Whole-cell proteome reference maps of an extreme thermophile, Thermus thermophilus HB8.

Thermus thermophilus HB8 is a model microorganism for industrial applications because of its thermophilic enzymes, and for basic bacteriology to understand the coordination of the biological functions of the genome-encoded enzymes at the cellular level. Here, we present 2DE reference maps of T. thermophilus HB8 in the pH ranges 4-7 and 6-11 obtained with whole-cell lysates. PMF analysis using MALDI-TOF-MS and MS/MS analysis using nano-scale LC and quadrupole TOF-MS identified 258 different proteins among the 306 protein spots on 2DE gels. Functional classification indicated that 56%, 16%, and 14% of the identified proteins were related to metabolism, genetic information process, and cellular process, respectively. Detailed classification of the metabolism-related proteins suggested that during the exponential phase, amino acid and carbohydrate metabolism are major metabolic processes, whereas nucleotide and lipid metabolism are minor ones. On the other hand, volume quantification analysis revealed that proteins involved in the translational process, nucleotide metabolism, and central carbon metabolism were most abundantly expressed in the exponential phase.

Color regulation and stabilization of chromophore by Cys69 in photoactive yellow protein active center.

Model compounds of PYP chromophore were synthesized and characterized to investigate the role of the Cys69 residue in the active center, which has the intramolecular NHOC hydrogen bond to the conjugated carbonyl oxygen and thioester linkage of the chromophore. The results of UV-vis and (13)C NMR spectroscopy of the model compounds indicated that they delocalized the negative charge of the chromophore and increased the contribution of the quinoid-like resonance structure in the phenolate anion state. Thus, the Cys69 residue plays an important role in the regulation of the color and the stabilization of the chromophore anion in the active center.

Mass spectrometric analysis using ruthenium (II)-labeling for identification of glycosyl hydrolase product.

Analysis of products digested by glycosyl hydrolases helps understanding of the hydrolysis mechanism and the substrate recognition in the enzymes. We developed a new universal technique, which consists of ruthenium (II) complex labeling and mass spectrometry analysis, to identify the reducing sugars released from oligosaccharides by enzymatic digestion. This method was applied to enzymatic digestion by chitinase and cellulase of the hyperthermophilic archaea Pyrococcus fusiosus and Pyrococcus horikoshii respectively.

Novel photosystem involving protonation and deprotonation processes modelled on a PYP photocycle.

The synthesis of novel ortho-coumaric acid derivatives, with an amide group linked with an olefin moiety, which introduced photoinduced switching of the intramolecular hydrogen bonds is presented. An intramolecular OH...O=C hydrogen bond formed in a Z-phenol compound was switched to an intramolecular NH...O hydrogen bond in Z phenolate state via deprotonation. The pK(a) value of the Z-phenol derivative was lower than that of E-phenol, and a novel photocycle system involving protonation and deprotonation processes was achieved.

Dinuclear zinc(II) complex with novel tripodal polyamine ligand: synthesis, structure and kinetic study of carboxy ester hydrolysis.

An imidazole-containing tripodal polyamine ligand N(1)-(2-aminoethyl)-N(1)-(2-imidazol-1-ylethyl)-ethane-1,2-diamine (L) was prepared and its dinuclear zinc(II) complex [Zn(L)(H(2)O)](2)(ClO(4))(4).4H(2)O (1) was obtained and examined as a catalyst for the hydrolysis of 4-nitrophenyl acetate (NA). X-ray crystal structure analysis of the complex revealed that the complex features a dinuclear cation unit with a Zn...Zn distance of 8.34A and both Zn(II) centers adopt distorted trigonal-bipyramid geometry. The solution complexation investigation performed at 25 degrees C by means of potentiometric titration revealed that the mononuclear species [ZnL](2+) is predominating in the pH rage of 7.0-9.7 in the solution and the pK(a1) for the Zn-bound water is 8.50+/-0.01. Complex 1 promoted hydrolysis of NA showed a second-order rate constant of 0.046+/-0.004 M(-1)s(-1) at pH 9.0 in 10% (v/v) CH(3)CN aqueous solution at 25 degrees C. The pH-rate profile for the second-order rate constant of NA hydrolysis with complex 1 gave a sigmoidal curve. And the results show that in the hydrolysis process the two Zn(II) centers of the dinuclear deprotonated species do not cooperate with each other and the Zn-bound hydroxide servers as reactive nucleophile toward the ester.

Highly oriented aragonite nanocrystal-biopolymer composites in an aragonite brick of the nacreous layer of Pinctada fucata.

13C CP/MAS NMR and FE/TEM measurements of the aragonite brick of the nacreous layer of Pinctada fucata indicate that it assembles with highly oriented aragonite nanocrystals, which are regulated by biopolymers.

Association of Oxo-Molybdenum Dithiolene Complexes with a Multiamide Additive and Its Influence on the Ease of O-Atom Transfer.

To regulate the reactivity of dithiolene dioxomolybdenum(VI) complexes, two systems were studied: (1) chelate recognition with a diamide additive, 1,3-bis(isobutyrylamino)benzene (1); (2) a dithiolene complex with four amide substituents, (n-Pr(4)N)(2)[Mo(IV)O{S(2)C(2)(CONH(2))(2)}(2)].0.5(i-PrOH).DMF (2) (S(2)C(2)(CONH(2))(2) = 1,2-dicarbamoylethylene-1,2-dithiolate). Both amide compounds, the additive 1 and the complex 2, were designed to form NH.S and/or NH.O hydrogen bonding with thiolate ligands in (Mo(VI)O(2))(2+) and (Mo(IV)O)(2+) complexes. Titration analyses indicate the presence of a 1:1 complex in CH(2)Cl(2) solution. This association causes changes in solubility and reactivity of the molybdenum complexes. When the additive was mixed with a DMSO-d(6) solution of (NEt(4))(2)[Mo(IV)O{S(2)C(2)(COOMe)(2)}(2)] (3) (S(2)C(2)(COOMe)(2) = 1,2-di(methoxycarbonyl)ethylene-1,2-dithiolate), the rate of reduction of Me(3)NO was increased by a factor of 6. An acceleration was also observed for compound 2. It appeares that NH.S hydogen bonding is modulating the system.

Novel Rubredoxin Model Tetrathiolato Iron(II) and Cobalt(II) Complexes Containing Intramolecular Single and Double NH.S Hydrogen Bonds.

Simple rubredoxin model complexes with o-(acylamino)benzenethiolato and 2,6-(diacylamino)benzenethiolato were synthesized and characterized by (1)H NMR, IR, and electrochemical properties. The structures of (NEt(4))(2)[Fe(II)(S-o-t-BuCONHC(6)H(4))(4)].2EtCN (3a), (NEt(4))(2)[Co(II)(S-o-t-BuCONHC(6)H(4))(4)].2EtCN (3b), and (PPh(4))(2)[Co(II){S-2,6-(CF(3)CONH)(2)C(6)H(3)}(4)].Et(2)O (5b') were determined by X-ray analysis. The complexes have intramolecular single or double NH.S hydrogen bonds. The mean Fe-S and Co-S distances are significantly shorter than those of non-hydrogen-bonding complexes, [M(SPh)(4)](2-) (M = Fe(II), Co(II)), by 0.024 Å (3a), 0.032 Å (3b), and 0.029 Å (5b'), respectively. (NMe(4))(2)[Fe(II)(S-o-CH(3)CONHC(6)H(4))(4)] (2a) and (NMe(4))(2)[Fe(II){S-2,6-(CH(3)CONH)(2)C(6)H(3)}(4)] (6a) have remarkably positive shifted Fe(III)/Fe(II) redox couples by the single and double NH.S hydrogen bond, respectively. The shifts are +0.25 for 2a and +0.56 for 6a from the redox potential of [Fe(II)(SPh)(4)](2-), which indicate additivity of the effect of the NH.S hydrogen bond. The formation and the structure of a novel chelating amido(thiolato) complex (PPh(4))(2)[Co(II){S-2,6-(CF(3)CONH)(2)C(6)H(3)}(2)(S-2-CF(3)CONH-6-CF(3)CONC(6)H(3))].Et(2)O (9) are also described.

An Amide-Linked Ferrocene Dimer, [(CH(3)CONHC(5)H(4))Fe(C(5)H(4)CONHC(5)H(4))Fe(C(5)H(4)CONHCH(3))]. Formation of Inter- and Intramolecular NH.O=C Hydrogen Bonds.

An amide-linked ferrocene dimer, [(CH(3)CONHC(5)H(4))Fe(C(5)H(4)CONHC(5)H(4))Fe(C(5)H(4)CONHCH(3))] (7), and a monomer, [(CH(3)CONHC(5)H(4))Fe(C(5)H(4)CONHCH(3))] (5), were synthesized, and their unique structures were determined by X-ray analysis. Amide planes are stacked with respect to each other in both crystal and dilute solution. These complexes form intermolecular NH.O=C hydrogen bonds in the crystal. On the other hand, they form intramolecular NH.O=C hydrogen bonds in a dilute solution as established by IR and (1)H NMR measurements. Spectral data suggest the presence of flapping structures in solution. Crystal data for 5: triclinic, P&onemacr;, a = 9.358(3) Å, b = 9.947(4) Å, c = 7.946(3) Å, alpha = 106.86(3) degrees, beta = 113.15(3) degrees, gamma = 76.59(3) degrees, V = 644.9(5) Å(3), Z = 2. 7.(1)/(2)EtCN: orthorhombic, Pna2(1), a = 7.467(4) Å, b = 14.048(4) Å, c = 23.400(5) Å, Z = 4.

Structure and Properties of [Fe(4)S(4){2,6-bis(acylamino)benzenethiolato-S}(4)](2)(-) and [Fe(2)S(2){2,6-bis(acylamino)benzenethiolato-S}(4)](2)(-): Protection of the Fe-S Bond by Double NH.S Hydrogen Bonds.

Iron-sulfur clusters containing a singly or doubly NH.S hydrogen-bonded arenethiolate ligand, [Fe(4)S(4)(S-2-RCONHC(6)H(4))(4)](2)(-) (R = CH(3), t-Bu, CF(3)), [Fe(4)S(4){S-2,6-(RCONH)(2)C(6)H(3)}(4)](2)(-), [Fe(2)S(2)(S-2-RCONHC(6)H(4))(4)](2)(-) (R = CH(3), t-Bu, CF(3)), and [Fe(2)S(2){S-2,6-(RCONH)(2)C(6)H(3)}(4)](2)(-), were synthesized as models of bacterial [4Fe-4S] and plant-type [2Fe-2S] ferredoxins. The X-ray structures and IR spectra of (PPh(4))(2)[Fe(4)S(4){S-2,6-(CH(3)CONH)(2)C(6)H(3)}(4)].2CH(3)CN and (NEt(4))(2)[Fe(2)S(2){S-2,6-(t-BuCONH)(2)C(6)H(3)}(4)] indicate that the two amide NH groups at the o,o'-positions are directed to the thiolate sulfur atom and form double NH.S hydrogen bonds. The NH.S hydrogen bond contributes to the positive shift of the redox potential of not only (Fe(4)S(4))(+)/(Fe(4)S(4))(2+) but also (Fe(4)S(4))(2+)/(Fe(4)S(4))(3+) in the [4Fe-4S] clusters as well as (Fe(2)S(2))(2+)/(Fe(2)S(2))(3+) in the [2Fe-2S] clusters. The doubly NH.S hydrogen-bonded thiolate ligand effectively prevents the ligand exchange reaction by benzenethiol because the two amide NH groups stabilize the thiolate by protection from dissociation.

Dinuclear Calcium Complexes with Intramolecularly NH.O Hydrogen-Bonded Dicarboxylate Ligands.

A novel dinuclear calcium complex, [Ca(2){(2-OCO-3-CH(3)C(6)H(3)NHCO)(2)C(CH(3))(2)}(2)(CH(3)OH)(6)] (1), was synthesized as a structural model of 8-coordinated Ca(II) ions in the double calcium-binding site of thermolysin. The complex has four NH.O hydrogen bonds between the amide NH and the carboxylate oxygen anion. Two types of bridging coordination of the carboxylate ligand to Ca(II) were found in 1. The amide NH forms a strong NH.O hydrogen bond with the anionic oxygen of the two carboxylate oxygens. A ligand-exchange reaction between the dinuclear calcium complex and eight equimolar amounts of 2,4,6-trimethylbenzoic acid or 2-CH(3)-6-t-BuCONHC(6)H(3)COOH indicates that the NH.O hydrogen bond prevents the dissociation of the Ca-O bond.

Role of the Invariant Peptide Fragment Forming NH.S Hydrogen Bonds in the Active Site of Cytochrome P-450 and Chloroperoxidase: Synthesis and Properties of Cys-Containing Peptide Fe(III) and Ga(III) (Octaethylporphinato) Complexes as Models.

The primary sequence of Cys-X-Gly-Y- (X, hydrophobic residue; Y, hydrophilic residue) is highly conserved in cytochrome P-450s. The amide NHs of Leu, Gly, and X are assumed to form NH.S hydrogen bonds which are also found in the active site fragment, Cys-Pro-Ala-Leu, of chloroperoxidase (CPO). [Fe(III)(OEP)(Z-cys-Leu-Gly-Leu-OMe)] (OEP, octaethylporphinato; Z, benzyloxycarbonyl) and [Fe(III)(OEP)(Z-cys-Pro-Ala-Leu-OMe)] were synthesized as P-450 and CPO model complexes containing the invariant amino acid fragment of the active site. The corresponding gallium(III) complexes were also synthesized to investigate the solution structures using two-dimensional (2D) NMR experiments because the Ga(III) ion is similar to the Fe(III) ion in the ionic radii and in the coordination geometry. The solution structures of the peptide part of the gallium complexes indicate that the invariant fragments maintain a beta I-turn-like conformation and then form NH.S hydrogen bonds between S(gamma)Cys and NH of the third and fourth amino acid residues. The hydrogen bonds have also been confirmed by the (2)H NMR spectra of N(2)H-substituted Fe(III) peptide complexes. The Fe(III)/Fe(II) redox potentials of the Fe(III) complexes indicate that the NH.S hydrogen bonds in the fragments causes a slight positive shift of the redox potential. The tri- and tetrapeptide Fe(III) complexes containing the invariant fragments of P-450 are kinetically stable at 30 degrees C in CH(2)Cl(2). In contrast, [Fe(III)(OEP)(Z-cys-Leu-OMe)] decomposed to give [Fe(II)(OEP)] (22%) and the corresponding disulfide immediately in CD(2)Cl(2) at 30 degrees C for 1 h. These results indicate that the invariant fragments involving the hydrogen bonds cause the stabilization of the high-spin Fe(III) resting state rather than the positive shift of Fe(III)/Fe(II) redox potential.

Novel dense organic-lanthanide hybrid architectures: syntheses, structures and magnetic properties.

Six novel three-dimensional (3D) dense organic-lanthanide(III) frameworks with formula {[Ln(HBPTCA)(H2O)].3H2O}n [Ln = La (1), Ce (2), Sm (3)] and [Ln4(BPTCA)3(H2O)4]n [Ln = Tb (4), Dy (5), Ho (6)] were obtained by reactions of the corresponding lanthanide nitrate salt with 4,4'-bipyridine-2,2',6,6'-tetracarboxylic acid (H4BPTCA) under different conditions. Complexes 1-3 have the same structure with (4, 6(2))(2)(4(2), 6(10), 8(3)) topology, which is rare binodal (3, 6)-connecting rutile structure, while the complexes 4-6 also with the same structure have different topology of (4(2), 6)(4)(6, 8(2))(4)(4(3), 6(3))(4)(4(2), 6(4))(6)(4(4), 6(5), 8(5), 10). The results indicate that the reaction conditions have great influence on the structure of the resulted complexes in this system. In addition, the H4BPTCA was found to be an effective bridging ligand for construction of novel lanthanide-based dense hybrids, and two new coordination modes of the BPTCA4- were found in the complexes. The photoluminescent property of 4 and magnetic properties of 2, 5 and 6 were also investigated.

Zinc, cadmium, and mercury 1,2-benzenedithiolates with intramolecular NH...S hydrogen bonds.

Mononuclear Zn, Cd, and Hg 1,2-benzenedithiolates with intramolecular NH...S hydrogen bonds, [M(II){1,2-S2-3,6-(RCONH)2C6H2}2](2-) (R = CH 3, t-Bu; M = Zn, Cd, Hg), were synthesized and characterized by X-ray analysis and spectral measurements. The presence of intramolecular NH...S hydrogen bonds was established by the IR spectra. (199)Hg and (113)Cd nuclear magnetic resonance showed a stabilized four-thiolate coordinated structure and suggested the influence of the NH...S hydrogen bonds to ppi(Hg)-ppi(S) interactions. The NH stretching bands show that the NH...S hydrogen bonds in Cd and Hg complexes are stronger than those in the corresponding Zn complex. These results are supported by theoretical calculations. The experimental and theoretical results suggested that the NH...S hydrogen bond influences the efficient capture of toxic Cd and Hg ions by metallothioneins.

Manipulation of an intramolecular NH...O hydrogen bond by photoswitching between stable E/Z isomers of the cinnamate framework.

Novel carboxylic acid derivatives were synthesized, which allowed switching of the intramolecular distance between amide group and carboxylic oxygen atoms using E to Z photoisomerization of the cinnamate framework. An intramolecular NH...O hydrogen bond was formed in the Z carboxylate compound not only in solution but also in the solid state. The pK(a) value of the carboxylic acid was lowered as a consequence of the E/Z photoisomerization.

Selective isolation of N-terminal peptides from proteins and their de novo sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without regard to unblocking or blocking of N-terminal amino acids.

We have developed a new method to determine the N-terminal amino acid sequences of proteins, regardless of whether their N-termini are modified. This method consists of the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling of sulfo-NHS-SS-biotin to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease followed by oxidation with performic acid; (4) specific isolation of N-terminal peptides from digests using DITC resins; (5) de novo sequence analysis of the N-terminal peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the CAF (chemically assisted fragmentation) method or tandem mass spectrometric (MS/MS) analysis according to unblocked or blocked peptides, respectively. By employing DITC resins instead of avidin resins used in our previous method (Yamaguchi et al., Rapid Commun. Mass Spectrom. 2007; 21: 3329), it has been possible to isolate selectively N-terminal peptides from proteins regardless of modification of N-terminal amino acids. Here we propose a universal method for N-terminal sequence analysis of proteins.

Silver(I) complexes with oxazoline-containing tripodal ligands: structure variation via counter anions and reaction conditions.

Seven new silver(I) complexes of the formula [Ag2(L)2(CF3SO3)2] (1), [Ag2(L)2(CH3SO3)2] (2) [Ag2(L)2](BF4)2 (3), [Ag3(L)2(NO3)2]NO3.5H2O (4), [Ag2(L)(NO3)2].CH3OH (5), [Ag2(L)2](ClO4)2 (6) and [Ag3(L)2(CH3CN)3](ClO4)3 (7) have been synthesized by reactions of 1,3,5-tris(2-oxazolinyl)benzene (L) with varied silver(I) salts under different conditions. The influences of counter anions and reaction conditions on the structure of the complexes are discussed. Three complexes , 1, 2 and 3 with two kinds of different 1D chain structures were obtained under the same synthetic conditions by using different silver(I) salts, and the ligand L was found to adopt bis-monodentate (1 and 2) and tris-monodentate (3) coordination modes respectively. On the other hand, by using the same silver(I) nitrate or silver(I) perchlorate but different reaction solvents, 4 and 5 or 6 and 7 were isolated respectively. Complexes 4and 5 have different 1D chain structures, and 6 is isostructural with . However, 7 is a tri-nuclear, propeller-shaped M3L2 supramolecular capsule in which L adopts a cis,cis,cis-conformation, while the ligand L in 3-6 has cis,trans,trans-conformation. The results revealed that the nature of the counter anions, such as their size, coordination ability and coordination mode, and the reaction conditions all have great impact on the structure of the complexes. The complexes were also characterized by electrospray mass spectrometry. Furthermore, complex 7 exhibited modest second-harmonic-generation (SHG) efficiency.