Synthetic Biology
Course for Master Students
Cover image from: https://www.technologynetworks.com/drug-discovery/blog/how-is-synthetic-biology-shaping-the-future-of-drug-discovery-340290
part 3
Cover image from: https://www.technologynetworks.com/drug-discovery/blog/how-is-synthetic-biology-shaping-the-future-of-drug-discovery-340290
�Synthesis of artificial proteins
�Chemists have long been able to polymerize amino acids, but the amino acids connect in a disordered manner, resulting in products that bear little resemblance to natural proteins. However, it is possible to combine amino acids in a specified order, allowing the production of some biologically active proteins, including insulin. The process is quite complex, and thus, it is only successful in obtaining proteins containing around a hundred amino acids in their molecules. Synthetic biology is not only involved in the synthesis of xeno-nucleotides and xeno-nucleic acids but also in the synthesis of artificial biologically active proteins.
Experiments with artificial proteins have been conducted to investigate the impact of alternating polar and nonpolar amino acids in a chain on the spatial arrangement of proteins. For a beta-sheet on the protein surface, an alternating "one by one" pattern is required - ABAABAAB, ensuring that all polar groups face in one direction, and nonpolar groups face in the opposite direction. For an alpha-helix, the repetition of 7-amino acid fragments AABBAAB or AAABAAV is necessary, so that one side of the helix is polar and exposed to water, while the other side is nonpolar and internal.
Five nonpolar amino acids (valine, leucine, isoleucine, phenylalanine, methionine) are largely interchangeable among themselves. Glycine and proline stand apart as they do not fit into the alpha-helix or beta-sheet structures; instead, they are found in regions of sharp turns in the protein chain between helices or strands of beta-sheets. The other thirteen amino acids are polar and face water, making them virtually interchangeable as well. Thus, amino acids can be classified into three groups - "polar," "nonpolar," and "turning." This classification enables calculations of the three-dimensional structure of the protein. Experiments show that if a protein is compactly folded, it possesses some enzymatic activity. Moreover, short proteins may exhibit multiple enzymatic activities.
� In order for a polypeptide chain in a cell to become functionally active, it must undergo a process known as folding. Folding is the process of protein assembly. It is a physical process through which a protein chain acquires its three-dimensional structure, a conformation that makes it a biologically functional protein molecule.
chaperons
Chaperones - from the French "chaperone" - governess. Chaperones monitor the conformational state of a protein from the beginning of translation. They assist the synthesized polypeptide chain in finding the correct form, control and accelerate the folding process - the formation of the native conformation, stabilize partially denatured protein molecules. They sort and transport proteins across membranes, aid in the assembly of oligomeric proteins, and participate in the conformational switching of proteins from inactive to active states. Artificial proteins may not be active.
�Chaperonins are complex proteins composed of multiple subunits, resembling barrels with lids. Chaperonins bind to fully unfolded or partially unfolded protein molecules, shield hydrophobic groups, and pass the protein to the chaperonin. The chaperonin delivers the protein into the cavity of the barrel. After proper folding, the lid opens, and the functional protein molecule is released from the chaperonin. Chaperones and chaperonins can repair damaged protein structures. If repair is unsuccessful, the protein must be degraded in the proteasome - another complex protein that forms a cavity. According to molecular mass, all chaperones can be divided into six main groups:
chaperons
Chaperones are divided into constitutive proteins (whose high basal synthesis is not dependent on stress conditions in the organism's cells) and inducible ones, whose synthesis is weak under normal conditions but sharply increases when the cell is subjected to stress.
Inducible chaperones belong to the "heat shock proteins," whose rapid synthesis is observed in practically all cells exposed to any form of stress. The term "heat shock proteins" originated because these proteins were initially discovered in cells exposed to high temperatures. The subunits of chaperonins belong to the group of large molecular chaperones (60 kDa) and assemble into a toroidal structure consisting of two rings. During a heat shock in the cell, the content of these proteins, known as heat shock proteins (hsp), increases.
They include:
� High-molecular-weight chaperones are ATP-dependent, while the activity of small HSPs is ATP-independent. Genetic and biochemical data have shown that ATP hydrolysis is a crucial element in the activity of HSP70 chaperones. Proteins of this family bind to intermediate peptides through cycles of ATP binding and hydrolysis, and the subsequent exchange of ADP/ATP is accompanied by the release of peptides. HSP70 molecules contain two conserved domains - the N-terminal ATP-binding domain (45 kDa) and the C-terminal domain (15 kDa) that binds hydrophobic peptides. Between them lies a more variable region, the alpha-helical "lid." ATP-dependent HSP70 (with the "lid" open) freely interacts with immature or misfolded peptides, causing conformational changes that activate ATPase and enhance association with the HSP40 co-chaperone, promoting a transition to the ADP-dependent ("lid" closed) form.
Protein engineering
Another approach is directed evolution of proteins, which does not require a thorough understanding of the structure and functioning of protein molecules. In this method, libraries of random amino acid sequences are created, from which initially, by one method or another, polypeptide chains with the necessary properties are selected. Subsequently, with the use of random mutagenesis, new protein libraries are generated and utilized in further selection processes.
� Redesign is a method in protein engineering where a known amino acid sequence of proteins or enzymes is used, and through targeted mutagenesis, only specific amino acid residues are replaced. This process results in the creation of new artificial protein structures.
Rational protein design is based on the conscious utilization of the laws governing the spatial structure of proteins and the mechanisms of enzymatic catalysis to create new macromolecules with the desired properties. Constructing a protein molecule de novo involves obtaining a sequence that does not occur in nature. Additionally, this method is used to redesign known proteins to alter their properties and biological activity. Through redesign, mini-proteins are created, preserving the essential qualities of natural molecules, and enzymes with modified substrate specificity are obtained. Such enzymes exhibit greater stability in extreme conditions, known as extremozymes.
Computational protein modeling is one of the key stages in the application of protein engineering methods. Significant advancements in genome sequencing of organisms, including the decoding of the human genome, have spurred the development of computational structural biology (in silico biology). Empirical rules and algorithms for predicting the three-dimensional structure of proteins have been developed based on the analysis of experimentally determined protein structures. These form the foundation for molecular modeling of protein spatial structures. The Protein Data Bank (PDB) contains more than 15,000 spatial structures of various proteins. The atomic coordinates of a homologous protein can be used as a spatial matrix for homology modeling.
The mechanism of alanine scanning
Recently, the procedure of site-directed mutagenesis has become one of the routine techniques in molecular biology. Several companies manufacture standard reagent kits for site-directed mutagenesis and concurrently develop new innovative methodologies. One of the most effective methods is the "QuikChange" method by "Stratagene" (USA). A distinctive feature of the "QuikChange" system is that the primers must be mutually complementary. The mutation efficiency using the "QuikChange" method is at least 80%. This system is particularly suitable for introducing single nucleotide substitutions. Another system proposed by "Stratagene," called "ExSite," is optimal for introducing mutations such as deletions and insertions, with an efficiency of not less than 60%.
� In 2004, a group of scientists led by Baker D. synthesized a synthetic protein named Top 7. Initially, the protein was computationally modeled from its primary to tertiary structure. Subsequently, they chemically synthesized the protein. Top 7 consists of 93 amino acid residues. Significant financial resources were invested in creating this protein. Folding simulation was performed using the specialized computer program Folding@Home. Modeling the folding of such a small polypeptide required the participation of over half a million computers. On a single computer, modeling this protein would have taken about a hundred years. X-ray structural analysis revealed that the protein closely resembled the computer model and exhibited high stability. The spatial structure of this protein consists of two α-helices packed with five antiparallel β-sheets. Such a spatial structure of a protein does not exist in nature.
�The spatial structure of the Top 7 protein:
A - Computer model; B - Structure after synthesis.
A protein that does not exist in nature was synthesized. The first of such protein structures, albebetin, consists of two repeating elements, αββ, and forms a 4-stranded antiparallel β-sheet. Albebetin was modified to incorporate a biologically active segment of the interferon into its structure.
The current state of protein engineering opens broad perspectives for both fundamental molecular biology and its practical applications. Protein engineering employs a complex set of modern approaches for the structural-functional analysis of proteins, enabling the introduction of targeted mutations into any protein sites and the study of the structure and intricate mechanisms of enzyme functioning.
Using structural analysis of proteins and computer modeling, new mutant proteins can be created for medical purposes, such as cytokines with enhanced biological activity, modified antibodies, and so on. By expressing in vivo mutant proteins with altered properties (enhanced or weakened action, elimination of phosphorylation sites, proteolysis, etc.), it is possible to create a new approach in gene therapy. Improving protein stability through targeted changes in their structure expands their applications in various biotechnology fields. A new direction for protein engineering could be its integration with microelectronics for the development of novel biosensors.
� Created reporter proteins are protein molecules that allow assessing the rate of biosynthesis, the content of specific protein molecules, tracking their movement in the intracellular space, or isolating hard-to-reach proteins individually from a complex mixture. By attaching β-galactosidase as a reporter protein to the C-terminus of the analyzed protein, it is possible to purify the recombinant protein based on β-galactosidase activity or by tracing its antigenic determinants using immunological methods.
�The drug should contain an active ingredient to achieve a physiological effect and a ligand that recognizes the receptor on the surface of target cells. Additionally, it must have structural elements recognized by the body's transport system for delivering drugs to target cells. There should also be a spacer region necessary for separating the active ingredient from other functional parts of the drug after its delivery. This ideal scheme is implemented in the natural exotoxin of Pseudomonas aeruginosa. It is highly toxic and effective itself.
By structure, it is a protein consisting of a single polypeptide chain with a length of 613 amino acids organized into three functional domains. The N-terminal domain Ia (amino acid residues 1-252) is necessary for interacting with the receptors of Os2-macroglobulin on the surface of target cells (prototype of ligand for targeted drug action). The functions of domain Ib (amino acid residues 365-404) are unknown, and it can be removed from the toxin polypeptide chain without loss of activity. Domain II (amino acid residues 253-364) ensures efficient transport of the toxin into the cell cytosol (drug transport system), and domain III (amino acid residues 405-613) carries out ADP-ribosylation of the elongation factor in translation EF2, leading to translational inhibition and cell death of target cells.
Structure of the Pseudomonas toxin (numbers indicate the positions of amino acid residues).
�The generalized scheme of the targeted drug structure.
In addition to Pseudomonas exotoxin A as an active agent in hybrid toxins, successfully applied toxins include diphtheria toxin, tumor necrosis factor, and the A-chain of ricin. As the A-protein selectively interacts with the constant (Fc) regions of immunoglobulins of the G class in many mammals, such hybrid toxins, paired with an immunoglobulin generated against any cell surface antigen, selectively bind to these cells and kill them.
Immunotoxins represent another potential anti-tumor agent and can be used against cells expressing specific antigens on their surface. Currently, some immunotoxins have undergone clinical trials and are actively used in clinical practice in the USA and Western Europe for the treatment of oncological and autoimmune diseases.
abzymes
"Abzymes" are antibodies that possess enzymatic activity. In 1968, Lerner and Schultz demonstrated that antibodies can exhibit enzymatic activity. Therefore, a new direction emerges in protein engineering for the construction of novel enzymes based on immunoglobulins.
Antibody molecules resemble enzymes as they can form stable complexes with low-molecular-weight compounds called haptens. Haptens are low-molecular-weight compounds that lack immunogenicity. This concept was introduced by Landsteiner. However, unlike enzymes, antibodies do not participate in the metabolism of the antigen, and their function does not involve the transformation of the antigen into a transitional state.
In addition to artificial abzymes, natural abzymes have also been discovered. Some antibodies are known to generate hydrogen peroxide from singlet oxygen, enabling them to perform a protective function. Certain antibodies can possess DNAase and RNAase activities, and these have been identified in the sera of individuals with autoimmune diseases.
American researchers have synthesized an artificial protein molecule that is normally absent in the body and is capable of providing better protection against the human immunodeficiency virus (HIV) than the individual's own immune system. They created an unusual antibody-like molecule composed of a CD4 receptor fragment and a mimic of the small N-terminal portion of the CCR5 receptor, linked to the Fc fragment of human IgG. This construction is referred to as eCD4-Ig by the authors.
In the United Kingdom and Japan, a new synthetic protein, TPX2, has been developed to significantly enhance the effectiveness of cancer therapy. It is similar to the natural protein TRX2 but more resistant to chemotherapy. This protein mimics its natural counterpart found in the human body, preventing its destruction by the immune system. The natural protein TRX2 is a phosphoprotein involved in apoptosis, the cell cycle, cell division, mitosis, and acetylation. Research is being conducted on combining TRX2 with the protein Aurora-A to enhance cancer therapy. Aurora-A belongs to the mitotic serine/threonine kinases and plays a role in mitosis and meiosis. Its activation occurs through phosphorylation, which could significantly improve the effectiveness of cancer therapy.
�In Germany, using the method of artificial evolution, researchers have obtained an enzyme capable of identifying the HIV provirus in the cell's genome and excising it. The foundation for this enzyme is the protein Cre (cyclic recombinase), belonging to the class of so-called site-specific recombinases. This protein is widely used by biologists for various DNA manipulations.
Intensive research in the field of protein engineering has led to the discovery of new molecular mechanisms in stabilizing protein molecules, including intramolecular ionic interactions. Additionally, computer modeling programs for predicting the secondary and tertiary structures of proteins have been developed, enabling the creation of stable and active protein globules. Particularly productive are the methods of directed evolution of protein molecules, which enhance the thermal stability of proteins and enzymes without compromising their activity. The development of new methods for the synthesis and stabilization of proteins and enzymes, such as surface expression of proteins on cells, site-specific covalent attachment of proteins to various carriers, introduction of cross-links into crystalline enzymes, as well as methods of directed evolution of proteins and rational design, will allow the creation of protein molecules for biotechnology and other fields.
End of part 3