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PubMed2001

Synthesis, characterization, and electrochemistry of cis-oxothio- and cis-bis(thio)tungsten(VI) complexes of hydrotris(3,5-dimethylpyrazol-1-yl)borate.

The complexes TpWO2X react with sulfiding agents such as B2S3 or P4S10 to give the oxothio- and bis(thio)tungsten(VI) complexes TpWOSX (X = Cl(-)) and TpWS2X [X = Cl(-), S2PPh2(-); Tp = hydrotris(3,5-dimethylpyrazol-1-yl)borate]. The reaction of TpWS2Cl with (i) PPh3 in pyridine and (ii) dimethyl sulfoxide affords TpWOSCl in good overall yield. The chloro complexes undergo metathesis with alkali metal salts to yield species of the type TpWOSX and TpWS2X [X = OPh(-), SPh(-), SePh(-), (-)-mentholate]. The diamagnetic complexes exhibit NMR spectra indicative of C(1) (TpWOSX) or C(s) (TpWS2X) symmetry and IR spectra consistent with terminal oxo and thio ligation (nu(W=O), 940-925 cm(-1); nu(W=S) or nu(WS2), 495-475 cm(-1)). Crystals of (R,S)-TpWOS[(-)-mentholate] are monoclinic, space group P2(1), with a = 11.983(2) A, b = 18.100(3) A, c = 13.859(3) A, beta = 91.60(2) degrees, V = 3004.6(8) A(3), and Z = 4. Crystals of TpWS2(OPh)-CH2Cl2 are orthorhombic, space group Pbca, with a = 16.961(4) A, b = 33.098(7) A, c = 9.555(2) A, V = 5364(2) A(3), and Z = 8. The mononuclear, distorted-octahedral tungsten centers are coordinated by a tridentate Tp ligand, an alkoxy or aryloxy ligand, and two terminal chalcogenide ligands. The average W=O and W=S distances are 1.726(7) and 2.125(2) A, respectively, and the O=W=S and S=W=S angles 102.9(3) and 102.9(1) degrees, respectively. The tungsten and sulfur X-ray absorption spectra of TpWOSCl and TpWS2Cl are consistent with the presence of terminal pi-bonded thio ligands in both complexes. The thio complexes generally undergo a reversible one-electron reduction at potentials significantly more positive than their oxo analogues. The chemical, spectroscopic, and electrochemical properties of the complexes are heavily influenced by the presence of W=S pi frontier orbitals.

Aldehyde Oxidoreductases↗
PubMed2002

Sterically hindered carboxylate ligands support water-bridged dimetallic centers that model features of metallohydrolase active sites.

The synthesis and characterization of carboxylate-bridged dimetallic complexes are described. By using m-terphenyl-derived carboxylate ligands, a series of dicobalt(II), dicobalt(III), dinickel(II), and dizinc(II) complexes were synthesized. The compounds are [Co(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (1), [Co(2)(mu-OH(2))(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (2a-c), [Co(2)(mu-OH)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (3), [Ni(2)(mu-O(2)CAr(Tol))(4)L(2)] (4), [Ni(2)(mu-HO...H)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (5), and [Zn(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (6), where Ar(Tol)CO(2)H = 2,6-di(p-tolyl)benzoic acid and L = pyridine, THF, or N,N-dibenzylethylenediamine. Structural analysis of these complexes revealed that additional bridging ligands can be readily accommodated within the [M(2)(mu-O(2)CAr(Tol))(2)](2+) core, allowing a wide distribution of M...M distances from 2.5745(6) to 4.0169(9) A. Unprecedented bridging units [M(2)(mu-OH(2))(2)(mu-O(2)CR)(2)](n+) and [M(2)(mu-HO...H)(2)(mu-O(2)CR)(2)](n+) were identified in 2a-c and 5, respectively, in which strong hydrogen bonding accommodates shifts of protons from bridging water molecules toward the dangling oxygen atoms of terminal monodentate carboxylate groups. Such a proton shift along the O...H...O coordinate attenuates the donor ability of the anionic carboxylate ligand, which can translate into increased Lewis acidity at the metal centers. Such double activation of bridging water molecules by a Lewis acidic metal center and a metal-bound general base may facilitate the reactivity of metallohydrolases such as methionine aminopeptidase (MAP).

Aminopeptidases↗
PubMed2003

Computational study of the non-heme iron active site in superoxide reductase and its reaction with superoxide.

The ferrous square-pyramidal [Fe(NHis)4(SCys)] site of superoxide reductases (SORs) has been shown to reduce superoxide at a nearly diffusion-controlled rate. The final products of the reaction are hydrogen peroxide and the ferric hexacoordinated SOR site, with a carboxylate group from a conserved glutamate serving as the sixth ligand trans to the cysteine sulfur. A transient intermediate absorbing at approximately 600 nm in the reaction of the ferrous pentacoordinated site with superoxide has been proposed to be a ferric-(hydro)peroxo complex (Coulter, E.; Emerson, J.; Kurtz, D. M., Jr.; Cabelli, D. J. Am. Chem. Soc. 2000, 122, 11555-11556.). In the present study, DFT and ZINDO/S-CI results are shown to support the description of the 600-nm intermediate as an end-on, low-spin ferricperoxo or--hydroperoxo complex. Side-on peroxo coordination was found to be significantly less stable than end-on because of constraints on the imidazole ligand ring orientations imposed mostly by the protein. The modeled ferric-hydroperoxo complex had a decidedly nonplanar CysC beta-S-Fe-O-O geometry that appears to be imposed by the same constraints. A single prominent visible absorption of the (hydro)peroxo model is shown to be due mainly to a CysS-->Fe(III) pi charge transfer (CT) transition with a minor portion of His-->Fe(III) pi CT character and very little peroxo-->Fe(III) CT character. On the basis of calculations of models with various mono- and diprotonated peroxo ligands, protonation of the iron-bound peroxo oxygen is a key step in the decay of the ferric(hydro)peroxo complex favoring release of hydrogen peroxide over cleavage of the O-O bond, as occurs in the heme structural analogue, cytochrome P450.

Binding Sites↗
PubMed2001

A dipolar coupling based strategy for simultaneous resonance assignment and structure determination of protein backbones.

A new approach for simultaneous protein backbone resonance assignment and structure determination by NMR is introduced. This approach relies on recent advances in high-resolution NMR spectroscopy that allow observation of anisotropic interactions, such as dipolar couplings, from proteins partially aligned in field ordered media. Residual dipolar couplings are used for both geometric information and a filter in the assembly of residues in a sequential manner. Experimental data were collected in less than one week on a small redox protein, rubredoxin, that was 15N enriched but not enriched above 1% natural abundance in 13C. Given the acceleration possible with partial 13C enrichment, the protocol described should provide a very rapid route to protein structure determination. This is critical for the structural genomics initiative where protein expression and structural determination in a high-throughput manner will be needed.

Models, Molecular↗
PubMed2003

Observation of Fe-H/D modes by nuclear resonant vibrational spectroscopy.

Metal-hydrogen bonding is important in chemistry and catalysis, but H atoms are often difficult to observe, especially in metalloproteins. In this work we show that Fe-H interactions can be probed by nuclear resonance vibrational spectroscopy at the 14.4 keV 57Fe nuclear resonance. An important advantage of this method, compared to Raman and IR spectroscopy, is the selectivity for modes that involve 57Fe motion. We present data on the FeS4 site in rubredoxin and the [FeH(D)6]2- ion. Prospects for studying more complex systems are discussed.

Hydrogen↗
PubMed2005

De novo design of a redox-active minimal rubredoxin mimic.

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins and the de novo design of helical bundles that bind metal ion cofactors. However, the design of metalloproteins containing beta-structure is less well developed, despite the frequent occurrence of beta-conformations in natural metalloproteins. Here, we describe the design and construction of a beta-protein, RM1, that forms a stable, redox-active 4-Cys thiolate Fe(II/III) site analogous to the active site of rubredoxin. The protein folds into a beta-structure in the presence and absence of metal ions and binds Fe(II/III) to form a redox-active site that is stable to repeated cycles of oxidation and reduction, even in an aerobic environment.

Binding Sites↗
PubMed2004

Discovery of the chemical function of glycosidases: design, synthesis, and evaluation of mass-differentiated carbohydrate libraries.

[reaction: see text] Discovery of the catalytic chemical function of the many putative glycosidases coded in genomes currently relies on individual testing of possible substrates, usually as their p-nitrophenol conjugate. Herein, we present an alternative chemical proteomics approach using a synthetic mass-differentiated heat-stable substrate library with mass spectrometry readout. Library components do not serve as reaction inhibitors and both primary and secondary enzyme substrates can be delineated.

Carbohydrates↗
PubMed1999

Improved segmental isotope labeling of proteins and application to a larger protein.

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13C/15N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.

ATP-Binding Cassette Transporters↗
PubMed1997

A model for the unusual kinetics of thermal denaturation of rubredoxin.

The thermal denaturation of the simple, redox-active iron protein rubredoxin is characterized by a slow, irreversible decay of the characteristic red color of the iron center at elevated temperatures in the presence of oxygen at pH 7.8. The denaturation rate is essentially constant and the time period for complete bleaching is nearly independent of protein concentration. These two characteristics of the kinetics can be fit by a simple self-catalyzed kinetics model consisting of the combination of a first-order decay and catalysis by some product of that decay, i.e., dP/dt = k1[A] + (k2[P][A])/(K(m) + [A]), where A is native rubredoxin, P, is unspecified product, k1 is a first-order rate constant, and k2 and K(m) are the catalytic constants. In order for the second term to be of this simple form over the full course of a decay, the model must include the condition that the reaction is effectively irreversible. This model has properties which suggest other biological roles in regulation (changes in k1 or k2 can dramatically modulate the kinetics), in timing (titer-independent fixed reaction time), and in self-activation reactions. At one extreme (k1 >> k2) the kinetics becomes exponential, but at the other extreme (k2 >> k1) they show a dramatic and rapid terminal increase after a lag period. Some obvious possible roles in the kinetics of programmed cell death, prion disease, and protease autoactivation are discussed.

Catalysis↗
PubMed2004

Insights into ABC transport in archaea.

In archaea, ATP-binding cassette (ABC) transporters play a crucial role in substrate uptake, export, and osmoregulation. Archael substrate-binding-protein-dependent ABC transporters are equipped with a very high affinity for their cognate substrates which provide these organisms with the ability to efficiently scavenge substrates from their environment even when present only at low concentration. Further adaptations to the archaeal way of life are especially found in the domain organization and anchoring of the substrate-binding proteins to the membrane. Examination of the signal peptides of binding proteins of 14 archael genomes showed clear differences between euryarchaeotes and crenarchaeotes. Furthermore, a profiling and comparison of ABC transporters in the three sequenced pyrococcal strains was performed.

ATP-Binding Cassette Transporters↗
PubMed2005

Hot from the vent.

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DNA-Directed DNA Polymerase↗
PubMed1999

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

Archaeoglobus fulgidus↗
PubMed2006

Crystal structure of an H/ACA box ribonucleoprotein particle.

H/ACA ribonucleoprotein particles (RNPs) are a family of RNA pseudouridine synthases that specify modification sites through guide RNAs. They also participate in eukaryotic ribosomal RNA processing and are a component of vertebrate telomerases. Here we report the crystal structure, at 2.3 A resolution, of an entire archaeal H/ACA RNP consisting of proteins Cbf5, Nop10, Gar1 and L7ae, and a single-hairpin H/ACA RNA, revealing a modular organization of the complex. The RNA upper stem is bound to a composite surface formed by L7ae, Nop10 and Cbf5, and the RNA lower stem and ACA signature motif are bound to the PUA domain of Cbf5, thereby positioning middle guide sequences so that they are primed to pair with substrate RNA. Furthermore, Gar1 may regulate substrate loading and release. The structure rationalizes the consensus structure of H/ACA RNAs, suggests a functional role of each protein, and provides a framework for understanding the mechanism of RNA-guided pseudouridylation, as well as various cellular functions of H/ACA RNP.

Archaeal Proteins↗
PubMed2007

Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter.

Secondary transporters are integral membrane proteins that catalyse the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of Glt(Ph), a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-beta-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound alpha-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.

Amino Acid Motifs↗