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1. Molecular Engineering of Cyclic Azobenzene‐Peptide Hybrid Ligands for the Purification of Human Blood Factor VIII via Photo‐Affinity Chromatography

   https://doi.org/10.1002/adfm.202213881  

   Prodromou, Raphael; Moore, Brandyn_David; Chu, Wenning; Deal, Halston; San_Miguel, Adriana; Brown, Ashley_Carson; Daniele, Michael_Angelo‐Anthony; Pozdin, Vladimir_Aleksandrovich; Menegatti, Stefano (January 2023, Advanced Functional Materials)

   Abstract The use of benign stimuli to control the binding and release of labile biologics for their isolation from complex feedstocks is a key goal of modern biopharmaceutical technology. This study introduces cyclic azobenzene‐peptide (CAP) ligands for the rapid and discrete photo‐responsive capture and release of blood coagulation factor VIII (FVIII). A predictive method—based on amino acid sequence and molecular architecture of CAPs—is developed to correlate the conformation ofcis/trans‐CAP photo‐isomers to FVIII binding and release. Combined in silico ‐ in vitro analysis of FVIII:peptide interactions guide the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G‐cyclo_AZOB[Lys‐YYKHLYN‐Lys]‐G on translucent chromatographic beads, features high binding capacity (>6 mg of FVIII per mL of resin) and rapid photo‐isomerization kinetics (τ < 30 s) when exposed to 420–450 nm light at the intensity of 0.1 W cm⁻². The adsorbent purifies FVIII from a recombinant harvest using a single mobile phase, affording high product yield (>90%), purity (>95%), and blood clotting activity. The CAPs introduced in this report demonstrate a novel route integrating gentle operational conditions in a rapid and efficient bioprocess for the purification of life‐saving biotherapeutics. 

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2. Probing the Ni ²⁺ ‐selective Response of Fluorescent Probe NiSensor‐1 with the NiCast Photocaged Complex ^{† ‡}

   https://doi.org/10.1111/php.13567  

   Hickey, Erin E.; Kennedy, Daniel P.; Gwizdala, Celina; Basa, Prem N.; Müller, Peter; MacDonald, John; Burdette, Shawn C. (December 2021, Photochemistry and Photobiology)

   Abstract CTEA (N,N‐bis[2‐(carboxylmethyl)thioethyl]amine) is a mixed donor ligand that has been incorporated into multiple fluorescent sensors such as NiSensor‐1 that was reported to be selective for Ni²⁺. Other metal ions such as Zn²⁺do not produce an emission response in aqueous solution. To investigate the coordination chemistry and selectivity of this receptor, we prepared NiCast, a photocage containing the CTEA receptor. Cast photocages undergo a photoreaction that decreases electron density on a metal‐bound aniline nitrogen atom, which shifts the binding equilibrium toward unbound metal ion. The unique selectivity of CTEA was examined by measuring the binding affinity of NiCast and the CTEA receptor for Ni²⁺, Zn²⁺, Cd²⁺and Cu²⁺under different conditions. In aqueous solution, Ni²⁺binds more strongly to the aniline nitrogen atom than Cd²⁺; however, in CH₃CN, the change in affinity virtually disappears. The crystal structure of [Cu(CTEA)], which exhibits a Jahn–Teller–distorted square pyramidal structure, was also analyzed to gain more insight into the underlying coordination chemistry. These studies suggest that the fluorescence selectivity of NiSensor‐1 in aqueous solution is due to a stronger interaction between the aniline nitrogen atom and Ni²⁺compared to other divalent metal ions except Cu²⁺. 

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3. Spectroscopic and metal binding properties of a de novo metalloprotein binding a tetrazinc cluster

   https://doi.org/10.1002/bip.23229  

   Chino, Marco; Zhang, Shao‐Qing; Pirro, Fabio; Leone, Linda; Maglio, Ornella; Lombardi, Angela; DeGrado, William F. (September 2018, Biopolymers)

   Abstract De novodesign provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron–sulfur clusters, the design of tetranuclear clusters with oxygen‐rich environments remains in its infancy. In previous work, we described the design of homotetrameric four‐helix bundles that bind tetra‐Zn²⁺clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal‐binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the correspondingapo‐proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn²⁺in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in theapostate, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster. 

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4. Melanization slows the rapid movement of fungal necromass carbon and nitrogen into both bacterial and fungal decomposer communities and soils

   https://doi.org/10.1128/msystems.00390-23  

   Maillard, François; Michaud, Talia J; See, Craig R; DeLancey, Lang C; Blazewicz, Steven J; Kimbrel, Jeffrey A; Pett-Ridge, Jennifer; Kennedy, Peter G (June 2023, mSystems)

   Wolfe, Benjamin E (Ed.)

   ABSTRACT Microbial necromass contributes significantly to both soil carbon (C) persistence and ecosystem nitrogen (N) availability, but quantitative estimates of C and N movement from necromass into soils and decomposer communities are lacking. Additionally, while melanin is known to slow fungal necromass decomposition, how it influences microbial C and N acquisition as well as elemental release into soils remains unclear. Here, we tracked decomposition of isotopically labeled low and high melanin fungal necromass and measured¹³C and¹⁵N accumulation in surrounding soils and microbial communities over 77 d in a temperate forest in Minnesota, USA. Mass loss was significantly higher from low melanin necromass, corresponding with greater¹³C and¹⁵N soil inputs. A taxonomically and functionally diverse array of bacteria and fungi was enriched in¹³C and/or¹⁵N at all sampling points, with enrichment being consistently higher on low melanin necromass and earlier in decomposition. Similar patterns of preferential C and N enrichment of many bacterial and fungal genera early in decomposition suggest that both microbial groups co-contribute to the rapid assimilation of resource-rich soil organic matter inputs. While overall richness of taxa enriched in C was higher than in N for both bacteria and fungi, there was a significant positive relationship between C and N in co-enriched taxa. Collectively, our results demonstrate that melanization acts as a key ecological trait mediating not only fungal necromass decomposition rate but also necromass C and N release and that both elements are rapidly co-utilized by diverse bacterial and fungal decomposers in natural settings. IMPORTANCERecent studies indicate that microbial dead cells, particularly those of fungi, play an important role in long-term carbon persistence in soils. Despite this growing recognition, how the resources within dead fungal cells (also known as fungal necromass) move into decomposer communities and soils are poorly quantified, particularly in studies based in natural environments. In this study, we found that the contribution of fungal necromass to soil carbon and nitrogen availability was slowed by the amount of melanin present in fungal cell walls. Further, despite the overall rapid acquisition of carbon and nitrogen from necromass by a diverse range of both bacteria and fungi, melanization also slowed microbial uptake of both elements. Collectively, our results indicate that melanization acts as a key ecological trait mediating not only fungal necromass decomposition rate, but also necromass carbon and nitrogen release into soil as well as microbial resource acquisition. 

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5. Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu ⁺ supplants entropically favoured Zn ²⁺ to form Cu ₄ ⁺ clusters under physiological conditions

   https://doi.org/10.1039/d2sc00676f  

   Mehlenbacher, Matthew R.; Elsiesy, Rahma; Lakha, Rabina; Villones, Rhiza Lyne; Orman, Marina; Vizcarra, Christina L.; Meloni, Gabriele; Wilcox, Dean E.; Austin, Rachel N. (January 2022, Chemical Science)

   Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d 10 metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn 2+ and Cu + binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn 2+ binding was measured by chelation titrations of Zn 7 MT-3, while Cu + binding was measured by Zn 2+ displacement from Zn 7 MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant ( K ) and the change in enthalpy (Δ H ) and entropy (Δ S ) for these metal ions binding to MT-3. Zn 2+ was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn 7 MT-2 with Cu + revealed that both MT isoforms have similar Cu + affinities and binding thermodynamics, indicating that Δ H and Δ S are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu 4 + –thiolate clusters when Cu + displaces Zn 2+ under physiological conditions. Comparison of the Zn 2+ and Cu + binding thermodynamics reveal that enthalpically-favoured Cu + , which forms Cu 4 + –thiolate clusters, displaces the entropically-favoured Zn 2+ . These results provide a detailed thermodynamic analysis of d 10 metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper. 

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