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1. Tailored Polypeptide Star Copolymers for 3D Printing of Bacterial Composites Via Direct Ink Writing

   https://doi.org/10.1002/adma.202207542  

   Murphy, Robert_D; Garcia, Ronnie_V; Oh, Seung_J; Wood, Tanner_J; Jo, Kyoo_D; Read_de_Alaniz, Javier; Perkins, Ed; Hawker, Craig_J (December 2022, Advanced Materials)

   Abstract Hydrogels hold much promise for 3D printing of functional living materials; however, challenges remain in tailoring mechanical robustness as well as biological performance. In addressing this challenge, the modular synthesis of functional hydrogels from 3‐arm diblock copolypeptide stars composed of an inner poly(l‐glutamate) domain and outer poly(l‐tyrosine) or poly(l‐valine) blocks is described. Physical crosslinking due to ß‐sheet assembly of these star block copolymers gives mechanical stability during extrusion printing and the selective incorporation of methacrylate units allows for subsequent photocrosslinking to occur under biocompatible conditions. This permits direct ink writing (DIW) printing of bacteria‐based mixtures leading to 3D objects with high fidelity and excellent bacterial viability. The tunable stiffness of different copolypeptide networks enables control over proliferation and colony formation for embeddedEscherichia colibacteria as demonstrated via isopropyl ß‐d‐1‐thiogalactopyranoside (IPTG) induction of green fluorescent protein (GFP) expression. This translation of molecular structure to network properties highlights the versatility of these polypeptide hydrogel systems with the combination of writable structures and biological activity illustrating the future potential of these 3D‐printed biocomposites. 

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2. Profiling tyrosine kinase substrate recognition using bacterial peptide display and deep sequencing

   https://doi.org/10.64898/2025.12.28.696772  

   Lee, Minhee; Shah, Neel H (December 2025, bioRxiv)

   Tyrosine kinases control a wide range of cell signaling pathways that are central to human physiology, and they are dysregulated in a variety of human diseases, most notably cancers. Our understanding of tyrosine kinase biology hinges upon a clear delineation of their protein substrates. Thus, much effort has been invested into defining the substrate specificities of tyrosine kinases, which is partly driven by recognition of the amino acid sequences surrounding the phospho-acceptor tyrosine residues. Numerous methods have been developed to profile tyrosine kinase sequence recognition, and these approaches have collectively demonstrated that different tyrosine kinases have distinct substrate sequence preferences. Here, we describe one such method that combines bacterial peptide display and deep sequencing to study tyrosine kinase substrate preferences. Our approach enables rapid measurement of relative phosphorylation efficiencies for thousands of peptides simultaneously. The genetically-encoded nature of the peptide libraries used with this method allows for facile and cheap construction of libraries tailored to answer a variety questions. Notably, our approach is compatible with genetic code expansion via Amber codon suppression, which allows for the construction and screening of libraries containing non-canonical amino acids. Importantly, results from this assay correlate strongly with quantitative measurements of enzyme kinetics, they corroborate previously reported tyrosine kinase substrate preferences, and they can reveal new insights into tyrosine kinase substrate specificity. 

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3. Tyrosine, cysteine, and proton coupled electron transfer in a ribonucleotide reductase-inspired beta hairpin maquette

   https://doi.org/10.1039/C9CC04067F  

   McCaslin, Tyler G.; Pagba, Cynthia V.; Hwang, Hyea; Gumbart, James C.; Chi, San-Hui; Perry, Joseph W.; Barry, Bridgette A. (August 2019, Chemical Communications)

   Tyrosine residues act as intermediates in proton coupled electron transfer reactions (PCET) in proteins. For example, in ribonucleotide reductase (RNR), a tyrosyl radical oxidizes an active site cysteine via a 35 Å pathway that contains multiple aromatic groups. When singlet tyrosine is oxidized, the radical becomes a strong acid, and proton transfer reactions, which are coupled with the redox reaction, may be used to control reaction rate. Here, we characterize a tyrosine-containing beta hairpin, Peptide O, which has a cross-strand, noncovalent interaction between its single tyrosine, Y5, and a cysteine (C14). Circular dichroism provides evidence for a thermostable beta-turn. EPR spectroscopy shows that Peptide O forms a neutral tyrosyl radical after UV photolysis at 160 K. Molecular dynamics simulations support a phenolic/SH interaction in the tyrosine singlet and radical states. Differential pulse voltammetry exhibits pH dependence consistent with the formation of a neutral tyrosyl radical and a p K a change in two other residues. A redox-coupled decrease in cysteine p K a from 9 (singlet) to 6.9 (radical) is assigned. At pD 11, picosecond transient absorption spectroscopy after UV photolysis monitors tyrosyl radical recombination via electron transfer (ET). The ET rate in Peptide O is indistinguishable from the ET rates observed in peptides containing a histidine and a cyclohexylalanine (Cha) at position 14. However, at pD 9, the tyrosyl radical decays via PCET, and the decay rate is slowed, when compared to the histidine 14 variant. Notably, the decay rate is accelerated, when compared to the Cha 14 variant. We conclude that redox coupling between tyrosine and cysteine can act as a PCET control mechanism in proteins. 

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4. Chemical Modification of Silk Proteins via Palladium‐Mediated Suzuki−Miyaura Reactions

   https://doi.org/10.1002/macp.202300307  

   Santen, Racine M.; Owens, Kayla M.; Echague, Keith C.; Murphy, Amanda R. (October 2023, Macromolecular Chemistry and Physics)

   Abstract Suzuki−Miyaura cross‐coupling reactions are used to modify the tyrosine residues onBombyx morisilkworm silk proteins using a water‐soluble palladium catalyst. First, model reactions using tyrosine derivatives are screened to determine optimal reaction conditions. For these reactions, a variety of aryl boronic acids, solvents, buffers, and temperature ranges are explored. Qualitative information on the reaction progress is collected via high‐performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR). Optimized reactions are then applied to silk proteins. It is demonstrated the ability to modify silk fibroin in solution by first iodinating the tyrosine residues on the protein, and then carrying out Suzuki‐Miyaura reactions with a variety of boronic acid derivatives. Modification of silk is confirmed with NMR, ion‐exchange chromatography (IEC), UV‐vis, and infrared spectroscopy (IR). 

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5. Role of cytosolic, tyrosine‐insensitive prephenate dehydrogenase in Medicago truncatula

   https://doi.org/10.1002/pld3.218  

   Schenck, Craig A.; Westphal, Josh; Jayaraman, Dhileepkumar; Garcia, Kevin; Wen, Jiangqi; Mysore, Kirankumar S.; Ané, Jean‐Michel; Sumner, Lloyd W.; Maeda, Hiroshi A. (May 2020, Plant Direct)

   Abstract l‐Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4‐hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrA_a), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrA_p), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterizedTnt1‐transposon mutants ofMtPDH1(pdh1) inMedicago truncatulato investigate PDH function. The pdh1mutants lackedPDHtranscript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence thatMtPDH1is responsible for the PDH activity detected inM. truncatula. Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity ofpdh1 mutants were not significantly reduced compared with wild‐type (Wt) during symbiosis with nitrogen‐fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume‐specific specialized metabolism. 

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