Characterization of a synthetic peroxodiiron(III) protein model complex by nuclear resonance vibrational spectroscopy

The vibrational spectrum of an η[superscript 1],η[superscript 1]-1,2-peroxodiiron(III) complex was measured by nuclear resonance vibrational spectroscopy and fit using an empirical force field analysis. Isotopic 18O2 labelling studies revealed a feature involving motion of the {Fe2(O2)}[superscript 4+] core that was not previously observed by resonance Raman spectroscopy.National Institute of General Medical Sciences (U.S.) (GM-032134

Vesicular Zinc Promotes Presynaptic and Inhibits Postsynaptic Long-Term Potentiation of Mossy Fiber-CA3 Synapse

The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits a form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions, vesicular zinc is critical to proper function of hippocampal circuitry in health and disease.National Institute of General Medical Sciences (U.S.) (Grant GM065519

Vesicular Zinc Promotes Presynaptic and Inhibits Postsynaptic Long-Term Potentiation of Mossy Fiber-CA3 Synapse

The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits a form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions, vesicular zinc is critical to proper function of hippocampal circuitry in health and disease.National Institute of General Medical Sciences (U.S.) (Grant GM065519

Oxidation Reactions Performed by Soluble Methane Monooxygenase Hydroxylase Intermediates Hperoxo and Q Proceed by Distinct Mechanisms

Soluble methane monooxygenase is a bacterial enzyme that converts methane to methanol at a carboxylate-bridged diiron center with exquisite control. Because the oxidizing power required for this transformation is demanding, it is not surprising that the enzyme is also capable of hydroxylating and epoxidizing a broad range of hydrocarbon substrates in addition to methane. In this work we took advantage of this promiscuity of the enzyme to gain insight into the mechanisms of action of Hperoxo and Q, two oxidants that are generated sequentially during the reaction of reduced protein with O2. Using double-mixing stopped-flow spectroscopy, we investigated the reactions of the two intermediate species with a panel of substrates of varying C−H bond strength. Three classes of substrates were identified according to the rate-determining step in the reaction. We show for the first time that an inverse trend exists between the rate constant of reaction with Hperoxo and the C−H bond strength of the hydrocarbon examined for those substrates in which C−H bond activation is rate-determining. Deuterium kinetic isotope effects revealed that reactions performed by Q, but probably not Hperoxo, involve extensive quantum mechanical tunneling. This difference sheds light on the observation that Hperoxo is not a sufficiently potent oxidant to hydroxylate methane, whereas Q can perform this reaction in a facile manner. In addition, the reaction of Hperoxo with acetonitrile appears to proceed by a distinct mechanism in which a cyanomethide anionic intermediate is generated, bolstering the argument that Hperoxo is an electrophilic oxidant that operates via two-electron transfer chemistry.National Institute of General Medical Sciences (U.S.) (grant GM032134)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM08334

Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway

Stopped-flow kinetic investigations of soluble methane monooxygenase (sMMO) from M. capsulatus (Bath) have clarified discrepancies that exist in the literature regarding several aspects of catalysis by this enzyme. The development of thorough kinetic analytical techniques has led to the discovery of two novel oxygenated iron species that accumulate in addition to the well-established intermediates H[subscript peroxo] and Q. The first intermediate, P*, is a precursor to H[subscript peroxo] and was identified when the reaction of reduced MMOH and MMOB with O[subscript 2] was carried out in the presence of ≥540 μM methane to suppress the dominating absorbance signal due to Q. The optical properties of P* are similar to those of H[subscript peroxo], with ε[subscript 420] = 3500 M[superscript −1] cm[superscript −1] and ε[subscript 720] = 1250 M[superscript −1] cm[superscript −1]. These values are suggestive of a peroxo-to-iron(III) charge-transfer transition and resemble those of peroxodiiron(III) intermediates characterized in other carboxylate-bridged diiron proteins and synthetic model complexes. The second identified intermediate, Q*, forms on the pathway of Q decay when reactions are performed in the absence of hydrocarbon substrate. Q* does not react with methane, forms independently of buffer composition, and displays a unique shoulder at 455 nm in its optical spectrum. Studies conducted at different pH values reveal that rate constants corresponding to P* decay/H[subscript peroxo] formation and H[subscript peroxo] decay/Q formation are both significantly retarded at high pH and indicate that both events require proton transfer. The processes exhibit normal kinetic solvent isotope effects (KSIEs) of 2.0 and 1.8, respectively, when the reactions are performed in D[subscript 2]O. Mechanisms are proposed to account for the observations of these novel intermediates and the proton dependencies of P* to H[subscript peroxo] and H[subscript peroxo] to Q conversion.National Institute of General Medical Sciences (U.S.) (Grant GM032134)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM08334

Multiple Roles of Component Proteins in Bacterial Multicomponent Monooxygenases: Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas sp. OX1

Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In this study, we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H2O2) generation. Single-turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was 50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single-turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to H2O2 formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed.National Institute of General Medical Sciences (U.S.) (grant GM032134)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM08334)CEINGE - Biotecnologie Avanzate (Italy

Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research.

Antibody-based therapeutics and recombinant protein reagents are often produced in mammalian expression systems, which provide human-like post-translational modifications. Among the available mammalian cell lines used for recombinant protein expression, Chinese hamster ovary (CHO)-derived suspension cells are generally utilized because they are easy to culture and tend to produce proteins in high yield. However, some proteins purified from CHO cell overexpression suffer from clipping and display undesired non-human post translational modifications (PTMs). In addition, CHO cell lines are often not suitable for producing proteins with many glycosylation motifs for structural biology studies, as N-linked glycosylation of proteins poses challenges for structure determination by X-ray crystallography. Hence, alternative and complementary cell lines are required to address these issues. Here, we present a robust method for expressing proteins in human embryonic kidney 293 (HEK293)-derived stable pools, leading to recombinant protein products with much less clipped species compared to those expressed in CHO cells and with higher yield compared to those expressed in transiently-transfected HEK293 cells. Importantly, the stable pool generation protocol is also applicable to HEK293S GnTI- (N-acetylglucosaminyltransferase I-negative) and Expi293F GnTI- suspension cells, facilitating production of high yields of proteins with less complex glycans for use in structural biology projects. Compared to HEK293S GnTI- stable pools, Expi293F GnTI- stable pools consistently produce proteins with similar or higher expression levels. HEK293-derived stable pools can lead to a significant cost reduction and greatly promote the production of high-quality proteins for diverse research projects

Improving the catalytic performance of an artificial metalloenzyme by computational design

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η(5)-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5(-)), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46-64-fold improved affinity for the iridium pianostool complex [(η(5)-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model

VERITAS: Harnessing the power of nomenclature in biologic discovery

ABSTRACTWe are entering an era in which therapeutic proteins are assembled using building block-like strategies, with no standardized schema to discuss these formats. Existing nomenclatures, like AbML, sacrifice human readability for precision. Therefore, considering even a dozen such formats, in combination with hundreds of possible targets, can create confusion and increase the complexity of drug discovery. To address this challenge, we introduce Verified Taxonomy for Antibodies (VERITAS). This classification and nomenclature scheme is extensible to multispecific therapeutic formats and beyond. VERITAS names are easy to understand while drawing direct connections to the structure of a given format, with or without specific target information, making these names useful to adopt in scientific discourse and as inputs to machine learning algorithms for drug development