Chemogenetic and optogenetic control of post-translational modifications through genetic code expansion

Post-translational modifications (PTMs) of proteins extensively diversify the biological information flow from the genome to the proteome and thus have profound pathophysiological implications. Precise dissection of the regulatory networks of PTMs benefits from the ability to achieve conditional control through external optogenetic or chemogenetic triggers. Genetic code expansion provides a unique solution by allowing for site-specific installation of functionally masked unnatural amino acids (UAAs) into proteins, such as enzymes and enzyme substrates, rendering them inert until rapid activation through exposure to light or small molecules. Here, we summarize the most recent advances harnessing this methodology to study various forms of PTMs, as well as generalizable approaches to externally control nodes-of-interest in PTM networks.     

Increasing the chemical space of proteins in living cells via genetic code expansion

In recent years it has become possible to genetically encode an expanded set of designer amino acids with tailored chemical and physical properties (dubbed unnatural amino acids, UAAs) into proteins in living cells by expanding the genetic code. Together with developments in chemistries that are amenable to and selective within physiological settings, these strategies have started to have a big impact on biological studies, as they enable exciting in cellulo applications. Here we highlight recent advances to covalently stabilize transient protein–protein interactions and capture enzyme substrate-complexes in living cells using proximity-triggered and residue-selective photo-induced crosslinking approaches. Furthermore, we describe recent efforts in controlling enzyme activity with photocaged UAAs and in extending their application to a variety of enzymatic scaffolds. In addition, we discuss the site-specific incorporation of UAAs mimicking post-translational modifications (PTMs) and approaches to generate natively-linked ubiquitin–protein conjugates to probe the role of PTMs in modulating complex cellular networks.     

Synthetase polyspecificity as a tool to modulate protein function

The site-specific incorporation of unnatural amino acids (UAAs) into proteins in bacteria is made possible by the evolution of aminoacyl-tRNA synthetases that selectively recognize and aminoacylate the amino acid of interest. Recently we have discovered that some of the previously evolved aaRSs display a degree of polyspecificity and are capable of recognizing multiple UAAs. Herein we report the polyspecificity of an aaRS evolved to encode a comarin containing amino acid. This polyspecificity was then exploited to introduce several UAAs into the fluorophore of GFP, altering its photophysical properties.     

High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.     

Crucial Optimization of Translational Components towards Efficient Incorporation of Unnatural Amino Acids into Proteins in Mammalian Cells

The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.     

Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids

We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) N^ε-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2–6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of N^ε-Boc-l-Lysine (BocK) and N^ε-propargyl-l-Lysine (PrK) by 2–5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.     

Direct protein–protein conjugation by genetically introducing bioorthogonal functional groups into proteins

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein–protein conjugates has gained research interest. In this study, site-specific protein–protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein–protein conjugates and protein complexes of biochemical significance.     

An expanding genetic code

A general method was recently developed that makes it possible to genetically encode unnatural amino acids (UAAs) with diverse physical, chemical or biological properties in Escherichia coli, yeast, and mammalian cells. Over 30 UAAs have been cotranslationally incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding tRNA-synthetase pair. A key feature of this methodology is the orthogonality between the new translational components and their endogenous host counterparts. Specifically, the codon for the UAA should not encode a common amino acid; neither the new tRNA nor cognate aminoacyl tRNA synthetase should cross-react with any endogenous tRNA-synthetase pairs; and the new synthetase should recognize only the UAA and not any of the 20 common amino acids. This methodology provides a powerful tool for exploring protein structure and function both in vitro and in vivo, as well as generating proteins with new or enhanced properties.     

A Robust Platform for Unnatural Amino Acid Mutagenesis in E. coli Using the Bacterial Tryptophanyl-tRNA synthetase/tRNA pair

We report the development of a robust user-friendly Escherichia coli (E. coli) expression system, derived from the BL21(DE3) strain, for site-specifically incorporating unnatural amino acids (UAAs) into proteins using engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS)-tRNA^Trp pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNA^Trp pair in BL21(DE3) E. coli with an orthogonal counterpart from Saccharomyces cerevisiae, and reintroducing it into the resulting altered translational machinery tryptophanyl (ATMW-BL21) E. coli strain as an orthogonal nonsense suppressor. The resulting expression system benefits from the favorable characteristics of BL21(DE3) as an expression host, and is compatible with the broadly used T7-driven recombinant expression system. Furthermore, the vector expressing the nonsense-suppressing engineered EcTrpRS-tRNA^Trp pair was systematically optimized to significantly enhance the incorporation efficiency of various tryptophan analogs. Together, the improved strain and the optimized suppressor plasmids enable efficient UAA incorporation (up to 65% of wild-type levels) into several different proteins. This robust and user-friendly platform will significantly expand the scope of the genetically encoded tryptophan-derived UAAs.     

Rewiring translation - Genetic code expansion and its applications

With few minor variations, the genetic code is universal to all forms of life on our planet. It is difficult to imagine that one day organisms might exist that use an entirely different code to translate the information of their genome. Recent developments in the field of synthetic biology, however, have opened the gate to their creation. The genetic code of several organisms has been expanded by the heterologous expression of evolved aminoacyl-tRNA synthetase/tRNA(CUA) pairs that mediate the incorporation of unnatural amino acids in response to amber codons. These UAAs introduce exciting new features into proteins, such as spectroscopic probes, UV-inducible crosslinkers, and functional groups for bioorthogonal conjugations or posttranslational modifications. Orthogonal ribosomes provide a parallel translational machinery in Escherichia coli that has lost its evolutionary constraints. Evolved variants of these ribosomes translate amber or quadruplet codons with massively enhanced efficiency. Here, I review these recent developments emphasizing their tremendous potential to facilitate biochemical and cell biological studies.     

Oxovanadium(IV) complexes of carbohydrates: A brief overview

In this brief review the most recent studies and the most relevant aspects of the complexes generated by interaction of carbohydrates and related molecules with the oxovanadium(IV) cation, VO²⁺, are presented and discussed. The survey includes complexes of mono-, di- and polysaccharides, and of other molecules related to simple sugars. First studies with conduritols and related molecules are also described. Moreover, complexes of ascorbic and quinic acids and of some peculiar flavonoids are also included. Some comments on the general physicochemical properties of these complexes are made and their biological activities and effects are also briefly discussed.     

Satellite Dnas in the Embryos of Various Species of the Genus Drosophila

A tentative evolutionary pattern has been found for two classes of the multiple satellite DNA's found in the genus Drosophila. The satellite DNA's from five Drosophila species (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) were analyzed and found to fall into three arbitrary CsCl buoyant density classes: Class I, rho = 1.661-1.669 g cm(-3), DNA molecules composed of primarily dA and dT moieties; Class II, rho = 1.685 and rho = 1.692, DNA molecules of low GC content; and Class III, rho = 1.711, a DNA of high GC composition. The dAT satellite DNA's appear in all the species studied except D. hydei, the species of most recent evolutionary divergence, whereas the heavy satellite appears only in the two species of most recent divergence, D. virilis and D. hydei.     

Interaction of photons with molecules – cross-sections for photoabsorption, photoionization, and photodissociation

A survey is given of recent progress in measurements of photoabsorption, photoionization, and photodissociation cross-sections of molecules in the wavelength range of photons in the vacuum ultraviolet (VUV), where the optical oscillator-strengths of most molecules are predominantly distributed. Some remarks are presented on molecules in the condensed phase. Particular emphasis is placed on the current understanding of spectroscopy and dissociation dynamics of molecules in the superexcited states which are produced through the interaction of photons in this wavelength range. In the VUV range, most of the observed superexcited states are assigned to high-Rydberg states which are vibrationally (and/or rotationally), doubly, or inner-core excited, and converge to each of the ion states. Received: 2 September 1998 / Accepted in revised form: 10 September 1999     

Recent Efforts in Identification of Privileged Scaffolds as Antiviral Agents

Viral infections are the most important health concern nowadays to mankind, which is unexpectedly increasing the health complications and fatality rate worldwide. The recent viral infection outbreak developed a pressing need for small molecules that can be quickly deployed for the control/treatment of re-emerging or new emerging viral infections. Numerous viruses, including the human immunodeficiency virus (HIV), hepatitis, influenza, SARS-CoV-1, SARS-CoV-2, and others, are still challenging due to emerging resistance to known drugs. Therefore, there is always a need to search for new antiviral small molecules that can combat viral infection with new modes of action. This review highlighted recent progress in developing new antiviral molecules based on natural product-inspired scaffolds. Herein, the structure-activity relationship of the FDA-approved drugs along with the molecular docking studies of selected compounds have been discussed against several target proteins. The findings of new small molecules as neuraminidase inhibitors, other than known drug scaffolds, Anti-HIV and SARS-CoV are incorporated in this review paper.     

Site-specific in vitro and in vivo incorporation of molecular probes to study G-protein-coupled receptors

Chemical modification of proteins has a rich history in biochemistry and chemical biology. However, studies of membrane protein function, especially in cases where functional expression is low and purification and reconstitution are not feasible, present unique challenges. Heptahelical G-protein-coupled receptors (GPCRs) are a particularly important class of cell-surface receptors that represent targets of more than a quarter of all therapeutic drugs. Understanding with chemical precision how GPCRs function in biological membranes remains a central problem in biology. Recently a number of creative strategies have been developed that allow site-specific attachment of chemical probes or tags directly on expressed receptors or on biologically active peptide ligands or substrates. One particularly important advance is the genetic encoding of unnatural amino acids (UAAs) with unique small bioorthogonal tags using amber codon suppression in mammalian cells. This method should allow site-specific labeling of GPCRs with various molecular probes to facilitate cell-based studies of protein-protein or protein-ligand interactions and the visualization of conformational changes using fluorescence spectroscopy or single-molecule imaging.     

SELEX and the evolution of genomes

The interrupted genome structures of complex multicellular organisms have most likely changed the evolution of the regulation of metabolism and development. Wasted intron sequences make regulation of gene expression in (for example) mammals appear to be unnecessarily complicated. The recent discoveries that globular RNA molecules are very much like the antigen-combining sites of antibodies suggest that intronic RNA may be used to help solve the problems raised by this complexity.     

Bioreactors for plant cells: hardware configuration and internal environment optimization as tools for wider commercialization

Mass production of value-added molecules (including native and heterologous therapeutic proteins and enzymes) by plant cell culture has been demonstrated as an efficient alternative to classical technologies [i.e. natural harvest and chemical (semi)synthesis]. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up plant cell culture-based processes (most notably to produce paclitaxel) and several commercial processes have been established so far. The choice of a suitable bioreactor design (or modification of an existing commercially available reactor) and the optimization of its internal environment have been proven as powerful tools toward successful mass production of desired molecules. This review highlights recent progress (mostly in the last 5 years) in hardware configuration and optimization of bioreactor culture conditions for suspended plant cells.