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1. Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy

   https://doi.org/10.1101/2024.10.29.620861  

   Lee, Song-Yi; Roh, Heegwang; Gonzalez-Perez, David; Mackey, Mason R; Kim, Keun-Young; Hoces, Daniel; McLaughlin, Colleen N; Adams, Stephen R; Nguyen, Khanh; Luginbuhl, David J; et al (October 2024, bioRxiv)

   Abstract Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H₂O₂. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O₂instead of toxic H₂O₂, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID. 

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2. Characterization and effect of metal ions on the formation of the Thermus thermophilus Sco mixed disulfide intermediate

   https://doi.org/10.1002/pro.3502  

   Lopez, Liezelle C.; Mukhitov, Nikita; Handley, Lindsey D.; Hamme, Cristina S.; Hofman, Cristina R.; Euers, Lindsay; McKinney, Jennifer R.; Piers, Amani D.; Wadler, Ellen; Hunsicker‐Wang, Laura M. (October 2018, Protein Science)

   Abstract The Sco protein fromThermus thermophilushas previously been shown to perform a disulfide bond reduction in the Cu_Aprotein fromT. thermophilus, which is a soluble protein engineered from subunit II of cytochromeba₃oxidase that lacks the transmembrane helix. The native cysteines onTtSco andTtCu_Awere mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine inTtCu_Aand subsequent release upon incubation with the complementaryTtSco protein demonstrated the formation of the mixed disulfide intermediate. The cysteine ofTtSco that attacks the disulfide bond in the targetTtCu_Aprotein was determined to beTtSco Cysteine 49. This cysteine is likely more reactive than Cysteine 53 due to a higher degree of solvent exposure. Removal of the metal binding histidine, His 139, does not change MDI formation. However, altering the arginine adjacent to the reactive cysteine in Sco (Arginine 48) does alter the formation of the MDI. Binding of Cu²⁺or Cu⁺toTtSco prior to reaction withTtCu_Awas found to preclude formation of the mixed disulfide intermediate. These results shed light on a mechanism of disulfide bond reduction by theTtSco protein and may point to a possible role of metal binding in regulating the activity. ImportanceThe function of Sco is at the center of many studies. The disulfide bond reduction in Cu_Aby Sco is investigated herein and the effect of metal ions on the ability to reduce and form a mixed disulfide intermediate are also probed. 

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3. Reaction mechanism for atmospheric pressure plasma treatment of cysteine in solution

   https://doi.org/10.1088/1361-6463/ace196  

   Polito, Jordyn; Herrera Quesada, María J.; Stapelmann, Katharina; Kushner, Mark J. (July 2023, Journal of Physics D: Applied Physics)

   Abstract Mechanisms for the cold atmospheric plasma (CAP) treatment of cells in solution are needed for more optimum design of plasma devices for wound healing, cancer treatment, and bacterial inactivation. However, the complexity of organic molecules on cell membranes makes understanding mechanisms that result in modifications (i.e. oxidation) of such compounds difficult. As a surrogate to these systems, a reaction mechanism for the oxidation of cysteine in CAP activated water was developed and implemented in a 0-dimensional (plug-flow) global plasma chemistry model with the capability of addressing plasma-liquid interactions. Reaction rate coefficients for organic reactions in water were estimated based on available data in the literature or by analogy to gas-phase reactions. The mechanism was validated by comparison to experimental mass-spectrometry data for COST-jets sustained in He/O₂, He/H₂O and He/N₂/O₂mixtures treating cysteine in water. Results from the model were used to determine the consequences of changing COST-jet operating parameters, such as distance from the substrate and inlet gas composition, on cysteine oxidation product formation. Results indicate that operating parameters can be adjusted to select for desired cysteine oxidation products, including nitrosylated products. 

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4. Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform

   https://doi.org/10.1093/synbio/ysac008  

   Sakai, Andrei; Deich, Christopher R; Nelissen, Frank H; Jonker, Aafke J; Bittencourt, Daniela M; Kempes, Christopher P; Wise, Kim S; Heus, Hans A; Huck, Wilhelm T; Adamala, Katarzyna P; et al (February 2022, Synthetic Biology)

   Abstract Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract 

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5. Aromatic Residue Positioning Influences Helical Peptoid Structure in Aqueous Solution

   https://doi.org/10.1055/a-2238-5394  

   Javed, Jwwad M; Scukas, Katherine; Nguyen, Michelle T; Fuller, Amelia A (January 2024, Synlett)

   Abstract Water-soluble peptidomimetics, including peptoids, are promising functional surrogates for biologically relevant, amphiphilic, helical peptides. Twenty amphiphilic peptoid hexamers with predicted helical structures were designed, prepared, and studied using circular dichroism (CD) spectroscopy. The site-specific contributions of aromatic and charged residues to the helical structure of peptoid hexamers in aqueous solution was evaluated, revealing that aromatic residue positioning most significantly impacts structure. 

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