Uno Ferro,a de novo Designed Protein,Binds Transition Metals with High Affinity and Stabilizes Semiquinone Radical Anion

Metalloenzymes often utilize radicals in order to facilitate chemical reactions. Recently, DeGrado and co-workers have discovered that model proteins can efficiently stabilize semiquinone radical anion produced by oxidation of 3,5-di-tert-butylcatechol (DTBC) in the presence of two zinc ions. Here, we show that the number and the nature of metal ions have relatively minor effect on semiquinone stabilization in model proteins, with a single metal ion being sufficient for radical stabilization. The radical is stabilized by both metal ion, hydrophobic sequestration, and interactions with the hydrophilic residues in the protein interior resulting in a remarkable, nearly 500 mV change in the redox potential of the SQ ^{. −}/catechol couple compared to bulk aqueous solution. Moreover, we have created 4G-UFsc, a single metal ion-binding protein with pm affinity for zinc that is higher than any other reported model systems and is on par with many natural zinc-containing proteins. We expect that the robust and easy-to-modify DFsc/UFsc family of proteins will become a versatile tool for mechanistic model studies of metalloenzymes.     

Identification of novel vaccine candidates against carbapenem resistant Klebsiella pneumoniae: A systematic reverse proteomic approach

Klebsiella pneumoniae is declared as antibiotic resistant by WHO, with the critical urgency of developing novel antimicrobial therapeutics as drug resistance is the second most dangerous threat after terrorism. Besides many attempts still, there is no effective vaccine available against K. pneumoniae. By utilizing all the available proteomic data we prioritized the novel proteins ideal for vaccine development using bioinformatics tools and techniques. Among the huge data, eight proteins passed all the barriers and were considered ideal candidates for vaccine development. These include: copper silver efflux system outer membrane protein (CusC), outer membrane porin protein (OmpN), Fe⁺⁺ enterobactin transporter substrate binding protein (fepB), zinc transporter substrate binding protein (ZnuA), ribonuclease HI, tellurite resistant methyltransferase (the B), and two uncharacterized hypothetical proteins (WP_002918223 and WP_002892366). These proteins were also subjected to epitope analysis and were found best for developing subunit vaccine against K. pneumoniae. The study shows that the potential vaccine targets are sufficiently efficient being virulent, of outer membranous origin and can be proposed for the DNA third-generation vaccines development that would help to cope up infections caused by multidrug-resistant K. pneumoniae.     

Closed loop folding units from structural alignments: Experimental foldons revisited

Nonoverlapping closed loops of around 25–35 amino acids formed via nonlocal interactions at the loop ends have been proposed as an important unit of protein structure. This hypothesis is significant as such short loops can fold quickly and so would not be bound by the Leventhal paradox, giving insight into the possible nature of the funnel in protein folding. Previously, these closed loops have been identified either by sequence analysis (conservation and autocorrelation) or studies of the geometry of individual proteins. Given the potential significance of the closed loop hypothesis, we have explored a new strategy for determining closed loops from the insertions identified by the structural alignment of proteins sharing the same overall fold. We determined the locations of the closed loops in 37 pairs of proteins and obtained excellent agreement with previously published closed loops. The relevance of NMR structures to closed loop determination is briefly discussed. For cytochrome c, cytochrome b₅₆₂ and triosephophate isomerase, independent folding units have been determined on the basis of hydrogen exchange experiments and misincorporation proton‐alkyl exchange experiments. The correspondence between these experimentally derived foldons and the theoretically derived closed loops indicates that the closed loop hypothesis may provide a useful framework for analyzing such experimental data. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Molecular mechanics study of myelin basic protein peptide 87-118: Some local energy minima

Myelin basic protein (MBP) is the major extrinsic protein of the myelin sheath in the central nervous system. It is this protein that is destroyed in such demyelinating diseases as multiple sclerosis. We have examined the predicted structures of one segment of MBP using the molecular mechanics program ECEPP83 developed by Scheraga and co-workers as modified by Chuman, Momany, and Schafer. We have focused upon a segment, 87-118, containing the Pro-Pro-Pro sequence (residues 100–102), which has been predicted from standard algorithms to exist in a hairpin loop connecting anti-parallel beta-strands. Several local energy minima have been found and reported. Tripoline sequences are not rare in proteins, but their structure and function is still uncertain.     

Zinc chloride‐catalyzed one‐pot,three‐component synthesis of 5,8‐dihydro‐5,8‐dioxo‐4H‐chromene derivatives

An efficient zinc chloride‐catalyzed one‐pot synthesis of 5,8‐dihydro‐5,8‐dioxo‐4H‐chromene derivatives have been achieved by the reaction of 2,5‐dihydroxy‐6‐undecyl‐1,4‐bezoquinone, cyanothioacetamide, and aromaticaldehyde, in EtOH at room temperature. The structures of the products were characterized by IR, ¹H‐NMR, mass spectra, and elemental analyses. J. Heterocyclic Chem., (2011).     

Conformational statistics of polymer chain terminally attached to wall (III) —NRW model loop chain

When the two end groups of a linear polymer chain are absorbed on a solid surface, the polymer chain forms the “loop” conformation. Investigation has been made on the conformational statistics of a model loop chain by the normal random walk (NRW) on a lattice confined in the half-infinite space. Based on the conformational distribution function of the NRW model tail chain, it is easy to deduce an analytical formula expressing the conformational number of the model loop chain. It was found that the ratio of the conformational number of the model loop chain to that of the free chain varies with the power functionN ^-2/3 when the chain lengthN→οο. The same result -was obtained by means of the recursion equation. The ratio of the mean square end-to-end distanceh ² for the model loop chain to its mean square bond lengthl ² is 2N/3. Compared with the free chain with the same lengthN, the mean square end-to-end distance of the model loop chain contracts to a certain extent. The basic relationships deduced were supported by the exact enumeration and Monte Carlo simulations. Project supported by the National Natural Science Foundation of China.     

Unveiling the binding interaction of zinc (II) complexes of homologous Schiff-base ligands on the surface of BSA protein: A combined experimental and theoretical approach

Four new zinc (II) complexes [Zn (HL¹H)Br₂] (1), [Zn (HL¹H)Cl₂] (2), [Zn₂(HL²)Br₃] (3), and [Zn (HL²)Cl] (4) have been synthesized by adopting template synthetic strategy and utilizing two homologous Schiff base ligands (H₂L¹ = 4-bromo-2-{[2-(2-hydroxyethylamino)-ethylimino]-methyl}-6-methoxyphenol, H₂L² = 4-bromo-2-{[3-(2-hydroxyethylamino)propylimino]methyl}-6-methoxyphenol), differing in one -CH₂- unit in the ligating backbone, by adopting template synthetic strategy. All the complexes have been characterized by single crystal X-ray diffraction analysis as well as by other routine physicochemical techniques. Ligand mediated structural variations have been observed and rationalized by density functional theoretical (DFT) calculations. Interaction of the complexes 1–4 with Bovine Serum Albumin protein (BSA) has been studied by different spectroscopic techniques. A complete thermodynamic profile (ΔH^o, ΔS^o and ΔG^o) was evaluated initially from the change in absorption and fluorescence spectra upon addition of BSA to the complexes. Appreciable binding constant values in the range ~ 0.94–4.51 × 10⁴ M⁻¹ indicate efficient binding tendency of the complexes to BSA with the sequence 1 ≅ 2 > 3 ≅ 4. Circular dichroism (CD), isothermal calorimetric titration experiments, molecular docking and molecular dynamics have been performed to gain deep insight into the binding regions of complex 1 to BSA. Experimental evidences suggest an interaction of zinc complexes at the surface of BSA protein and this particular binding has been exploited to determine unknown concentration of BSA protein. For this purpose complex 1 was explored as a BSA protein quantification tool.     

Pentane‐1,5‐diaminium dibromide

The crystal structure of the title salt, C₅H₁₆N₂²⁺·2Br⁻, with Z = 12 and more unusually Z′ = 3, forms part of a small group of crystal structures in the Cambridge Structural Database that are ammonium bromide salts. One of the diaminium cation chains in the asymmetric unit exhibits positional disorder, which was modelled using a suitable disorder model. This compound also exhibits organic–inorganic layering in its packing arrangement that is typical of this class of compound. An extensive complex three‐dimensional hydrogen‐bonding network is also identified. The hydrogen bonds evident in this crystal structure were identified as being most likely strong charge‐assisted hydrogen bonds.     

Magnetic Multistability in an Anion-Radical Pimer

Radical pimers are the simplest and most important models for studying charge-transfer processes and provide deep insight into π-stacked organic materials. Notably, radical pimer systems with magnetic bi- or multistability may have important applications in switchable materials, thermal sensors, and information-storage media. However, no such systems have been reported. Herein, we describe a new pimer consisting of neutral N-(n-propyl) benzene triimide ([BTI-3C]) and its anionic radical ([BTI-3C]^−.) that exhibits rare magnetic multistability. The crystalline pimer was readily synthesized by reduction of BTI-3C with cobaltocene (CoCp₂). The transition occurred with a thermal hysteresis loop that was 27 K wide in the range of 170–220 K, accompanied by a smaller loop with a width of 25 K at 220–242 K. The magnetic multistability was attributed to slippage of the π-stacked BTI structures and entropy-driven conformational isomerization of the side propyl chains in the crystalline state during temperature variation.     

NMR and Molecular Genetics as Tools for Investigating Protein Interactions in Membrane Systems

In our laboratory, we have applied the tools of nuclear magnetic resonance (NMR) spectroscopy and molecular genetics to investigate the structural and dynamic properties of membrane-associated proteins and their interactions with membrane components. There are two general classes of membrane proteins, i.e., intrinsic and peripheral ones. For the intrinsic membrane proteins, we have chosen the membranebound D-lactate dehydrogenase (D-LDH) of Escherichia coli as a model to study protein-lipid interactions in membranes. D-LDH is a respiratory enzyme of molecularweight 65, 000 containing flavin adenine dinucleotide (FAD) as a cofactor. The activity of purified D-LDH is enhanced up to 100-fold by lipids and detergents. The gene for D-LDH has been sequenced, and production of the enzyme amplified up to 300-times normal levels. We have biosynthetically incorporated 5-fluorotryptophan (5F-Trp) into D-LDH and studied the five Trp residues by ¹⁹F-NMR spectroscopy. In order to gain additional information using ¹⁹F-NMR, site-specific, oligonucleotide-directed mutagenesis has been used to insert a sixth Trp into D-LDH at various positions throughout the 571-amino acid chain. These mutant D-LDHs are being characterized biochemically and through NMR. For peripheral membrane proteins, we have chosen two periplasmic binding proteins, histidine-binding protein J (J protein) of Salmonella Typhimurium and glutamine-binding protein (GlnBP) of E. coli as models to investigate the structure-function relationship in periplasmic binding protein-mediated active transport systems. These two proteins both have molecular weights of approximately 25, 000. By using mutant J proteins and GlnBPs and site-specific, oligonucleotide-directed mutagenesis techniques, we have assigned several resonances to specific amino acid residues. We are investigating the relationship between ligand-induced conformational changes in these two proteins and their roles in the active transport of ligand across the cell membrane. We have found that a combination of isotopic labeling, biochemistry, molecular biology, and NMR is a very useful approach to investigate various interactions of membrane-associated protein systems.     

Synthesis and characterization of group 12 complexes of N,N-methyl phenyl-N,N-butyl phenyl dithiocarbamate

Zn(II), Cd(II), and Hg(II) complexes of N-methyl-N-phenyl dithiocarbamate (L¹) and N-butyl-N-phenyl dithiocarbamate (L²) formulated as ML¹L² have been synthesized and characterized by elemental analysis, FT–IR, ¹H- and ¹³C-NMR spectroscopic techniques. Single-crystal X-ray structures of the Zn(II) and Hg(II) complexes are also reported. X-ray crystal structures revealed that in the zinc(II) complex, the dithiocarbamate is chelating and bridging, forming eight-member rings, while the Hg complex is monomeric with bidentate dithiocarbamate. In both complexes, the metals are in distorted tetrahedral geometry and the methyl and butyl groups of the dithiocarbamates exhibit compositional disorder between two positions.     

Non‐Uniform Sampling and J‐UNIO Automation for Efficient Protein NMR Structure Determination

High‐resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments and automation of data handling therefore attracts continued interest. Here, non‐uniform sampling (NUS) of 3D heteronuclear‐resolved [¹H,¹H]‐NOESY data yielded two‐ to three‐fold savings of instrument time for structure determinations of soluble proteins. With the 152‐residue protein NP_372339.1 from Staphylococcus aureus and the 71‐residue protein NP_346341.1 from Streptococcus pneumonia we show that high‐quality structures can be obtained with NUS NMR data, which are equally well amenable to robust automated analysis as the corresponding uniformly sampled data.     

Synthesis of 1-substituted benzimidazole metal complexes and structural characterization of dichlorobis(1-phenyl-1 H -benzimidazole- κN 3)cobalt(II) and dichlorobis (1-phenyl-1 H -benzimidazole- κN 3)zinc(II)

The Co(II), Zn(II), Ni(II), Cu(II), and Fe(II) complexes of 1-phenylbenzimidazole were synthesized and characterized by NMR and elemental analyses. The crystal structures of dichlorobis(1-phenyl-1?H-benzimidazole-κN³ )cobalt(II) and dichlorobis(1-phenyl-1?H-benzimidazole-κN³ )zinc(II) have been determined by single-crystal X-ray diffraction.     

Syntheses and structures of Zn(II) and Ni(II) complexes of 4-N-(acetylacetone amine)acetophenone thiosemicarbazone

A new ligand, 4-N-(acetylacetone amine)acetophenone thiosemicarbazone (HL, 1), was synthesized by condensation of p-aminoacetophenone with thiosemicarbazide and acetylacetone. Treatment of HL with zinc acetate and nickel acetate afforded ZnL₂ (2) and NiL₂?·?DMF (3). The crystal structures of 1, 2 and 3 have been determined by single-crystal X-ray diffraction. 2 and 3 have similar structures, in which metal atom is coordinated in a distorted tetrahedral configuration, and L^? coordinates to zinc(II) or nickel(II) through the azomethine nitrogen and the thiolato sulfur atom.     

Labeling Strategy and Signal Broadening Mechanism of Protein NMR Spectroscopy in Xenopus laevis Oocytes

We used Xenopus laevis oocytes, a paradigm for a variety of biological studies, as a eukaryotic model system for in‐cell protein NMR spectroscopy. The small globular protein GB1 was one of the first studied in Xenopus oocytes, but there have been few reports since then of high‐resolution spectra in oocytes. The scarcity of data is at least partly due to the lack of good labeling strategies and the paucity of information on resonance broadening mechanisms. Here, we systematically evaluate isotope enrichment and labeling methods in oocytes injected with five different proteins with molecular masses of 6 to 54 kDa. ¹⁹F labeling is more promising than ¹⁵N, ¹³C, and ²H enrichment. We also used ¹⁹F NMR spectroscopy to quantify the contribution of viscosity, weak interactions, and sample inhomogeneity to resonance broadening in cells. We found that the viscosity in oocytes is only about 1.2 times that of water, and that inhomogeneous broadening is a major factor in determining line width in these cells.     

Magnetic Multistability in an Anion‐Radical Pimer

Radical pimers are the simplest and most important models for studying charge‐transfer processes and provide deep insight into π‐stacked organic materials. Notably, radical pimer systems with magnetic bi‐ or multistability may have important applications in switchable materials, thermal sensors, and information‐storage media. However, no such systems have been reported. Herein, we describe a new pimer consisting of neutral N‐(n‐propyl) benzene triimide ([BTI‐3C]) and its anionic radical ([BTI‐3C]^?.) that exhibits rare magnetic multistability. The crystalline pimer was readily synthesized by reduction of BTI‐3C with cobaltocene (CoCp₂). The transition occurred with a thermal hysteresis loop that was 27 K wide in the range of 170–220 K, accompanied by a smaller loop with a width of 25 K at 220–242 K. The magnetic multistability was attributed to slippage of the π‐stacked BTI structures and entropy‐driven conformational isomerization of the side propyl chains in the crystalline state during temperature variation.     

Unexpected Coordination Modes of the Tris(imidazolyl)phosphane Oxide Ligand 4‐TIPOiPr in the Chloro Complexes of Zinc,Cobalt and Nickel

The coordination chemistry of the water soluble phosphane oxide ligand tris[2‐isopropylimidazol‐4(5)‐yl]phosphane oxide, 4‐TIPO^iPr, has been explored. A variety of 3d‐metal halide complexes have been prepared and the crystal structures of the solvates [(4‐TIPO^iPr)ZnCl₂]·MeOH·¹/₂dioxane ( 1 ·MeOH·¹/₂dioxane), [(4‐TIPO^iPr)CoCl₂]·H₂O·2dioxane ( 2 ·H₂O·2dioxane) and [(4‐TIPO^iPr)₂Ni(MeOH)₂]Cl₂·2MeOH ( 3 ·2MeOH) have been determined. All three structures show unprecedented coordination modes of the 4‐TIPO^iPr ligand. Both zinc and cobalt complexes are coordinated in a bidentate κ²N fashion, whereas the nickel atom is coordinated by two ligands in a κN,O mode using one imidazolyl substituent and the P=O oxygen atom.     

Structure and coordination in mono and dinuclear Zn(II)-pyrrolidine dithiocarbamate complexes

The structures of the chelate Zn(PDTC)₂ and its dimeric form Zn₂(PDTC)₄ are investigated theoretically at B3LYP/cc-pVDZ level. The natural bond orbital (NBO) analysis has been performed to explore the metal–ligand coordination of these chelates. In Zn(PDTC)₂, the sulfur atoms mainly use 3p sub-shells to coordinate with mixed (4s + 4p _x  + 4p _y  + 4p _z ) orbital of zinc having sp ³ hybridization. In Zn₂(PDTC)₄, each zinc atom coordinates with one terminal and two bridging PDTC ligands. The contribution of bridging sulfur atoms in chelation is much more than terminal sulfurs. The bridging sulfur atoms use 3s and 3p sub-shells to coordinate with 4s and 4p sub-shells of metal center zinc. The charge transfer interactions between sulfur and metal center involving 4d, 5s, and 5p sub-shells of zinc are much feeble compared to those involving 4s and 4p sub-shells of zinc.     

Synthesis,structure, protein binding,and cytotoxicity of zinc(II) complexes with tridentate NNO Schiff-base ligands

Mononuclear and trinuclear zinc(II) complexes (1 and 2) with tridentate NNO Schiff-base ligands (HL¹?=?N-2-pyridiylmethylidene-4-chloro-2-hydroxy-phenylamine, HL²?=?N-2-pyridiylmethylidene-2-hydroxy-5-chloro-phenylamine) have been synthesized and characterized by single-crystal X-ray diffraction and elemental analysis. The binding properties of zinc(II) complexes with calf thymus DNA (CT-DNA) and HSA were investigated by UV–visible, fluorescence, and circular dichroism spectra. The zinc(II) complexes bind significantly to CT-DNA by intercalation and bind to protein HSA through a static quenching mechanism. The in vitro cytotoxicity of the complexes on human tumor cells lines was assessed by 3-(4,5-dimathylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Hoechst 33258 staining experiments.