Manganese is perhaps the most electronically versatile element, yet the redox properties, reactivity, and catalytic applications of low-valent Mn0/Mn-I complexes remain underexplored due to the propensity for Mn0 to dimerize, quenching high-energy metalloradicals. We report a series of redox-active monometallic MnI, Mn0 and Mn-I complexes containing a BH2-bridged dicarbene, characterized using a suite of experimental and cutting-edge computational (DFT) methods. Slow electron transfer kinetics at MnI/0 are observed, with computations and electrochemical simulations in excellent agreement with experimental values. Despite the lack of steric bulk at the BH2-bridged Mn0, the tBu groups at the dicarbene provide adequate steric protection to prevent dimerization, with percent buried volume (%V bur) serving as a valuable steric ranking tool. We also show that a %V bur > 83% prevents dimerization for a diverse array of Mn0 complexes from the literature. Ligand sterics of BPh2-and BH2-bridged complexes dictate reaction outcomes when MnI and Mn-I are exposed to nucleophiles and electrophiles, respectively, while Mn0 facilitates the radical cycloisomerization catalysis of 6-iodo-1-hexene at room temperature. This work underscores the importance of ligand sterics in rationalizing reactivity patterns at Mn and provides valuable insights for designing chelating ligands that can selectively leverage MnI/0/‑I states in redox-mediated catalytic reactions.

Inhibition of enzyme catalysis is critical for regulating cellular metabolism. Gentisate and salicylate 1,2-dioxygenases (GDO and SDO) are primary enzymes in the key gentisic pathway, which perform O2-dependent degradation of aromatic compounds with the aid of a ferrous cofactor. Here, we report on the inhibition of the reaction catalysis of GDO and SDO by natural amino acids. Steady-state Michaelis-Menten studies monitoring the rate of the reaction in the presence of 18 amino acids show that l-histidine primarily inhibits SDO- and GDO-mediated catalysis. In addition, l-threonine and l-asparagine also inhibit the SDO-catalyzed chemical reaction with a diminished capacity. All amino acids competitively inhibit the catalysis suggesting their binding in the catalytic cavity. While l-histidine exhibits inhibition with a binding affinity of 80 μM (SDO) and 200 μM (GDO), d-histidine does not influence the rate of the chemical reaction, demonstrating that the chirality of the amino acid is critical for supporting optimal interactions that enable binding into the catalytic pocket. Lack of inhibition by small amino acids such as l-glycine suggests that the binding of the inhibitor is mediated by both the metal-ligating backbone carboxylate group and favorable interactions between the side chain and the catalytic cavity. Using nitric oxide as a surrogate for O2 and EPR spectroscopy, we show that inhibitor binding does not obstruct the access of O2 to the active site. In addition, EPR results demonstrate that unlike substrate binding, which introduces distortions, inhibitor binding does not alter the geometry of the metal center.

Antimicrobial protein S100A12 sequesters Zn(II) via a His3Asp motif to inhibit pathogens during infection. Here, UV-vis and NMR spectroscopies and molecular dynamics (MD) simulations are used to gain molecular insight into the Zn(II) chelation properties of S100A12 under pH conditions relevant to infection and inflammation. UV-vis measurements show that binding of Zn(II) to apo S100A12 exhibits a sigmoidal dependence with pH, beginning its decline at pH 7.0 and vanishing at pH 4.0. In the Ca(II)-bound protein, a similar sigmoidal curve is found to exhibit an acidic shift, an effect not attributed to a Ca(II)-induced pKa suppression of the His3Asp scaffold. NMR measurements show that upon lowering the pH, resonances exhibit nonlinear migration trends with pH, suggesting the occurrence of several proton binding events, consistent with the sigmoidal pH dependence of Zn(II) binding. Analysis of the NMR chemical shifts versus pH shows that both apo- and Ca(II)-bound protein undergo conformational changes exhibiting spatial correlations, which are dispersed across the polypeptide in the apo protein and confined to discrete regions in Ca(II)-S100A12. MD simulations show the formation of a strong salt bridge within the His3Asp scaffold of the apo protein upon protonation of Zn(II)-ligating histidines. Geometric constraints imposed by Ca(II) in the Ca(II)-bound protein hinder the formation of similar salt bridges until protonation of a histidine residue external to the His3Asp and near Ca(II) takes place. Overall, our experimental and computational results support a scenario where protonation of the His3Asp motif triggers the loss of Zn(II) binding and yields protonated histidine residues prone to form stable salt bridges. In Ca(II)-S100A12, formation of stable salt bridges at pH 7 is hindered compared to the apo form, thus extending Zn(II)-binding affinity to lower pH.

Cupin dioxygenases such as salicylate 1,2-dioxygense (SDO) perform aromatic C-C bond scission via a 3-His motif tethered iron cofactor. Here, transient kinetics measurements are used to monitor the catalytic cycle of SDO by using a nitro-substituted substrate analog, 3-nitrogentisate. Compared to the natural substrate, the nitro group reduces the enzymatic kcat by 500-fold, thereby facilitating the detection and kinetic characterization of reaction intermediates. Sums and products of reciprocal relaxation times derived from kinetic measurements were found to be linearly dependent on O2 concentration, suggesting reversible formation of two distinct intermediates. Dioxygen binding to the metal cofactor takes place with a forward rate of 5.9x103 M-1 s-1: two orders of magnitude slower than other comparable ring-cleaving dioxygenses. Optical chromophore of the first intermediate is distinct from the in situ generated SDO Fe(III)-O2 & sdot;- complex but closer to the enzyme-substrate precursor. Salicylate dioxygenase performs aromatic C-C bond scission of its substrates, salicylate and gentisate, with the aid of iron cofactor tethered to a 3-His binding motif. By utilizing a nitro substituted substrate analog, which attenuates the enzymatic kcat by 500-fold, detection and kinetics characterization two reaction intermediates was performed with stopped-flow optical absorption spectroscopy. image

Gentisate and salicylate 1,2-dioxygenases (GDO and SDO) facilitate aerobic degradation of aromatic rings by inserting both atoms of dioxygen into their substrates, thereby participating in global carbon cycling. The role of acid-base catalysts in the reaction cycles of these enzymes is debatable. We present evidence of the participation of a proton shuffler during catalysis by GDO and SDO. The pH dependence of Michaelis-Menten parameters demonstrates that a single proton transfer is mandatory for the catalysis. Measurements at variable temperatures and pHs were used to determine the standard enthalpy of ionization (Delta H (ion)degrees) of 51 kJ/mol for the proton transfer event. Although the observed apparent pK a in the range of 6.0-7.0 for substrates of both enzymes is highly suggestive of a histidine residue, Delta H-ion degrees establishes an arginine residue as the likely proton source, providing phylogenetic relevance for this strictly conserved residue in the GDO family. We propose that the atypical 3-histidine ferrous binding scaffold of GDOs contributes to the suppression of arginine pK a and provides support for this argument by employing a 2-histidine-1-carboxylate variant of the enzyme that exhibits elevated pKa. A reaction mechanism considering the role of the proton source in stabilizing key reaction intermediates is proposed.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

Vanadium and oxygen centers are prevalent in a plethora of inorganic complexes ranging from functional materials to biological systems and pharmaceutical agents. Nuclear magnetic resonance (NMR) spectroscopy can provide a detailed characterization of electronic and coordination environments of vanadium and oxygen centers. Compared to the more common and relatively well-studied NMR nuclei such as 1 H, 13 C, 15 N and 31 P, 17 O, and 51 V NMR studies often suffer from sensitivity and resolution challenges owing to their unique nuclear properties. Despite these limitations, several detailed studies undertaken in the literature illustrate that extensive characterization of these centers can be performed by NMR, particularly in the solid state, leading to critical evaluation of the properties of the system under consideration. These studies underscore the development in NMR methodologies and hardware, which has allowed for 17 O and 51 V NMR measurements to become more widely available to researchers. In this chapter, we present an overview of the recent applications of 17 O and 51 V solid state NMR spectroscopy to investigate oxygen and vanadium centers in inorganic complexes relevant to materials and biological systems.

Calgranulin C performs antimicrobial activity in the human immune response by sequestering Zn(II). This biological function is afforded with the aid of two structurally distinct Ca(II)-binding EF hand motifs, wherein one of which bears an unusual amino acid sequence. Here, we utilize solution state NMR relaxation measurements to investigate the mechanism of Ca(II)-modulated enhancement of Zn(II) sequestration by calgranulin C. Using C13 /N15 CPMG dispersion experiments we have measured pH-dependent major and minor state populations exchanging on micro-to-millisecond timescale. This conformational exchange takes place exclusively in the Ca(II)-bound state and can be mapped to residues located in the EF-I loop and the linker between the tandem EF hands. Molecular dynamics (MD) simulations spanning nano-to-microsecond timescale offer insights into the role of pH-dependent electrostatic interactions in EF-hand dynamics. Our results suggest a pH-regulated dynamic equilibrium of conformations that explore a range of "closed" and partially "open" sidechain configurations within the Zn(II) binding site. We propose a novel mechanism by which Ca(II) binding to a non-canonical EF loop regulates its flexibility and tunes the antimicrobial activity of calgranulin C.

Fluorine Doped Tin Oxide (FTO) electrode was fabricated with reduced Graphene Oxide (rGO) for sensitive detection of Japanese encephalitis virus (JEV) non-structural 1 (NS1) protein. Beforehand, in-silico 3D structure, stability, and docking of recombinant JEV NS1 antigen (NS1-Ag) and antibody (Ab) was evaluated. The recombinant NS1 Ag of 42 kDa was produced in-house by successful cloning into pET-28a(+) plasmid and further expressed using BL21 Escherichia coli (E. coli) cells. The NS1 Ag was used to raise polyclonal antibodies (Ab) and both were characterized via Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western Blot, Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF), and Enzyme-Linked Immunosorbent Assay (ELISA). Further characterisation of all binding events such as rGO synthesis, and its conjugation with NS1 Ab, and NS1 Ag were confirmed through Fourier-Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy, Energy Dispersive X-Ray Analysis (EDX), Scanning Electron Microscopy (SEM), Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). The fabricated FTO electrode was optimised for various parameters such as pH, response time, temperature, concentration, and scan rate. The detection of JEV NS1 Ag was performed in buffer (LOD- 0.92 fM) as well in spiked serum (LOD- 1.3 fM) samples. The JEV NS1 Ab showed negligible cross-reactivity with other flaviviral NS1 Ag, provided a rapid response within 5 s, and remained stable up to 4 weeks. Furthermore, the fabricated immunosensor may be a potential candidate for further miniaturisation for accurate and early diagnosis of JEV in clinical samples.

Ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus strains is posing new COVID-19 diagnosis and treatment challenges. To help efforts to meet these challenges we examined data acquired from proteomic analyses of human SARS-CoV-2-infected cell lines and samples from COVID-19 patients. Initially, 129 unique peptides were identified, which were rigorously evaluated for repeats, disorders, polymorphisms, antigenicity, immunogenicity, toxicity, allergens, sequence similarity to human proteins, and contributions from other potential cross-reacting pathogenic species or the human saliva microbiome. We also screened SARS-CoV-2-infected NBHE and A549 cell lines for presence of antigenic peptides, and identified paratope peptides from crystal structures of SARS-CoV-2 antigen-antibody complexes. We then selected four antigen peptides for docking with known viral unbound T-cell receptor (TCR), class I and II peptide major histocompatibility complex (pMHC), and identified paratope sequences. We also tested the paratope binding affinity of SARS-CoV T- and B-cell peptides that had been previously experimentally validated. The resultant antigenic peptides have high potential for generating SARS-CoV-2-specific antibodies, and the paratope peptides can be directly used to develop a COVID-19 diagnostics assay. The presented genomics and proteomics-based in-silico approaches have apparent utility for identifying new diagnostic peptides that could be used to fight SARS-CoV-2.

S100A12 is a member of the Ca(II) binding S100 family of proteins that functions within the human innate immune system. As a metal‐chelator, S100A12 binds both Ca(II) and Zn(II) at structurally distinct sites. Ca(II) binding enhances the Zn(II) binding affinity and the mechanism of the mode of action of Ca(II) is not fully understood. Here, using solution and solid state NMR spectroscopy, we have conducted structural characterization of S100A12 upon Ca(II) and Zn(II) binding. We demonstrate that distinct conformational changes are introduced in helix II and the hinge domain of the protein upon Zn(II) binding to Ca(II)‐S100A12, suggesting that both ions modulate the structure of S100A12. Moreover, Ca(II)‐S100A12 exhibits a broader pH range for Zn(II) binding compared to the apo protein by restricting conformational changes to Zn(II) binding residue, D25. In conclusion, our results indicate that Ca(II) stabilizes Zn(II) binding affinity by modulating structural changes. These findings will be of value for understanding of the nutrient‐sequestration effect of neutrophils and the interaction between the S100A12 and other receptors such as receptor for advanced glycation endproducts (RAGE) and toll‐like receptor 4 (TLR).Support or Funding InformationThis work was supported by the City University of New York startup funds, the National Institutes of Health (R15GM131338) and PSC CUNY award (62234‐00 50) to Rupal Gupta. NMR data presented herein were collected (in part) at the City University of New York’s Advanced Science Research Center (CUNY ASRC) Biomolecular NMR Facility.MAS NMR spectra of apo and Zn2+‐S100A12. a) Left: DARR (14.1 T) and NCACX (16.4 T) 2D spectra apo S100A12 with resonance assignments; Right: strip plots from CBCACONH (blue) and HNCACB (orange) solution NMR spectra for residues T26‐S28. Signals belonging to an assigned residue in MAS and solution spectra are connected with the same color; b) overlay of Zn2+‐(orange) and apo‐(blue) S100A12 DARR spectra acquired at 14.1 T and; c)–e) expansions of b) with assignments of the perturbed residues.Figure 1

Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn2+ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co2+ as a surrogate. Apo-S100A12 exhibits strong Co2+ binding between pH 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca2+-S100A12 can retain nanomolar Co2+ binding up to pH 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side-chain protonation of the Co2+/Zn2+ binding histidine residues. Instead, the calcium-mediated modulation is achieved by restraining pH-dependent conformational changes to EF loop 1, which contains Co2+/Zn2+ binding Asp25. This calcium-induced enhancement of Co2+/Zn2+ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.

Chemical shift tensors obtained from solid-state NMR spectroscopy are very sensitive reporters of structure and dynamics in proteins. While accurate 13 C and 15 N chemical shift tensors are accessible by magic angle spinning (MAS) NMR, their quantum mechanical calculations remain challenging, particularly for 15 N atoms. Here we compare experimentally determined backbone 13 C α and 15 N H chemical shift tensors by MAS NMR with hybrid quantum mechanics/molecular mechanics/molecular dynamics (MD-QM/MM) calculations for the carbohydrate-binding domain of galectin-3. Excellent agreement between experimental and computed 15 N H chemical shift anisotropy values was obtained using the Amber ff15ipq force field when solvent dynamics was taken into account in the calculation. Our results establish important benchmark conditions for improving the accuracy of chemical shift calculations in proteins and may aid in the validation of protein structure models derived by MAS NMR.

Abstract This report details the synthesis and characterization of six new Mn(II) complexes coordinated to systematically varied 2,2',2''-nitrilotris(N-arylacetamidate) ligands (LR; R = NO2, Cl, Br, H, Me, and OMe). The complexes are synthesized as the di-tetramethylammonium salts [Me4N]2[MnLR(OAc)]. The nitro variant MnNO2 afforded crystals suitable for X-ray diffraction and its molecular structure is reported. We previously reported the crystal structures of FeNO2 and ZnNO2 and additionally report herein the synthesis and characterization of CoNO2. Using these four molecules, we conduct a brief comparison of the bond metrics to demonstrate that the primary difference governing structural changes is likely due to ionic crystal radii changes rather than electronic properties. The electrochemical properties of the MnR complexes were additionally explored with cyclic voltammetry, which revealed that the series is modulated by the various electronic substituents on the aryl groups of the ligands. The electrochemical studies also revealed, consistent with our previous report, that the acetate ligand on the MnR complexes is labile. Finally, a Hammett plot was constructed using the potentials obtained from cyclic voltammetry, which is compared with a few other similar transition metal complexes.

Gentisate 1,2-dioxygenases (GDOs) are non-heme iron enzymes that catalyze the oxidation of dihydroxylated aromatic substrate, gentisate (2,5-dihydroxybenzoate). Salicylate 1,2-dioxygenase (SDO), a member of the GDO family, performs the ring scission of monohydroxylated substrates such as salicylate, thereby oxidizing a broader range of substrates compared to GDOs. Although the two types of enzymes share a high degree of sequence similarity, the origin of substrate specificity between SDO and GDOs is not understood. We present electron paramagnetic resonance (EPR) investigation of ferrous-nitrosyl complexes of SDO and a GDO from the bacterium Corynebacterium glutamicum (GDOCg). The EPR spectra of these complexes, which mimic the Fe-substrate-O2 intermediates in the catalytic cycle, show unexpected differences in the substrate binding mode and the coordination geometry of the metal cofactor in the two enzymes. Binding of substrate to the ferrous center increases the symmetry of the Fe(II)–NO complex in SDO, while a reverse trend is observed in GDOCg where substrate ligation reduces the symmetry of the nitrosyl complex. Identical EPR spectra were obtained for the NO derivatives of a variant of GDOCg(A112G), which can oxidize salicylate, and wild-type GDOCg revealing that the A112G mutation does not alter the nature of the Fe-substrate-O2 ternary complex.

S100A12 is a member of the Ca2+ binding S100 family of proteins that functions within the human innate immune system. Zinc sequestration by S100A12 confers antimicrobial activity when the protein is secreted by neutrophils. Here, we demonstrate that Ca2+ binding to S100A12's EF-hand motifs and Zn2+ binding to its dimeric interface cooperate to induce reversible self-assembly of the protein. Solution and magic angle spinning nuclear magnetic resonance spectroscopy on apo-, Ca2+-, Zn2+-, and Ca2+,Zn2+-S100A12 shows that significant metal binding-induced chemical shift perturbations, indicative of conformational changes, occur throughout the polypeptide chain. These perturbations do not originate from changes in the secondary structure of the protein, which remains largely preserved. While the overall structure of S100A12 is dominated by Ca2+ binding, Zn2+ binding to Ca2+-S100A12 introduces additional structural changes to helix II and the hinge domain (residues 38-53). The hinge domain of S100A12 is involved in the molecular interactions that promote chemotaxis for human monocyte, acute inflammatory responses and generates edema. In Ca2+-S100A12, helix II and the hinge domain participate in binding with the C-type immunoglobulin domain of the receptor for advanced glycation products (RAGE). We discuss how the additional conformational changes introduced to these domains upon Zn2+ binding may also impact the interaction of S100A12 and target proteins such as RAGE.

We report dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) NMR spectroscopy in viral capsids from HIV-1 and bacteriophage AP205. Viruses regulate their life cycles and infectivity through modulation of their structures and dynamics. While static structures of capsids from several viruses are now accessible with near-atomic-level resolution, atomic-level understanding of functionally important motions in assembled capsids is lacking. We observed up to 64-fold signal enhancements by DNP, which permitted in-depth analysis of these assemblies. For the HIV-1 CA assemblies, a remarkably high spectral resolution in the 3D and 2D heteronuclear data sets permitted the assignment of a significant fraction of backbone and side-chain resonances. Using an integrated DNP MAS NMR and molecular dynamics (MD) simulation approach, the conformational space sampled by the assembled capsid at cryogenic temperatures was mapped. Qualitatively, a remarkable agreement was observed for the experimental 13C/15N chemical shift distributions and those calculated from substructures along the MD trajectory. Residues that are mobile at physiological temperatures are frozen out in multiple conformers at cryogenic conditions, resulting in broad experimental and calculated chemical shift distributions. Overall, our results suggest that DNP MAS NMR measurements in combination with MD simulations facilitate a thorough understanding of the dynamic signatures of viral capsids.