lys, Cys, Glu and Asp residues, which limits the measurable information. The combination of UAAs and mass spectrometry has greatly expanded the application of mass spectrometry:

(1) Encoding photocrosslinkers into proteins, which cross-link with adjacent residues under ultraviolet light. However, due to the poor selectivity and short half-life of the photocrosslinking reaction, mass spectrometry analysis is complicated and its application in capturing transient interactions is also limited.

(2) Chemical small molecule cross-linkers are mainly used in protein complexes that can stabilize interactions. These highly reactive small molecules used for cross-linking will affect the intracellular environment and may change the conformation or interaction of proteins.

(3) Endogenous cross-linking. The complex is locked by in situ covalent binding of interacting proteins, and the chemical reaction has biocompatibility and specificity in living systems and can proceed spontaneously, thereby achieving spontaneous and selective cross-linking of interacting proteins in living cells.

Fig. 1. Characterize direct protein interactions with unnatural amino acids (Nat Commun. 2024, 15: 5221).

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To incorporate UAAs into proteins, several strategies have been developed, with particular reliance on the adaptation of the natural translation machinery. The most prominent method involves the use of orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs. These engineered pairs do not cross-react with the host's native tRNA or RSs, allowing for the specific incorporation of UAAs at defined positions in the protein sequence. This is commonly achieved by suppressing stop codons or rare four-base codons. An additional layer of specificity can be added by using a mutually orthogonal ribosome that suggests further exclusion from the natural translational process.

• Structural Elucidation

UAAs can be tailored to include functional groups that are rarely or never found in natural amino acids, thereby providing unique handles for structural studies. For instance, UAAs containing photo-crosslinkable groups (e.g., benzophenone or diazirine rings) can form covalent bonds with proximal protein residues upon exposure to UV light. This capability allows for the 'freezing' of transient protein-protein interactions, which can then be analyzed by mass spectrometry or other structural biology techniques to provide insights into the interaction interfaces and conformational changes. Fluorescent UAAs are another powerful tool. By inserting these into proteins, researchers can visualize protein localization and interactions in live cells using fluorescence microscopy. This is particularly valuable for tracking dynamic processes and understanding the contextual behavior of protein complexes.

• Biophysical Characterization

Surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy can all benefit from the incorporation of UAAs. For example, UAAs containing sites for spin labeling enhance the sensitivity of electron paramagnetic resonance (EPR) studies, which can then be used to probe distances and conformational transitions within and between interacting proteins. Furthermore, UAAs designed to possess bioorthogonal reactive groups, such as azides or alkynes, allow for subsequent modification with probes and reporters. This strategy underpins click chemistry approaches to examine the interaction with high specificity and little perturbation to the system under study.

• Functional Analysis

UAAs also provide the means to engineer proteins with enhanced or novel functions. For example, by introducing a UAA with a reactive side chain not present in natural amino acids, particular points of interaction can be 'toggled'—modified under specific conditions to assess the impact of perturbations on function. This delineates critical residues and domains for protein-protein interactions and their exact roles within cellular processes. This functional interrogation can be extended to the creation of 'designer' protein interactions, where UAAs facilitate controlled protein dimerization through unique chemical handles. Such controlled systems are pivotal in synthetic biology and in therapeutic contexts, where engineered interactions can lead to the design of novel signaling pathways or targeted therapeutic agents.

BOC Sciences stands out in the biochemistry industry for its robust amino acid offering, particularly its comprehensive catalog of unnatural amino acids tailored for advanced scientific research. We utilize cutting-edge technologies and state-of-the-art synthetic methods to ensure the production of high-purity unnatural amino acids, which are critical for dissecting complex protein interaction mechanisms. These specialized amino acids enable researchers to introduce novel functional groups into proteins, facilitating detailed studies of protein function, dynamics, and interactions.

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