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.

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.

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.

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.

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.

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.

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.

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.

Specific isolation of N-terminal fragments from proteins and their high-fidelity de novo sequencing.

A new method to determine N-terminal amino acid sequences of multiple proteins at low pmol level by a parallel processing has been developed. The method contains the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling with sulfosucccimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate(sulfo-NHS-SS-biotin) to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease; (4) specific isolation of N-terminal fragments of proteins by affinity capture using the biotin-avidin system; (5) de novo sequence analysis of peptides by MALDI-TOF-/MALDI-TOF-PSD mass spectrometry with effective utilization of the CAF (chemically assisted fragmentation) method.1 This method is also effective for N-terminal sequencing of each protein in a mixture of several proteins, and for sequencing components of a multiprotein complex. It is expected to become an essential proteomics tool for identifying proteins, especially when used in combination with a C-terminal sequencing method.

Simultaneous detection of N-terminal fragment ions in a protein mixture using a ruthenium(II) complex.

Use of a bis(terpyridine)ruthenium(II) derivative as an N-terminal labeling reagent resulted in the simultaneous detection and individual determination of all the N-terminal fragments of the proteins in a mixture without requiring any separation. All of the N-termini of the guanidinated proteins were labeled selectively by the ruthenium complex (-CO-labeling). After chymotryptic digestion, the fragments were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and post-source decay (PSD). The -CO moiety exclusively enhanced N-terminal fragment ions in mass spectra and enabled easy N-terminal sequencing. In a mixture containing three different proteins (lysozyme, ubiquitin, and insulin), all of the N-terminal fragment ions labeled with the ruthenium complex were found to produce uniformly intense peaks without the detection of the other unlabeled fragments. The N-terminal sequences of these ions were determined individually by PSD analysis. Application to unknown proteins from Thermus thermophilus HB8 with two-dimensional electrophoretic separation resulted in the successful determination of the N-terminal sequence and easy identification of the target protein.

New metal-organic frameworks with large cavities: selective sorption and desorption of solvent molecules.

Five novel transition metal complexes [Cd(II) (3)(tpba-2)(2)(SCN)(6)].6 THF.3 H(2)O (1), [Cu(II) (3)(tpba-2)(2)(SCN)(6)].6 THF.3 H(2)O (2), [Ni(II) (3)(tpba-2)(2)(SCN)(6)].6 THF.3 H(2)O (3), [Cd(II) (2)(tpba-2)(SCN)(3)]ClO(4) (4), [Cu(I) (3)(SCN)(6)(H(3)tpba-2)] (5) [TPBA-2 = N',N'',N'''-tris(pyrid-2-ylmethyl)-1,3,5-benzenetricarboxamide, THF=tetrahydrofuran] were obtained by reactions of the corresponding transition metal salts with TPBA-2 ligand in the presence of NH(4)SCN using layering or solvothermal method, respectively. The results of X-ray crystallographic analysis showed that complexes 1, 2 and 3 are isostructural and have the same 2D honeycomb network structure with Kagomé lattice, in which all the M(II) (M = Cd, Cu, Ni) atoms are six-coordinated, and the TPBA-2 ligands adopt cis,cis,cis conformation while the thiocyanate anions act as terminal ligands. Capsule-like motifs are found in 1, 2 and 3, in which six THF molecules are hosted, and the results of XPRD and solid-state (13)C NMR spectral measurements showed that the compound 1 can selectively desorb and adsorb THF molecules occurring along with the re-establishment of its crystallinity. In contrast to 1, 2 and 3, complex 4 has different 2D network structure, resulting from TPBA-2 ligands with cis,trans,trans conformation, thiocyanate anions serving as end-to-end bridging ligands, and the incomplete replacement of perchlorate anions, which further link the 2D layers into 3D framework by the hydrogen bonds. In complex 5, the Cu(II) atoms are reduced to Cu(I) during the process of solvothermal reaction, and the Cu(I) atoms are connected by thiocyanate anions to form a 3D porous framework, in which the protonated TPBA-2 ligands are hosted in the cavities as templates.

High sequence-coverage detection of proteolytic peptides using a bis(terpyridine)ruthenium(II) complex.

The use of a bis(terpyridine)ruthenium(ii) complex for peptide labeling (Ru-CO labeling) supplied high intensity peaks in mass spectrometry (MS) analysis that overcame the contribution of protonation or sodiated adduction to peptides. Ru-CO-labeled insulin A- and B-chains were detected simultaneously in comparable peak abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The mass spectra of chymotryptic peptide fragments of Ru-CO-labeled insulin also simultaneously indicated both N-terminal fragment ions, and amino acid sequences were determined easily by matrix-assisted laser desorption/ionization post-source-decay (MALDI-PSD). The sensitivity of detecting Ru-CO-labeled peptide fragment ions was not dependent on the length or the sequences of the peptides. The Ru-CO labeling method was applied to tryptic myoglobin fragments. The method indicated that each fragment ion is detected nearly equal in abundance and enabled the desired fragment ions to be distinguished from matrix clusters or their in-source fragments in lower mass regions. The desired fragment ions can be found in the mass region higher than 670.70 (= Ru-CO). This method provided a high sequence coverage (96%) by peptide mass fingerprinting (PMF). Application of this method to a protein mixture (myoglobin, lysozyme and ubiquitin) successfully achieved high sequence-coverage characterization (>90%) of these proteins simultaneously.

Enhancement of MALDI-MS spectra of C-terminal peptides by the modification of proteins via an active ester generated in situ from an oxazolone.

For selective C-terminal derivatization of peptides and proteins, we have devised a method for activating the C-terminal carboxyl group by extending the oxazolone chemistry. A mixture of formic acid and acetic anhydride was found to be effective for the formation of an oxazolone, which was converted to an active ester in situ in the presence of a phenol or an N-hydroxide. In particular, the resulting active ester with pentafluorophenol facilitated the subsequent reaction with an amine and the hydrazine derivative to yield the C-terminal amide and hydrazide, respectively. The peptides thus coupled with arginine methyl ester or 2-hydrazino-2-imidazoline containing the guanidino moiety exhibited the positive-ion peaks in matrix-assisted laser desorption/ionization (MALDI) mass spectra with appreciably enhanced intensities. As expected from the reaction mechanism, the carboxyl groups of aspartic and glutamic acid residues were not modified, while the amino groups that could react with the activated peptides were concomitantly protected by formylation. The MALDI peaks corresponding to the C-terminal peptide fragments of proteins were specifically enhanced, discriminating against those from internal peptides that were not tagged with a positive charge. In favorable cases, the C-terminal peptide fragments were clearly discerned by MALDI-MS after chymotryptic digestion and were identified by their MALDI postsource decay analysis. Based on these results, we suggest a method for C-terminal sequencing of a protein.

Crystal structures and 77Se NMR spectra of molybdenum(IV) areneselenolates having intramolecular NH...Se hydrogen bonds.

Salts of the monooxomolybdenum(IV,V) areneselenolates having intramolecular NH...Se hydrogen bonds, [Mo(IV)O(Se-2-RCONHC6H4)4]2- (R = t-Bu, CH3, CF3) and [Mo(V)O(Se-2-t-BuCONHC6H4)4]-, were synthesized and characterized by 1H nuclear magnetic resonance (NMR), 77Se NMR, electron spin resonance (ESR), UV-visible spectra, X-ray analysis, and electrochemical measurements. 77Se-1H correlated spectroscopy (COSY) indicated a significant correlation between amide 1H and selenolate 77Se atoms through an NH...Se hydrogen bond with 1J(77Se-1H) = 5.4 Hz coupling. The hydrogen bonds contribute to the positive shift in the Mo(V)/Mo(IV) redox potential. In the crystal structure of (PPh4)2[Mo(IV)O(Se-2-CH3CONHC6H4)4], an NH...O=Mo hydrogen bond was found. Ab inito calculations support the presence of intramolecular NH...O=Mo and NH...Se hydrogen bonds.

Syntheses, structures, and photoluminescence properties of metal(II) halide complexes with pyridine-containing flexible tripodal ligands.

Seven coordination compounds, [Zn(L3)Cl2] . MeOH . H2O (1), [Mn(L3)2Cl2] . 0.5EtOH . 0.5H2O (2), [Cu3(L2)2Cl6] . 2DMF (3), [Cu3(L2)2Br6] . 4MeOH (4), [Hg2(L4)Cl4] (5), [Hg2(L4)Br4] (6), and [Hg3(L4)2I6] . H2O (7), were synthesized by the reactions of ligands 1,3,5-tris(3-pyridylmethoxyl)benzene (L3), 1,3,5-tris(2-pyridylmethoxyl)benzene (L2), and 1,3,5-tris(4-pyridylmethoxyl)benzene (L4) with the corresponding metal halides. All the structures were established by single-crystal X-ray diffraction analysis. In complexes 1 and 2, L3 acts as a bidentate ligand using two of three pyridyl arms to link two metal atoms to result in two different 1D chain structures. In complexes 3 and 4, each L2 serves as tridentate ligand and connects three Cu(II) atoms to form a 2D network structure. Complexes 5 and 6 have the same framework structure, and L4 acts as a three-connecting ligand to connect Hg(II) atoms to generate a 3D 4-fold interpenetrated framework, while the structure of complex 7 is an infinite 1D chain. The results indicate that the flexible ligands can adopt different conformations and thus can form complexes with varied structures. In addition, the coordination geometry of the metal atom and the species of the halide were found to have great impact on the structure of the complexes. The photoluminescence properties of the complexes were investigated, and the Zn(II), Mn(II) and Hg(II) complexes showed blue emissions in solid state at room temperature.

Cadmium(II) and copper(II) complexes with imidazole-containing tripodal polyamine ligands: pH and anion effects on carbon dioxide fixation and assembling.

A new Cd(II) complex [Cd3(L)3(mu3-CO3)](ClO4)4.2CH3CN (1) with two-dimensional (2D) network structure was obtained by reaction of an imidazole-containing tripodal polyamine ligand N1-(2-aminoethyl)-N1-(2-imidazolethyl)-ethane-1,2-diamine (L) with Cd(ClO4)2.6H2O at pH 9.0 in air. The carbonate anions (CO3(2-)) are from the hydration of the atmospheric carbon dioxide, which is the same as in the previously reported Cu(II) complex [Cu3(L)3(mu3-CO3)](ClO4)4.3CH3CN (2). However, the coordination mode of CO3(2-) in 1 is mu3-eta2:eta2:eta2 while the one in 2 is mu3-eta1:eta1:eta1. One-dimensional (1D) chain Cd(II) and Cu(II) complexes [Cd(L)Cl]ClO4.H2O (3) and [Cu(L)(H2O)](ClO4)2 (4) without CO3(2-) were prepared by a similar method as that for 1 and 2 except for the different reaction pH, namely, 3 and 4 were obtained at pH 7 while 1 and 2 were obtained at pH 9. In addition, when Cu(NO3)2 was used to react with L at pH 9, a unique 1D double-stranded helical chain complex [Cu(L)Cl]NO3.1.25H2O (5) was obtained. The results revealed that the reaction pH and the counteranion have great impact on the carbon dioxide absorption and hydration as well as on the assembling and structure of the complexes. The magnetic property of complex 2 was investigated in the temperature range of 1.8-300 K, and weak ferromagnetic coupling among the mu3-eta1:eta1:eta1-CO3(2-) bridged Cu(II) atoms was observed.

Dioxotungsten 1,2-benzenedithiolate complex stabilized by NH...S hydrogen bonds.

Novel dioxo-tungsten(VI) bis(1,2-benzenedithiolate) complexes with neighboring amide groups, as models for tungsten enzymes, (NEt4)2[W(VI)O2{1,2-S(2)-3,6-(RCONH)2C6H2}2] (R = CH3, t-Bu), were designed and synthesized. The presence of the NH...S hydrogen bond was confirmed through IR spectrometry and X-ray crystallographic analysis. In the W(VI)O2 complexes, the NH...S hydrogen bond trans to the oxo ligand is stronger than that cis to oxo. On the basis of comparisons with [W(VI)O2(1,2-S2C6H4)2](2-), the NH...S hydrogen bond positively shifted the W(VI)/W(V) redox potentials and depressed the reduction by benzoin or triphenylphosphine. These results suggest that the NH...S hydrogen bond stabilizes the oxo ligand through trans influence and regulates O-atom transfer in tungsten and molybdenum enzymes.

Metal-organic architectures of silver(I), cadmium(II), and copper(II) with a flexible tricarboxylate ligand.

Three novel metal-organic architectures, [Ag3(bta)].1.5H2O (1), [Cd3(bta)2(H2O)7].5H2O (2), and [Cu11(bta)6(Hbta)2(H2O)10].29H2O (3), were obtained by reactions of the corresponding metal salts with a flexible tripodal ligand, benzene-1,3,5-triacetic acid (H3bta), and their structures were determined by single-crystal X-ray diffraction studies. The results revealed that, in complexes 1 and 2, the carboxylate groups of the bta3- ligand adopted varied coordination modes to link metal atoms and further to form three-dimensional structures with open channels occupied by water molecules, while in complex 3, for the first time, the flexible H3bta acted as a secondary building unit to generate a novel nanometer-sized metallocage, which is composed of a Cu(II) paddle wheel (square secondary building units) and bta3-/Hbta2- organic links (triangular secondary building units). The photoluminescence properties of complexes 1 and 2 were investigated, and the results showed that 2 exhibited photoluminescence in the solid state at room temperature.

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.