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1. Non-covalent complexes of the peptide fragment Gly-Asn-Asn-Gln-Gln-Asn-Tyr in the gas-phase. Photodissociative cross-linking, Born–Oppenheimer molecular dynamics, and ab initio computational binding study

   https://doi.org/10.1039/c8cp06893c  

   Huang, Shu R. ; Liu, Yang ; Tureček, František ( January 2019 , Physical Chemistry Chemical Physics)

   Non-covalent complexes of the short amyloid peptide motif Gly-Asn-Asn-Gln-Gln-Asn-Tyr (GNNQQNY) with peptide counterparts that were tagged with a diazirine ring at the N-termini (*GNNQQNY) were generated as singly charged ions in the gas phase. Specific laser photodissociation (UVPD) of the diazirine tag in the gas-phase complexes at 355 nm generated transient carbene intermediates that underwent covalent cross-linking with the target GNNQQNY peptide. The crosslinking yields ranged between 0.8 and 4.5%, depending on the combinations of peptide C-terminal amides and carboxylates. The covalent complexes were analyzed by collision-induced dissociation tandem mass spectrometry (CID-MS 3 ), providing distributions of cross-links at the target peptide amino acid residues. A general preference for cross-linking at the target peptide Gln-4-Gln-5-Asn-6-Tyr-7 segment was observed. Born–Oppenheimer molecular dynamics calculations were used to obtain 100 ps trajectories for nine lowest free-energy conformers identified by ωB97X-D/6-31+G(d,p) gradient geometry optimizations. The trajectories were analyzed for close contacts between the incipient carbene atom and the X–H bonds in the target peptide. The close-contact analysis pointed to the Gln-5 and Tyr-7 residues as the most likely sites of cross-linking, consistent with the experimental CID-MS 3 results. Non-covalent binding in the amide complexes was evaluated by DFT calculations of structures and energies. Although antiparallel arrangements of the GNNQQNY and *GNNQQNY peptides were favored in low-energy gas-phase and solvated complexes, the conformations and peptide–peptide interface surfaces were found to differ from the secondary structure of the dry interface in GNNQQNY motifs of amyloid aggregates. 

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2. Synthesis and conformational preferences of peptides and proteins with cysteine sulfonic acid

   https://doi.org/10.1039/D3OB00179B  

   Bhatt, Megh R. ; Zondlo, Neal J. ( March 2023 , Organic & Biomolecular Chemistry)

   Cysteine sulfonic acid (Cys-SO3H; cysteic acid) is an oxidative post-translational modification of cysteine, resulting from further oxidation from cysteine sulfinic acid (Cys- SO2H). Cysteine sulfonic acid is considered an irreversible post-translational modification, which serves as a biomarker of oxidative stress that has resulted in oxidative damage to proteins. Cysteine sulfonic acid is anionic, as a sulfonate (Cys-SO –; cysteate), in the ionization state that 3 is almost exclusively present at physiological pH (pKa ~ –2). In order to understand protein structural changes that can occur upon oxidation to cysteine sulfonic acid, we analyzed its conformational preferences, using experimental methods, bioinformatics, and DFT-based computational analysis. Cysteine sulfonic acid was incorporated into model peptides for α-helix and polyproline II helix (PPII). Within peptides, oxidation of cysteine to the sulfonic acid proceeds rapidly and efficiently at room temperature in solution with methyltrioxorhenium (MeReO3) and H2O2. Peptides containing cysteine sulfonic acid were also generated on solid phase using trityl-protected cysteine and oxidation with MeReO3 and H2O2. Using methoxybenzyl (Mob)-protected cysteine, solid-phase oxidation with MeReO3 and H2O2 generated the Mob sulfone precursor to Cys-SO – within fully synthesized peptides. These two solid-phase methods allow the synthesis of peptides containing either Cys-SO – or Cys-SO – in a 32 practical manner, with no solution-phase synthesis required. Cys-SO – had low PPII propensity 3 for PPII propagation, despite promoting a relatively compact conformation in φ. In contrast, in a PPII initiation model system, Cys-SO – promoted PPII relative to neutral Cys, with PPII initiation similar to Cys thiolate but less than Cys-SO – or Ala. In an α-helix model system, Cys- 2 SO – promoted α-helix near the N-terminus, due to favorable helix dipole interactions and 3 favorable α-helix capping via a sulfonate-amide side chain-main chain hydrogen bond. Across all peptides, the sulfonate side chain was significantly less ordered than that of the sulfinate. Analysis of Cys-SO – in the PDB revealed a very strong propensity for local (i/i or i/i+1) side 3 chain-main chain sulfonate-amide hydrogen bonds for Cys-SO –, with > 80% of Cys-SO – 33 residues exhibiting these interactions. DFT calculations conducted to explore these conformational preferences indicated that side chain-main chain hydrogen bonds of the sulfonate with the intraresidue amide and/or with the i+1 amide were favorable. However, hydrogen bonds to water or to amides, as well as interactions with oxophilic metals, were weaker for the sulfonate than the sulfinate, due to lower charge density on the oxygens in the sulfonate. 

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3. Donor acceptor fluorophores: synthesis, optical properties, TD-DFT and cytotoxicity studies

   https://doi.org/10.1039/D0OB02313B  

   Essam, Zahraa M. ; Ozmen, Guliz Ersoy ; Setiawan, Dani ; Hamid, Riri Rizkianty ; Abd El-Aal, Reda M. ; Aneja, Ritu ; Hamelberg, Donald ; Henary, Maged ( March 2021 , Organic & Biomolecular Chemistry)

   null (Ed.)

   Donor–π-acceptor (D–π-A) fluorophores consisting of a donor unit, a π linker, and an acceptor moiety have attracted attention in the last decade. In this study, we report the synthesis, characterization, optical properties, TD-DFT, and cytotoxicity studies of 17 near infrared (NIR) D–π-A analogs which have not been reported so far to the best of our knowledge. These fluorophores have chloroacrylic acid as the acceptor unit and various donor units such as indole, benzothiazole, benzo[ e ]indole, and quinoline. The fluorophores showed strong absorption in the NIR (700–970 nm) region due to their enhanced intramolecular charge transfer (ICT) between chloroacrylic acid and the donor moieties connected with the Vilsmeier–Haack linker. The emission wavelength maxima of the fluorophores were in between 798 and 870 nm. Compound 20 with a 4-quinoline donor moiety showed an emission wavelength above 1000 nm in the NIR II window. The synthesized fluorophores were characterized by 1 H NMR and 13 C NMR, and their optical properties were studied. Time dependent density functional theory (TD-DFT) calculations showed that the charge transfer occurs from the donor groups (indole, benzothiazole, benzo[ e ]indole, and quinoline) to the acceptor chloroacrylic acid moiety. Fluorophores with [HOMO] to [LUMO+1] transitions were shown to possess a charge separation character. The cytotoxicity of selected fluorophores, 4 , 7 , 10 and 12 was investigated against breast cancer cell lines and they showed better activity than the anti-cancer agent docetaxel. 

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4. Production of Class II MHC Proteins in Lentiviral Vector‐Transduced HEK‐293T Cells for Tetramer Staining Reagents

   https://doi.org/10.1002/cpz1.36  

   Willis, Richard A. ; Ramachandiran, Vasanthi ; Shires, John C. ; Bai, Ge ; Jeter, Kelly ; Bell, Donielle L. ; Han, Lixia ; Kazarian, Tamara ; Ugwu, Kyla C. ; Laur, Oskar ; et al ( February 2021 , Current Protocols)

   Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed inE. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.

   This article was corrected on 19 July 2022. See the end of the full text for details.

   Basic Protocol 1: Lentivirus production and expression line creation

   Support Protocol 1: Six‐well assay for estimation of production cell line yield

   Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His‐tags

   Basic Protocol 2: Cultures for production of Class II MHC proteins

   Basic Protocol 3: Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatography

   Alternate Protocol 1: IMAC purification of His‐tagged Class II MHC

   Support Protocol 3: Protein concentration measurements and adjustments

   Support Protocol 4: Polishing purification by anion‐exchange chromatography

   Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation

   Basic Protocol 4: Peptide exchange

   Basic Protocol 5: Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry

   Alternate Protocol 2: Native isoelectric focusing to validate MHC‐II peptide loading

   Basic Protocol 6: Multimerization

   Basic Protocol 7: Staining cells with Class II MHC tetramers

    

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5. Emerging investigator series: characterization of silver and silver nanoparticle interactions with zinc finger peptides

   https://doi.org/10.1039/C9EN00065H  

   Park, Grace ; Amaris, Zoe N. ; Eiken, Madeline K. ; Baumgartner, Karl V. ; Johnston, Kathryn A. ; Williams, Mari A. ; Marckwordt, Jasmine G. ; Millstone, Jill E. ; Splan, Kathryn E. ; Wheeler, Korin E. ( January 2019 , Environmental Science: Nano)

   In biological systems, chemical and physical transformations of engineered silver nanomaterials (AgENMs) are mediated, in part, by proteins and other biomolecules. Metalloprotein interactions with AgENMs are also central in understanding toxicity, antimicrobial, and resistance mechanisms. Despite their readily available thiolate and amine ligands, zinc finger (ZF) peptides have thus far escaped study in reaction with AgENMs and their Ag( i ) oxidative dissolution product. We report spectroscopic studies that characterize AgENM and Ag( i ) interactions with two ZF peptides that differ in sequence, but not in metal binding ligands: the ZF consensus peptide CP-CCHC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Both ZF peptides catalyze AgENM (10 and 40 nm, citrate coated) dissolution and agglomeration, two important AgENM transformations that impact bioreactivity. AgENMs and their oxidative dissolution product, Ag( i )(aq), mediate changes to ZF peptide structure and metalation as well. Spectroscopic titrations of Ag( i ) into apo-ZF peptides show an Ag( i )–thiolate charge transfer band, indicative of Ag( i )–ZF binding. Fluorescence studies of the Zn( ii )–NCp_7 complex indicate that the Ag( i ) also effectively competes with the Zn( ii ) to drive Zn( ii ) displacement from the ZFs. Upon interaction with AgENMs, Zn( ii ) bound ZF peptides show a secondary structural change in circular dichroism spectroscopy toward an apo-like structure. The results suggest that Ag( i ) and AgENMs may alter ZF protein function within the cell. 

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