Unnatural Amino Acid Mutagenesis Research Articles

Tracking the intracellular itinerary of APP and de novo Aβ generation using click chemistry

AbstractBackgroundAmyloid‐beta (Aβ), is generated by sequential cleavage of the amyloid precursor protein (APP) by beta‐ and gamma‐secretase. The intracellular sites of cleavage are unknown due to the lack of tools that can detect de novo Aβ production.MethodUsing unnatural amino acid (UAA) mutagenesis and click chemistry, a single fluorophore (magenta) site‐specifically binds to Aβ within the APP construct. The C‐terminus of APP is fused with the green fluorescent protein mClover, allowing for a distinction between full‐length APP and Aβ, as the uncleaved APP will appear white (magenta+green) from the bifluorescence, contrasting with the magenta of Aβ alone when separated from the green mClover C‐terminus. Live cells expressing the APP‐mClover construct (green) were incubated with cell‐impermeable tetrazine‐AlexaFluor 594 (magenta) at 4°C, which click‐labeled the UAA on Aβ on cell surface APP. The cells were then incubated at 37°C, resuming endocytosis of bifluorescent APP.ResultAfter click‐labeling, the APP construct resides exclusively at the cell surface. Once warmed to 37°C, the intracellular bifluorescence increases with time, suggesting APP endocytosis (Fig 1). Using object‐based analyses, the magenta click‐label:green mClover ratio was determined from super‐resolution z‐stack images (Fig 2). The magenta:green ratio was lower in cells treated with gamma‐secretase inhibitor (GSI).ConclusionA qualitative and semi‐quantitative picture can be created of Aβ’s localization by following the separation of the bifluorescent signal into intraluminal magenta click‐label and membrane‐bound green mClover. This technology will be coupled with immunocytochemistry of subcellular markers, which will allow for further specificity on the location of Aβ travel within a cell, with regards to the endolysosomal pathway. Furthermore, this technology can be applied to examine Aβ within the unique cytoarchitecture of primary neurons which may help inform the design of new therapeutics in this cell type central to AD pathogenesis.

Controlling the interaction between CaMKII and Calmodulin with a photocrosslinking unnatural amino acid.

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild-type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium-dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV-induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto-inhibitory role of the regulatory segment, inter-subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKIIL308BzF in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.

Elucidating signal transduction pathway in light-activated adenylyl cyclase using unnatural amino acid mutagenesis.

There is extensive research which explores the photochemistry of Blue Light Using FAD photoreceptors, however there is limited literature available on how the signal is transduced from the BLUF domain to the functional/output domain. A detailed understanding of their operation is needed to direct applications such as the development of optogenetic devices. Here, we elucidate the signal transduction pathway of the photoactivated adenylate cyclase OaPAC, in which a BLUF domain is fused to an adenylate cyclase domain that catalyzes the light-dependent formation of cAMP from ATP. OaPAC has very low dark state activity and is therefore a promising starting point for optogenetic applications. We used unnatural amino acid mutagenesis to explore how light absorption by the N-terminal BLUF domain on the fs timescale leads to activation of the C-terminal adenylate cyclase domain. We incorporated the IR probe azidophenylalanine at F103, W90 and F131 using orthogonal aminoacyl-tRNA-synthetases. The FTIR light minus dark difference spectrum shows that the environment around F103 and W90 residues changes upon light activation, however, there is no change in the environment around F131 upon photoactaction. The enzymatic activity of these mutants was not affected due to the IR probe incorporation. We also replaced Y125 with fluorotyrosine analogues to increase the acidity of the phenolic hydroxyl group and probe the role of Y125 in signal transduction which forms an intersubunit hydrogen bond with N256 from the other monomer in the OaPAC dimer. OaPAC with 3-fluorotyrosine, 3,5-difluorotyrosine or 2,3,5-trifluorotyrosine incorporated at Y125 showed 10 fold higher activity in the dark compared to wild-type OaPAC, whereas Y125F and Y125S showed no dark state activity, implying that the intersubunit hydrogen bond between Y125 and N256 is critical for signal transduction.

Abstract 1305: Elucidating signal transduction pathway in light-activated adenylyl cyclase using unnatural amino acid mutagenesis

Abstract 1305: Elucidating signal transduction pathway in light-activated adenylyl cyclase using unnatural amino acid mutagenesis

Quantitative Analysis and Optimization of Site-Specific Protein Bioconjugation in Mammalian Cells.

Despite a range of covalent protein modifications, few techniques exist for quantification of protein bioconjugation in cells. Here, we describe a novel method for quantifying in cellulo protein bioconjugation through covalent bond formation with HaloTag. This approach utilizes unnatural amino acid (UAA) mutagenesis to selectively install a small and bioorthogonally reactive handle onto the surface of a protein. We utilized the fast kinetics and high selectivity of inverse electron-demand Diels-Alder cycloadditions to evaluate reactions of tetrazine phenylalanine (TetF) with strained trans-cyclooctene-chloroalkane (sTCO-CA) and trans-cyclooctene lysine (TCOK) with tetrazine-chloroalkane (Tet-CA). Following bioconjugation, the chloroalkane ligand is exposed for labeling by the HaloTag enzyme, allowing for straightforward quantification of bioconjugation via simple western blot analysis. We demonstrate the versatility of this tool for quickly and accurately determining the bioconjugation efficiency of different UAA/chloroalkane pairs and for different sites on different proteins of interest, including EGFP and the estrogen-related receptor ERRα.

Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK.

Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) Nϵ -L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.

Probing the Role of Aurora Kinase A Threonylation with Site-Specific Lysine Threonylation.

Protein post-translational modifications play central roles in regulating protein functions. Lysine threonylation is a newly discovered reversible post-translational modification. However, the biological effect of lysine threonylation on proteins remains largely elusive. Here we report a chemical biology approach for site-specific incorporation of Nε-threonyllysine into proteins with high efficiency and investigate the biological effect of lysine threonylation on Aurora kinase A. Using this unnatural amino acid mutagenesis approach, we find that threonylation of Lys162 of Aurora kinase A inhibits its kinase activity both in vitro and in vivo and that the inhibitory effect can be reversed by the deacetylase Sirtuin 3, which removes the threonylated group from the lysine. Additionally, threonylation of Aurora kinase A makes its substrate p53 more stable in the cell. Therefore, our study demonstrates that site-specific lysine threonylation is a powerful method for probing the biological effect of protein threonylation.

Recent advancements in enzyme engineering via site-specific incorporation of unnatural amino acids.

With increased attention to excellent biocatalysts, evolving methods based on nature or unnatural amino acid (UAAs) mutagenesis have become an important part of enzyme engineering. The emergence of powerful method through expanding the genetic code allows to incorporate UAAs with unique chemical functionalities into proteins, endowing proteins with more structural and functional features. To date, over 200 diverse UAAs have been incorporated site-specifically into proteins via this methodology and many of them have been widely exploited in the field of enzyme engineering, making this genetic code expansion approach possible to be a promising tool for modulating the properties of enzymes. In this context, we focus on how this robust method to specifically incorporate UAAs into proteins and summarize their applications in enzyme engineering for tuning and expanding the functional properties of enzymes. Meanwhile, we aim to discuss how the benefits can be achieved by using the genetically encoded UAAs. We hope that this method will become an integral part of the field of enzyme engineering in the future.

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)-tRNATrp pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNATrp 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-tRNATrp 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.

Genetic Code Expansion in the Engineered Organism Vmax X2: High Yield and Exceptional Fidelity.

We report that the recently introduced commercial strain of Vibrio natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 noncanonical α-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recodedEscherichia colistrains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of misincorporated natural α-amino acids at the UAG-programmed position, especially in cases where the ncAA is a moderate substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that the use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve the selectivity of an aaRS toward highly desired but suboptimal ncAA substrates.

A CDR-based approach to generate covalent inhibitory antibody for human rhinovirus protease

Covalent target modulation with small molecules has been emerging as a promising strategy for drug discovery. However, covalent inhibitory antibody remains unexplored due to the lack of efficient strategies to engineer antibody with desired bioactivity. Herein, we developed an intracellular selection method to generate covalent inhibitory antibody against human rhinovirus 14 (HRV14) 3C protease through unnatural amino acid mutagenesis along the heavy chain complementarity-determining region 3 (CDR-H3). A library of antibody mutants was thus constructed and screened in vivo through co-expression with the target protease. Using this screening strategy, six covalent antibodies with proximity-enabled bioactivity were identified, which were shown to covalently target HRV14-3C protease with high inhibitory potency and exquisite selectivity. Compared to structure-based rational design, this library-based screening method provides a simple and efficient way for the discovery and engineering of covalent antibody for enzyme inhibition.

Effects of Glutamate Arginylation on α-Synuclein: Studying an Unusual Post-Translational Modification through Semisynthesis

A variety of post-translational modifications (PTMs) are believed to regulate the behavior and function of α-synuclein (αS), an intrinsically disordered protein that mediates synaptic vesicle trafficking. Fibrils of αS are implicated in neurodegenerative disorders such as Parkinson's disease. In this study, we used chemical synthesis and biophysical techniques to characterize the neuroprotective effects of glutamate arginylation, a hitherto little characterized PTM in αS. We developed semisynthetic routes combining peptide synthesis, unnatural amino acid mutagenesis, and native chemical ligation (NCL) to site-specifically introduce the PTM of interest along with fluorescent probes into αS. We synthesized the arginylated glutamate as a protected amino acid, as well as a novel ligation handle for NCL, in order to generate full-length αS modified at various individual sites or a combination of sites. We assayed the lipid-vesicle binding affinities of arginylated αS using fluorescence correlation spectroscopy (FCS) and found that arginylated αS has the same vesicle affinity compared to control protein, suggesting that this PTM does not alter the native function of αS. On the other hand, we studied the aggregation kinetics of modified αS and found that arginylation at E83, but not E46, slows aggregation and decreases the percentage incorporation of monomer into fibrils in a dose-dependent manner. Arginylation at both sites also resulted in deceleration of fibril formation. Our study represents the first synthetic strategy for incorporating glutamate arginylation into proteins and provides insight into the neuroprotective effect of this unusual PTM.

Extension of Genetic Marker List Using Unnatural Amino Acid System: An Efficient Genomic Modification Strategy in Escherichia coli.

Genetic manipulations including chromosome engineering are essential techniques used to restructure cell metabolism. Lambda/Red (λ/Red)-mediated recombination is the most commonly applied approach for chromosomal modulation in Escherichia coli. However, the efficiency of this method is significantly hampered by the laborious removal of the selectable markers. To overcome the problem, the integration helper plasmid was constructed, pSBC1a-CtR, which contains Red recombinase, Cre recombinase, and exogenous orthogonal aminoacyl-transfer RNA (tRNA) synthetase/tRNA pairs, allows an unnatural amino acid (UAA) to be genetically encoded at the defined site of the antibiotic resistance gene-encoded protein. When UAAs are not in the culture medium, there was no expression in the antibiotic resistance gene-encoded protein. Accordingly, the next procedure of antibiotic gene excising is not needed. To verify this method, poxB gene was knocked out successfully. Furthermore, sequential deletion of three target genes (galR, ptsG, and pgi) was able to generate neurosporene-producing strain marked by high growth rate. Thus, the site-specific incorporation UAA mutagenesis system were used to control and expand the use of conditional selectable marker, and the technique is used to facilitate a rapid continuous genome editing in Escherichia coli.

Optical Control of Cellular ATP Levels with a Photocaged Adenylate Kinase.

We have developed a new tool for the optical control of cellular ATP concentrations with a photocaged adenylate kinase (Adk). The photocaged Adk is generated by substituting a catalytically essential lysine with a hydroxycoumarin-protected lysine through site-specific unnatural amino acid mutagenesis in both E. coli and mammalian cells. Caging of the critical lysine residue offers complete suppression of Adk's phosphotransferase activity and rapid restoration of its function both in vitro and in vivo upon optical stimulation. Light-activated Adk renders faster rescue of cell growth than chemically inducible expression of wild-type Adk in E. coli as well as rapid ATP depletion in mammalian cells. Thus, caging Adk provides a new tool for direct conditional perturbation of cellular ATP concentrations thereby enabling the investigation of ATP-coupled physiological events in temporally dynamic contexts.

Chemoenzymatic Semisynthesis of Phosphorylated α-Synuclein Enables Identification of a Bidirectional Effect on Fibril Formation.

Post-translational modifications (PTMs) impact the pathological aggregation of α-synuclein (αS), a hallmark of Parkinson's disease (PD). Here, we synthesize αS phosphorylated at tyrosine 39 (pY39) through a novel route using in vitro enzymatic phosphorylation of a fragment followed by ligation to form the full-length protein. We can execute this synthesis in combination with unnatural amino acid mutagenesis to include two fluorescent labels for Förster resonance energy transfer (FRET) studies. We determine the effect of pY39 on the aggregation of αS and compare our authentically phosphorylated material to the corresponding glutamate 39 "phosphomimetic." Intriguingly, we find that αS-pY39 can either accelerate or decelerate aggregation, depending on the fraction of phosphorylated protein. The αS-E39 mutant can qualitatively reproduce some, but not all, of these effects. FRET measurements and analysis of existing structures of αS help to provide an explanation for this phenomenon. Our results have important implications for the treatment of PD patients with tyrosine kinase inhibitors and highlight the importance of validating phosphomimetics through studies of authentic PTMs.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Protein labeling for FRET with methoxycoumarin and acridonylalanine.

Site-specific protein labeling can be used to monitor protein motions and interactions in real time using Förster resonance energy transfer (FRET). While there are many fluorophores available for protein labeling, few FRET pairs are suitable for monitoring intramolecular protein motions without being disruptive to protein folding and function. Here, we describe the synthesis and use of a minimally perturbing FRET pair comprised of methoxycoumarin maleimide (Mcm-Mal) and acridonylalanine (Acd). Acd can be incorporated into a protein through unnatural amino acid mutagenesis. Mcm-Mal is fluorogenic when reacted with cysteine and can label cysteine/Acd double mutant proteins. This labeling strategy provides an easy to install FRET pair with a working range or 15-40Å, making it ideal for monitoring most intramolecular motions. Additionally, Mcm/Acd FRET can be combined with tryptophan fluorescence for monitoring multiple protein motions via three color FRET.

A Strategic Approach for Fluorescence Imaging of Membrane Proteins in a Native-like Environment

Biological membranes are complex barriers in which membrane proteins and thousands of lipidic species participate in structural and functional interactions. Developing a strategic approach that allows uniform labeling of membrane proteins while maintaining a lipidic environment that retains functional interactions is highly desirable for invitro fluorescence studies. Herein, we focus on complementing current methods by integrating the powerful processes of unnatural amino acid mutagenesis, bioorthogonal labeling, and the detergent-free membrane protein solubilization based on the amphiphilic styrene-maleic acid (SMA) polymer. Importantly, the SMA polymer preserves a thermodynamically stable shellof phospholipids. The approach that we present is both rapid and generalizable providing a population of uniquely labeled membrane proteins in lipid nanoparticles for quantitative fluorescence-based studies.

Site-specific acylation of a bacterial virulence regulator attenuates infection.

Microbiota generates millimolar concentrations of short-chain fatty acids (SCFAs) that can modulate host metabolism, immunity and susceptibility to infection. Butyrate in particular can function as a carbon source and anti-inflammatory metabolite, but the mechanism by which it inhibits pathogen virulence has been elusive. Using chemical proteomics, we discovered that several virulence factors encoded by Salmonella pathogenicity island-1 (SPI-1) are acylated by SCFAs. Notably, a transcriptional regulator of SPI-1, HilA, was acylated on several key lysine residues. Subsequent incorporation of stable butyryl-lysine analogs using CRISPR-Cas9 gene editing and unnatural amino acid mutagenesis revealed that site-specific modification of HilA impacts its genomic occupancy, expression of SPI-1 genes and attenuates Salmonella enterica serovar Typhimurium invasion of epithelial cells as well as dissemination in vivo. Moreover, a multiple-site HilA lysine-acylation mutant strain of S. Typhimurium was resistant to butyrate-mediated suppression in vivo. Our results suggest prominent microbiota-derived metabolites may directly acylate virulence factors to inhibit microbial pathogenesis in vivo.

Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis

Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2. Our data, combined with previous studies, provide the molecular sequence of events in the coupled binding of sodium and aspartate to GltPh.