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(E)-1-Hydroxy-2-methylbut-2-enyl 4-diphosphate reductase (IspH) is a [Fe4S4] cluster-containing enzyme involved in isoprenoid biosynthesis in many bacteria as well as in malaria parasites and is an important drug target. Several inhibitors including amino and thiol substrate analogues, as well as acetylene and pyridine diphosphates, have been reported. Here, we investigate the mode of binding of four pyridine diphosphates to Escherichia coli IspH by using X-ray crystallography. In three cases, one of the iron atoms in the cluster is absent, but in the structure with (pyridin-3-yl)methyl diphosphate, the most potent pyridine-analogue inhibitor reported previously, the fourth iron of the [Fe4S4] cluster is present and interacts with the pyridine ring of the ligand. Based on the results of quantum chemical calculations together with the crystallographic results we propose a side-on η(2) coordination of the nitrogen and the carbon in the 2-position of the pyridine ring to the unique fourth iron in the cluster, which is in the reduced state. The X-ray structure enables excellent predictions using density functional theory of the (14)N hyperfine coupling and quadrupole coupling constants reported previously using HYSCORE spectroscopy, as well as providing a further example of the ability of such [Fe4S4]-containing proteins to form organometallic complexes.

We have obtained the structure of the bacterial diterpene synthase, tuberculosinol/iso-tuberculosinol synthase (Rv3378c) from Mycobacterium tuberculosis , a target for anti-infective therapies that block virulence factor formation. This phosphatase adopts the same fold as found in the Z- or cis-prenyltransferases. We also obtained structures containing the tuberculosinyl diphosphate substrate together with one bisphosphonate inhibitor-bound structure. These structures together with the results of site-directed mutagenesis suggest an unusual mechanism of action involving two Tyr residues. Given the similarity in local and global structure between Rv3378c and the M. tuberculosis cis-decaprenyl diphosphate synthase (DPPS; Rv2361c), the possibility exists for the development of inhibitors that target not only virulence but also cell wall biosynthesis, based in part on the structures reported here.

We show that copper-containing metal–organic nanoparticles (MONPs) are readily synthesized via Cu(II)-mediated intramolecular cross-linking of aspartate-containing polyolefins in water. In situ reduction with sodium ascorbate yields Cu(I)-containing MONPs that serve as highly efficient supramolecular catalysts for alkyne–azide “click chemistry” reactions, yielding the desired 1,4-adducts at low parts per million catalyst levels. The nanoparticles have low toxicity and low metal loadings, making them convenient, green catalysts for alkyne–azide “click” reactions in water. The Cu-MONPs enter cells and perform efficient, biocompatible click chemistry, thus acting as intracellular nanoscale molecular synthesizers.

The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and (2)H, (17)O, and (57)Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe-4S](2+) cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate's 3-CH(2)OH group has rotated away from the reduced iron-sulfur cluster, and the next, η(3)-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (4 and 5) follow the same reaction mechanism.

We report the inhibition of the Aquifex aeolicus IspH enzyme (LytB, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, EC 1.17.1.2) by a series of diphosphates and bisphosphonates. The most active species was an alkynyl diphosphate having IC(50) = 0.45 microM (K(i) approximately 60 nM), which generated a very large change in the 9 GHz EPR spectrum of the reduced protein. On the basis of previous work on organometallic complexes, together with computational docking and quantum chemical calculations, we propose a model for alkyne inhibition involving pi (or pi/sigma) "metallacycle" complex formation with the unique fourth Fe in the Fe(4)S(4) cluster. Aromatic species had less activity, and for these we propose an inhibition model based on an electrostatic interaction with the active site E126. Overall, the results are of broad general interest since they not only represent the first potent IspH inhibitors but also suggest a conceptually new approach to inhibiting other Fe(4)S(4)-cluster-containing proteins that are of interest as drug and herbicide targets.

We report the results of an investigation of the binding of a series of bisphosphonate drugs to human bone using 2H, 13C, 15N, and 31P nuclear magnetic resonance spectroscopy. The 31P NMR results show that the bisphosphonate groups bind irrotationally to bone, displacing orthophosphate from the bone mineral matrix. Binding of pamidronate is well described by a Langmuir-like isotherm, from which we deduce an approximately 30-38 A2 surface area per pamidronate molecule and a deltaG = -4.3 kcal mol(-1). TEDOR of [13C3, 15N] pamidronate on bone shows that the bisphosphonate binds in a gauche [N-C(1)] conformation. The results of 31P as well as 15N shift and cross-polarization measurements indicate that risedronate binds weakly, since it has a primarily neutral pyridine side chain, whereas zoledronate (with an imidazole ring) binds more strongly, since the ring is partially protonated. The results of 2H NMR measurements of side-chain 2H-labeled pamidronate, alendronate, zoledronate, and risedronate on bone show that all side chains undergo fast but restricted motions. In pamidronate, the motion is well simulated by a gauche+/gauche- hopping motion of the terminal -CH2-NH3(+) group, due to jumps from one anionic surface group to another. The results of double-cross polarization experiments indicate that the NH3(+)-terminus of pamidronate is close to the bone mineral surface, and a detailed model is proposed in which the gauche side-chain hops between two bone PO4(3-) sites.

We report the results of a solid-state NMR and quantum chemical investigation of the 13C gamma NMR chemical shifts in phenylalanine and tyrosine in dipeptides and proteins. Accurate computation of the experimental shifts is shown to require a good description of local electrostatic field effects, and we find the best results (R2=0.94, rmsd=1.6 ppm, range = 17.1 ppm, N=14) by using a self-consistent reaction field continuum model. There are no obvious correlations with phi, psi, chi 1, or chi2 torsion angles, unlike the results seen with other amino acids. There is, however, a linear relation between computed C gamma atomic charges and shifts for the 14 peptide as well as 18 protein residues investigated. This result is similar to the correlation reported in the 1960s between pi-electron density and 13C shifts for classical 4n + 2 (n=0, 1, 2) pi-electron aromatic species, such as cyclopentadienide and the tropylium cation, and in fact, we found that the shielding/atomic charge correlation seen in the peptides and proteins is virtually identical to that seen with a broad range of aromatic carbocations/carbanions. These results suggest the dominance of an electrostatic field polarization model in which increasing pi electron density results in an increase in C gamma atomic charge and increased shielding (of sigma 11 and sigma 22, perpendicular to the pi orbital) in Phe and Tyr, as well as in the other aromatic species. These results are of general interest since they demonstrate the importance of electrostatic field effects on Phe and Tyr C gamma chemical shifts in peptides and proteins and imply that inclusion of these effects will be necessary in order to interpret the shifts of other aromatic species, such as drug molecules, bound to proteins.

Bisphosphonates are a class of molecules in widespread use in treating bone resorption diseases and are also of interest as immunomodulators and anti-infectives. They function by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), but the details of how these molecules bind are not fully understood. Here, we report the results of a solid-state (13)C, (15)N, and (31)P magic-angle sample spinning (MAS) NMR and quantum chemical investigation of several bisphosphonates, both as pure compounds and when bound to FPPS, to provide information about side-chain and phosphonate backbone protonation states when bound to the enzyme. We then used computational docking methods (with the charges assigned by NMR) to predict how several bisphosphonates bind to FPPS. Finally, we used X-ray crystallography to determine the structures of two potent bisphosphonate inhibitors, finding good agreement with the computational results, opening up the possibility of using the combination of NMR, quantum chemistry and molecular docking to facilitate the design of other, novel prenytransferase inhibitors.

A general protocol for the structural characterization of paramagnetic molecular solids using solid-state NMR is provided and illustrated by the characterization of a high-spin Fe(II) catalyst precursor. We show how good NMR performance can be obtained on a molecular powder sample at natural abundance by using very fast (30 kHz) magic angle spinning (MAS), even though the individual NMR resonances have highly anisotropic shifts and very short relaxation times. The results include the optimization of broadband heteronuclear (proton-carbon) recoupling sequences for polarization transfer; the observation of single or multiple quantum correlation spectra between coupled spins as a tool for removing the inhomogeneous bulk magnetic susceptibility (BMS) broadening; and the combination of NMR experiments and density functional theory calculations, to yield assignments.

We report the first solid-state NMR, crystallographic, and quantum chemical investigation of the origins of the 13C NMR chemical shifts of the imidazole group in histidine-containing dipeptides. The chemical shift ranges for Cgamma and Cdelta2 seen in eight crystalline dipeptides were very large (12.7-13.8 ppm); the shifts were highly correlated (R2= 0.90) and were dominated by ring tautomer effects and intermolecular interactions. A similar correlation was found in proteins, but only for buried residues. The imidazole 13C NMR chemical shifts were predicted with an overall rms error of 1.6-1.9 ppm over a 26 ppm range, by using quantum chemical methods. Incorporation of hydrogen bond partner molecules was found to be essential in order to reproduce the chemical shifts seen experimentally. Using AIM (atoms in molecules) theory we found that essentially all interactions were of a closed shell nature and the hydrogen bond critical point properties were highly correlated with the N...H...O (average R2= 0.93) and Nepsilon2...H...N (average R2= 0.98) hydrogen bond lengths. For Cepsilon1, the 13C chemical shifts were also highly correlated with each of these properties (at the Nepsilon2 site), indicating the dominance of intermolecular interactions for Cepsilon1. These results open up the way to analyzing 13C NMR chemical shifts, tautomer states (from Cdelta2, Cepsilon1 shifts), and hydrogen bond properties (from Cepsilon1 shifts) of histidine residue in proteins and should be applicable to imidazole-containing drug molecules bound to proteins, as well.

We have obtained the carbon-13 nuclear magnetic resonance spectra of a series of tryptophan-containing peptides and model systems, together with their X-ray crystallographic structures, and used quantum chemical methods to predict the (13)C NMR shifts (or shieldings) of all nonprotonated aromatic carbons (C(gamma), C(delta 2) and C(epsilon 2). Overall, there is generally good accord between theory and experiment. The chemical shifts of Trp C(gamma) in several proteins, hen egg white lysozyme, horse myoglobin, horse heart cytochrome c, and four carbonmonoxyhemoglobins, are also well predicted. The overall Trp C(gamma) shift range seen in the peptides and proteins is 11.4 ppm, and individual shifts (or shieldings) are predicted with an rms error of approximately 1.4 ppm (R value = 0.86). Unlike C(alpha) and N(H) chemical shifts, which are primarily a function of the backbone phi,psi torsion angles, the Trp C(gamma) shifts are shown to be correlated with the side-chain torsion angles chi(1) and chi(2) and appear to arise, at least in part, from gamma-gauche interactions with the backbone C' and N(H) atoms. This work helps solve the problem of the chemical shift nonequivalences of nonprotonated aromatic carbons in proteins first identified over 30 years ago and opens up the possibility of using aromatic carbon chemical shift information in structure determination.

We report the results of density functional theory (DFT) calculations of the (57)Fe Mössbauer isomer shifts (delta(Fe)) for a series of 24 inorganic, organometallic, and metalloprotein/metalloporphyrin model systems in S = 0, (1)/(2), 1, (3)/(2), 2, and (5)/(2) spin states. We find an excellent correlation between calculation and experiment over the entire 2.34 mm s(-1) range of isomer shifts: a 0.07-0.08 mm s(-1) rms deviation between calculation and experiment (corresponding to 3-4% of the total delta(Fe) range, depending on the functionals used) with R(2) values of 0.973 and 0.981 (p0.0001). The best results are obtained by using the hybrid exchange-correlation functional B3LYP, used previously for (57)Fe Mössbauer quadrupole splittings and (57)Fe NMR chemical shifts and chemical shielding anisotropies. The relativistically corrected value of alpha, alpha(rel), converges with the large basis set used in this work, but the exact values vary somewhat with the methods used: -0.253 a(0)(3) mm s(-1) (Hartree-Fock; HF); -0.316 a(0)(3) mm s(-1) (hybrid HF-DFT; B3LYP), or -0.367 a(0)(3) mm s(-1) (pure DFT; BPW91). Both normal and intermediate spin state isomer shifts are well reproduced by the calculations, as is the broad range of delta(Fe) values: from [Fe(VI)O(4)](2-) (-0.90 mm s(-1) expt; -1.01 mm s(-1) calc) to KFe(II)F(3) (1.44 mm s(-1) expt; 1.46 mm s(-1) calc). Molecular orbital analyses of all inorganic solids as well as all organometallic and metalloporphyrin systems studied reveal that there are three major core MO contributions to rho(tot)(0), the total charge density at the iron nucleus (and hence delta(Fe)), that do not vary with changes in chemistry, while the valence MO contributions are highly correlated with delta(Fe) (R(2) = 0.915-0.938, depending on the functionals used), and the correlation between the valence MO contributions and the total MO contribution is even better (R(2) = 0.965-0.976, depending on the functionals used). These results are of general interest since they demonstrate that DFT methods now enable the accurate prediction of delta(Fe) values in inorganic, organometallic, and metalloporphyrin systems in all spin states and over a very wide range of delta(Fe) values with a very small rms error.

We have carried out a solid-state magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic investigation of the (13)C(alpha) chemical shielding tensors of alanine, valine, and leucine residues in a series of crystalline peptides of known structure. For alanine and leucine, which are not branched at the beta-carbon, the experimental chemical shift anisotropy (CSA) spans (Omega) are large, about 30 ppm, independent of whether the residues adopt helical or sheet geometries, and are in generally good accord with Omega values calculated by using ab initio Hartree-Fock quantum chemical methods. The experimental Omegas for valine C(alpha) in two peptides (in sheet geometries) are also large and in good agreement with theoretical predictions. In contrast, the "CSAs" (Deltasigma) obtained from solution NMR data for alanine, valine, and leucine residues in proteins show major differences, with helical residues having Deltasigma values of approximately 6 ppm while sheet residues have Deltasigma approximately 27 ppm. The origins of these differences are shown to be due to the different definitions of the CSA. When defined in terms of the solution NMR CSA, the solid-state results also show small helical but large sheet CSA values. These results are of interest since they lead to the idea that only the beta-branched amino acids threonine, valine, and isoleucine can have small (static) tensor spans, Omega (in helical geometries), and that the small helical "CSAs" seen in solution NMR are overwhelmingly dominated by changes in tensor orientation, from sheet to helix. These results have important implications for solid-state NMR structural studies which utilize the CSA span, Omega, to differentiate between helical and sheet residues. Specifically, there will be only a small degree of spectral editing possible in solid proteins since the spans, Omega, for the dominant nonbranched amino acids are quite similar. Editing on the basis of Omega will, however, be very effective for many Thr, Val, and Ileu residues, which frequently have small ( approximately 15-20 ppm) helical CSA (Omega) spans.

The trans hydrogen bond 3hJNC‘ coupling observed between peptide groups in proteins is shown to be mediated by a closed shell, noncovalent interaction between the donor hydrogen atom and the acceptor oxygen atom. The magnitude of 3hJNC‘ is shown to be an exponential function of the mutual penetration of the nonbonding van der Waals shells of the isolated donor and acceptor fragments. Our results also show that the magnitude of JFF, the through-space coupling between two nonbonded fluorine nuclei in organic molecules and in a protein, exhibits a similar exponential dependence upon penetration of nonbonding monomer charge densities. These results support the idea that the existence of electron-coupled nuclear spin−spin coupling requires neither a covalent bond nor an attractive electrostatic bond between the coupled nuclei. By relating the results of calculations using Bader's theory of Atoms in Molecules, (Bader, R. F. W. Atoms in Molecules−A Quantum Theory; Clarendon Press: Oxford, 1990) to these couplin...

By using density functional theory it is demonstrated that the long-range 19F−19F J-couplings (>3J) seen in small organic molecules can be calculated with good accuracy using small molecule fragments and in some cases complete molecules. The results reproduce the exponential distance dependence of J seen experimentally, demonstrate the dominance of the Fermi contact interaction, and rule out any significant covalent or through-bond contributions to J in these systems. The calculations also verify an experimentally observed 19F−19F J-coupling seen between two [6-F]Trp residues in the protein dihydrofolate reductase (for d = 2.98 A), where there is clearly no covalent bonding between the two 19F sites. The results also clarify the abnormally small J-couplings seen previously in phenanthrenes and cyclohexenes, which are shown by ab initio and molecular mechanics geometry optimizations to be due to conversion of the supposedly planar structures to more distorted but less sterically hindered structures. These ...

The influence of a series of different ligands (H, O, S, CN-, CO, and Ru), electrochemically adsorbed onto carbon-supported nanoscale Pt particles, has been investigated by 195Pt NMR, in an electrochemical environment. The NMR Knight shifts from atoms in the interior of the particles are invariant to surface ligand adsorption, as expected by the Friedel−Heine invariance theorem. However, the shifts of the surface and subsurface atoms vary over a ∼11000 ppm range for the adsorbates investigated. The Knight shifts of the surface and subsurface platinum atoms, obtained by using an NMR layer-model simulation, linearly map the electronegativity of the adsorbate. These observations permit the derivation of a correlation between the “healing length” of the Pt metal, a parameter indicating how fast the bulk metal properties are recovered when moving in from the surface, and the electronegativity of the adsorbate, providing a new probe of electrocatalyst and heterogeneous catalyst surface electronic structure.

We have investigated the charge density, ρ(r), its curvature, ∂2ρ/∂rij, the dipole moment, μ, and the electrostatic potential, Φ(r), in l-asparagine monohydrate by using high-resolution single-crystal X-ray crystallography and quantum chemistry. In addition, we have compared electric field gradient, ∇E, results obtained from crystallography and quantum chemistry with those obtained from single-crystal 14N nuclear magnetic resonance spectroscopy. A multipole model of the X-ray ρ(r) is compared to Hartree−Fock and density functional theory predictions, using two different large basis sets. The quality of the calculated charge densities is evaluated from a simultaneous comparison of eight Hessian-of-ρ(r) tensors at bond critical points between non-hydrogen atoms. These tensors are expressed in an icosahedral representation, which includes information on both tensor magnitude and orientation. The best theory-versus-experiment correlation is found at the B3LYP/6-311++G(2d,2p) level, which yields a slope of 1.0...

13C NMR shift and spin−lattice relaxation measurements have been used to investigate 13CO (ex MeOH) on fuel cell grade Pt electrodes (having average particle diameters of 2, 2.5, and 8.8 nm) in an electrochemical environment from 80 to 293 K at 8.47 and 14.1 T. The temperature dependence of the 13C spin−lattice relaxation rate, 1/T1, shows a Korringa relationship which is independent of magnetic field, for all three samples. However, the peak positions and the corresponding T1T values depend on particle size, with those of the 8.8 nm sample approaching values found for unsupported polycrystalline platinum black in an electrochemical environment (J. B. Day et al., J. Am. Chem. Soc. 1996, 118, 13046−13050). The 13C T1 is single exponential, independent of particle size and temperature, in contrast to previous results obtained on oxide-supported Pt−CO systems in a “dry” environment, in which relaxation was nonexponential at low temperatures, but exponential at high temperatures, suggesting strongly a quantum...

We have synthesized and studied via solid-state NMR, Mossbauer spectroscopy, single-crystal X-ray diffraction, and density functional theory the following Fe−O2 analogue metalloporphyrins: Fe(5,10,15,20-tetraphenylporphyrinate) (nitrosobenzene)(1-methylimidazole); Fe(5,10,15,20-tetraphenylporphyrinate) (nitrosobenzene)(pyridine); Fe(5,10,15,20-tetraphenylporphyrinate)(4-nitroso-N,N-dimethylaniline)(pyridine); Fe(2,3,7,8,12,13,17,18-octaethylporphyrinate) (nitrosobenzene)(1-methylimidazole) and Co(2,3,7,8,12,13,17,18-octaethylporphyrinate)(NO). Our results show that the porphyrin rings of the two tetraphenylporphyrins containing pyridine are ruffled while the other three compounds are planar: reasons for this are discussed. The solid-state NMR and Mossbauer spectroscopic results are well reproduced by the DFT calculations, which then enable the testing of various models of Fe−O2 bonding in metalloporphyrins and metalloproteins. We find no evidence for two binding sites in oxypicket fence porphyrin, chara...

We have synthesized and characterized via single-crystal X-ray diffraction methods iron(II), ruthenium(II), and osmium(II) carbonyl derivatives of (1-methylimidazole)(5,10,15,20-tetraphenylporphyri...

We have investigated the question of how CO ligands bind to iron in metalloporphyrins and metalloproteins by using a combination of nuclear magnetic resonance (NMR), 57Fe Mossbauer, and infrared sp...

The carbon-13 and oxygen-17 nuclear magnetic resonance spectroscopic shielding behavior, as well as the oxygen-17 nuclear quadrupole coupling constants (NQCC), in the four metal−CO systems Fe(CO)5, Fe2(CO)9, Ni2(η5-C5H5)2(CO)2, and Rh6(CO)16 have been investigated both experimentally and by density functional calculations. Characteristics of the spectroscopic observables and bonding for the most common types of metal−carbonyl coordination, μ1-, μ2-, and μ3-CO, may thus be compared in detail. There is generally very good agreement between the theoretical predictions and the experimental measurements, including the 17O shift predictions for Fe2(CO)9 and Rh6(CO)16 made previously. Interestingly, the bridging oxygen shift tensor in Fe2(CO)9 has its most deshielded component parallel to the C−O axis. This is highly unusual for carbonyl ligands, but is the normal behavior seen in organic carbonyl groups. To explain this and other observations, the computed shielding tensors and electric field gradients have bee...

We have investigated the 57Fe Mossbauer quadrupole splittings in the following compounds by using density functional theory, and in some cases via experiment: Fe(CO)3(cyclo-butadiene), Fe(CO)5, Fe(CO)3(1,4−butadiene), CpFe(CO)2Me, Fe(CO)3(propenal), CpFe(CO)2Cl, (CO)(pyridine)(DMGBPh2)2Fe(II) (DMG = dimethylglyoximato), (CO)(pyridine)(DMGBBN)2Fe(II) (BBN = 9-borabicyclo[3.3.1]nonane), (CO)(1-methylimidazole)(5,10,15,20-tetraphenylporphinato)Fe(II), (CO)(pyridine)(5,10,15,20-tetraphenyl-porphinato)Fe(II), (nitrosobenzene)(pyridine)(5,10,15,20-tetraphenylporphinato)Fe(II), (pyridine)2(5,10,15,20-tetraphenylporphinato)Fe(II), (1-methylimidazole)2(5,10,15,20-tetramesitylporphinato)Fe(II), and (trimethylphosphine)2(2,3,7,8,12,13,17,18-octaethylporphinato)Fe(II). The electric field gradients at iron were evaluated by using a locally dense basis approach: a Wachters' all electron representation for iron, a 6-311++G2d basis for all atoms directly bonded to iron, and either a 6-31G* basis for all other atoms or,...

We have investigated the carbon-13 solution nuclear magnetic resonance (NMR) chemical shifts of Cα, Cβ, and Cγ carbons of 19 valine residues in a vertebrate calmodulin, a nuclease from Staphylococcus aureus, and a ubiquitin. Using empirical chemical shift surfaces to predict Cα, Cβ shifts from known, X-ray φ,ψ values, we find moderate accord between prediction and experiment. Ab initio calculations with coupled Hartree−Fock (HF) methods and X-ray structures yield poor agreement with experiment. There is an improvement in the ab initio results when the side chain χ1 torsion angles are adjusted to their lowest energy conformers, using either ab initio quantum chemical or empirical methods, and a further small improvement when the effects of peptide-backbone charge fields are introduced. However, although the theoretical and experimental results are highly correlated (R2 ∼ 0.90), the observed slopes of ∼−0.6−0.8 are less than the ideal value of −1, even when large uniform basis sets are used. Use of density ...

The carbon-13 nuclear magnetic resonance shielding surfaces for the isotropic and anisotropic shielding components, σ11, σ22, and σ33, for Cα in N-formylglycine amide, and Cα and Cβ in N-formylalanine amide, N-formylvaline amide (χ1 = 180°, −60°, 60°), N-formylisoleucine amide (all χ1 = −60° conformers), N-formyl serine amide (χ1 = 74.3°), and N-formylthreonine amide (χ1 = 180°, −60°, 60°) have been computed at the Hartree−Fock level by using large, locally dense basis sets. The results for Cα in glycine and alanine show the expected ∼4−5 ppm increase in isotropic shielding of sheet over helical geometries, and the overall breadths of the shielding tensors are very similar for both helical and sheet fragments (|σ33 − σ11| ∼ 31−37 ppm). However, for each of the Cβ substituted amino acids (valine, isoleucine, serine, and threonine) our results for Cα indicate not only the expected ∼4−5 ppm increase in shielding of sheet fragments over helical ones but also a large increase in the overall tensor breadths for...

A simple method for the determination of backbone dihedral angles in peptides and proteins is presented. The chemical-shift anisotropies (CSA) of the central alanine α-carbon in powdered crystalline tripeptides, whose structures have been determined previously by X-ray crystallography, were measured by cross-polarization magic-angle-spinning nuclear magnetic resonance. The experimental CSA values were correlated with ab initio chemical-shielding calculations over Ramanchandran φ/ψ space on an N-formyl-l-alanine amide fragment. Using this correlation, φ/ψ probability surfaces for one of the tripeptides were calculated based only on the α-carbon CSA, allowing a prediction of backbone angles. Dihedral angles predicted by these calculations fall within ±12° of the values determined by crystallography. This approach should be useful in the determination of solid-state protein structure.

A density functional method has been used to successfully predict the isotropic 59Co nuclear magnetic resonance (NMR) chemical shifts of the following anionic, cationic and neutral Co(III) complexes: [Co(CN)6]3-, [Co(NH3)6]3+, [Co(NO2)6]3-, [Co(NH3)4CO3]+, Co(acac)3, and [Co(en)3]3+. Isotropic chemical shifts are well-reproduced by using Wachters' cobalt basis set and uniform 6-31G* basis sets on the light atoms, together with the use of the B3LYP hybrid functional. In addition, the principal elements of the 59Co shielding tensor (σ11, σ22, and σ33), the absolute shieldings of Co(CN)63- and Co(acac)3, and the Co−C bond length shielding derivative for Co(CN)63- are also in good agreement with previous experimental estimates. There are no obvious distinctions between the predicted shifts (or shielding tensor elements) of anionic and cationic complexes. The ability to successfully predict both shift trends, absolute shieldings, shielding tensor elements, and a vibrational shielding derivative for d6 transit...

We have prepared Lactobacillus casei dihydrofolate reductase containing biosynthetically incorporated (2S,4S)-5-fluoroleucine ([5-F]-Leu DHFR) and have obtained its 1H and 19F NMR spectra at 9.4 Tesla. The 19F spectrum of [5-F]-Leu DHFR showed 12 fairly sharp peaks (one containing two overlapped signals) for the 13 leucine residues in DHFR, covering a chemical shift range of 15 ppm. The large range of chemical shifts observed could not be explained solely in terms of the electrostatic field effects due to local charge fields and is thought to have a second contribution from side-chain conformational differences (γ-gauche effects) between different leucine residues, making 19F NMR of aliphatic amino acids in proteins a potentially useful new probe of protein structure.

We have investigated the effects of basis set quality on the accuracy and speed of ab initio calculations of Cat CB chemical shifts (or shieldings) of alanine and valine residues in proteins. Five basis set combinations were studied: a uniform 3-21G and a 4-31G/3-21G “locally dense” basis, both producing 88 contracted functions; a 6-31 lG+(2d)/3-21G and a 6-311G+(2d)/6-31G basis, each giving a total of 108 contracted functions; and a uniform STO-3G basis, giving 48 contracted functions. We find that results obtained by using small Gaussian basis sets correlate well with calculations performed using very large (6-31 lG++(2d,2~)/6-31G) basis sets, with only changes in slope and offset being required in order to bring results into excellent agreement, while the time necessary for the calculations is significantly reduced. This marked decrease in computational time using small, locally dense basis sets has permitted the first calculation of a three-dimensional chemical shielding hypersurface for valine. Chemical shifts predicted by using the @,v,xl shielding hypersurface are in good accord with chemical shift values detennined experimentally. These results open up new opportunities for the relatively rapid evaluation of amino acid shielding hypersurfaces for future use in protein structure prediction and refinement.

We show that protein structures in solution can be refined by using I3Ca and I3C@ chemical shifts. We investigated 12 alanine and 8 valine residues in the nuclease from Staphylococcus aureus and found that the 4, q, and x values of these residues are closer to the X-ray 4, q, and x values when nuclear Overhauser effect distance restraints and J-coupling torsion angle restraints are supplemented with chemical shift restraints. For 4 in particular, the rmsd versus the X-ray structure is -10" with chemical shift restraints versus -20" without chemical shift restraints. Both "fully-restrained" families of structures, those determined with and without chemical shift constraints, had small numbers of minor NOE violations. However, the chemical shift restrained structures were consistent with experimental Ala and Val I3Ca and I3C0 chemical shifts, whereas the structures determined without shift restraints yielded back calculated chemical shifts in poor accord with experiment. Carbon- 13 chemical shifts therefore appear to be of use in protein structure refinement when used in conjunction with chemical shift surfaces computed using ab initio methods.

IspH is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using hyperfine sublevel correlation (HYSCORE) spectroscopy, we show that pyridine inhibitors of IspH directly bind to the unique fourth Fe in the 4Fe-4S cluster, opening up new routes to inhibitor design, of interest in the context of both anti-bacterial as well as anti-malarial drug discovery.

Using gauge-including atomic orbital self-consistent field ab initio quantum chemical methods, we have computed the effects of bond lengths, bond angles, and torsion angles on the carbon- 13 chemical shielding of C α (and C β ) sites in model fragments for glycine, alanine, and valine residues in proteins. Predicted chemical shieldings are highly sensitive to bond length variations, and we show that it is essential to relax or energy minimize protein structures (to remove large errors associated with bond length uncertainties) in order to sucessfully predict experimental 13 C NMR spectra

We report the first successful calculation of the fluorine nuclear magnetic resonance spectrum of a protein, the galactose binding protein from Escherichia coli, labeled with ( 5-'9F) tryptophan. Our results indicate that the experimental l9F chemical shifts are dominated by weak, or long-range, electrical interactions, which can be calculated by using the responses of the shielding tensor elements to the uniform field components (&~d/dE,) and the nonuniform or gradient terms (t3uap/W,,), together with the average values of the fields (Ex) and field gradients (V,,) obtained from molecular dynamics (MD) trajectories. A series of "shielding trajectories", Au(Ex,Vxx,Vyy,Vzz)f(7), are obtained, and the mean values, A;, correlate well with the actual shift pattern and overall shift range observed experimentally (Luck, L. A.; Falke, J. J. Biochemistry 1991,30,42484256). The computed 19F NMR shielding of the pentapeptide Gly- Gly-(5-F)Trp-Gly-Gly in HzO is close to, but somewhat more shielded than, that of the denatured protein. Almost all computed 19F chemical shifts are upfield of the field-free value, in accord with the results of ab initio calculations. The chemical shifts calculated are sensitive to the charge chosen for F in the LRF-MD trajectories, and best agreement with experiment is obtained with qF = -0.25. Calculations on the Salmonella typhimurium galactose binding protein yield extremely similar chemical shift spectra, consistent with the -7% difference in amino acid composition. The uniform field components make the largest contributions to the shielding patterns observed, presumably because the gradient terms fall off more rapidly with distance. The exposed residue, Trp 284, has the largest amplitude of fluctuation associated with its shielding trajectory, possibly due to the rapid and random movement of neighboring water molecules. Trp 284 is highly shielded due to interaction with water, although other buried residues (e.g. Trp 133) may also be highly shielded, due to electric field effects within the protein. Our results imply that van der Waals interactions do not play a major role for fluorine shielding nonequivalence in proteins, since the experimental results can be reproduced by using solely the computed field and field gradient terms. The ability to predict protein NMR shielding patterns and ranges offers promise for structural analysis and also provides a way of validating different methods of computing protein electrostatics. In this respect, it is instructive to note that the charge fields from ionized surface groups are found to be largely shielded by the dielectric response of the solvent and the protein.

Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.

We present a molecular model of the major "conformational substates" observed by infrared spectroscopy in carbonmonoxyhemoglobins, myoglobins, and peroxidases, in tem of an electrical perturbation of the CO fundamental vibrational frequencies due to the possibility of Hez - Hdl tautomerism, and 180° C@-C' ring flips, of distal histidine residues. The model supports a previous interpretation of vibrational frequencies and nuclear magnetic resonance chemical shifts of CO ligands in heme proteins as originating in a weak electric field (dipole and quadrupole interactions with permanent dipole moments, dipole polarizabilities, and shielding polarizabilities), and opens up the possibility of future detailed molecular interpretations of both NMR chemical shifts (of 'H, I3C, and I5N), as well as IR data, on proteins and other macromolecules.

We report the results of an investigation of the interaction between Se and Ru in Ru−Se electrocatalysts showing high activity and methanol tolerance in the oxygen reduction reaction (ORR). The res...

We report the results of an ITC (isothermal titration calorimetry) investigation of the binding of six bisphosphonates to the enzyme farnesyl diphosphate synthase (FPPS; EC 2.5.1.10) from Trypanosoma brucei. The bisphosphonates investigated were zoledronate, risedronate, ibandronate, pamidronate, 2-phenyl-1-hydroxyethane-1,1-bisphosphonate, and 1-(2,2-bisphosphonoethyl)-3-iodo pyridinium. At pH = 7.4, both risedronate and the phenylethane bisphosphonate bind in an enthalpy-driven manner (DeltaH approximately -9 to 10 kcal mol-1), but the other four bisphosphonates bind in an entropy-driven manner (DeltaS varying from 31.2 to 55.1 cal K-1 mol-1). However, at pH = 8.5, zoledronate binding switches from entropy to enthalpy-driven. The DeltaG results are highly correlated with FPPS inhibition results obtained using a radiochemical assay (R2 = 0.85, N = 11, P < 0.001). The DeltaH and DeltaS results are interpreted in terms of a model in which bisphosphonates with charged side chains have positive DeltaH values, due to the enthalpic cost of desolvation (due to strong ion-dipole interactions) and, likewise, a positive DeltaS, due to an increase in water entropy (both ligand and protein associated) on ligand binding to FPPS: the hydrophobic effect. For the neutral side chains (risedronate at pH 7.4, 8.5 and zoledronate at pH 8.5, as well as the phenylethane bisphosphonate), binding is overwhelmingly enthalpy-driven, with the enhanced activity of the basic side chain containing species being attributable to their becoming protonated in the active site. Given the large size of the bisphosphonate market and the potential importance of the development of these compounds for cancer immunotherapy and anti-parasitic chemotherapy, these results are of broad general interest in the context of the development of new, potent, and selective FPPS inhibitors.

We report the experimental determination of the (13)C(alpha) chemical shift tensors of Ala, Leu, Val, Phe, and Met in a number of polycrystalline peptides with known X-ray or de novo solid-state NMR structures. The 700 Hz dipolar coupling between (13)C(alpha) and its directly bonded (14)N permits extraction of both the magnitude and the orientation of the shielding tensor with respect to the C(alpha)-N bond vector. The chemical shift anisotropy (CSA) is recoupled under magic-angle spinning using the SUPER technique (Liu et al., J. Magn. Reson. 2002, 155, 15-28) to yield quasi-static chemical shift powder patterns. The tensor orientation is extracted from the (13)C-(14)N dipolar modulation of the powder line shapes. The magnitudes and orientations of the experimental (13)C(alpha) chemical shift tensors are found to be in good accord with those predicted from quantum chemical calculations. Using these principal values and orientations, supplemented with previously measured tensor orientations from (13)C-(15)N and (13)C-(1)H dipolar experiments, we are able to predict the (phi, psi, chi(1)) angles of Ala and Val within 5.8 degrees of the crystallographic values. This opens up a route to accurate determination of torsion angles in proteins based on shielding tensor magnitude and orientation information using labeled compounds, as well as the structure elucidation of noncrystalline organic compounds using natural abundance (13)C NMR techniques.

We have used quantum chemical methods to predict 67Zn NMR chemical shifts as well as quadrupole coupling constants (CQ) in a series of biomimetic and inorganic zinc complexes. The 67Zn chemical shifts are predicted with an R2 = 0.975, corresponding to a 24.3 ppm or 6.7% error over the entire 365 ppm 67Zn chemical shift range. The 67Zn CQ values are predicted with an R2 = 0.991, corresponding to a 1.17 MHz or 3.0% error over the entire 38.75 MHz range. The 67Zn NMR shifts in a series of complexes containing N,O ligands are, in general, highly correlated with the number of oxygen ligands. The ability to compute 67Zn NMR shifts as well as CQ values opens up the possibility of using both of these properties in structure determination or refinement in proteins.

We report the crystallographic structures of the potent phosphoantigens Phosphostim (the bromohydrin of isopentenyl pyrophosphate) and E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate bound to the mevalonate pathway enzyme isopentenyl pyrophosphate/dimethylallyl pyrophosphate isomerase (IPPI). Racemic Phosphostim forms covalent complexes with IPPI: a 4-thioether with C67 and a 4-ester with E116. Only the E116 ester forms with the chiral species, S-Phosphostim, with the w.t. enzyme, while the C67 thioether forms with a mutant Y104F IPPI. The potent phosphoantigen HMBPP also binds to IPPI, but is only a weak ( approximately 50 muM) inhibitor. These results strongly support an SN2 reaction for inhibition of IPPI by Phosphostim, in contrast to the SN1 or concerted type of reaction found with epoxide inhibitors, which react at C-3, and are of general interest in the context of the development of novel mevalonate pathway inhibitors. They also provide clues as to the nature of the binding site of synthetic phosphoantigens in gammadelta T cell activation. In particular, both bromohydrin and epoxy phosphoantigens are potent, irreversible inhibitors of IPPI while HMBPP is only a weak inhibitor, ruling out an IPPI or IPPI-like target for HMBPP in gammadelta T cell activation.

We have carried out a series of density functional theory (DFT) calculations to predict the 57Fe Mössbauer quadrupole splittings (DeltaEQ) and isomer shifts (deltaFe) for the nitrosyl complex of isopenicillin N synthase with the substrate delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (IPNS.ACV.NO) and an {FeNO}7 (S = 3/2) model system, FeL(NO)(N3)2 (L = N,N',N' '-trimethyl-1,4,7-triazacyclononane). B3LYP predictions on the model compound are in almost exact agreement with experiment. The same DFT methods did not enable the prediction of the experimental DeltaEQ and deltaFe results for IPNS.ACV.NO when using the experimental protein crystal structure but did permit good predictions of DeltaEQ, deltaFe, and the asymmetry parameter (eta) when using a fully optimized structure. This optimized structure also enabled good predictions of the Mössbauer spectra of the photodissociation product of IPNS.ACV.NO. Mulliken and natural bonding orbital (NBO) spin density analyses indicate an electronic configuration of FeII (S = 2) anti-ferromagnetically coupled to NO (S = 1/2) in the protein as well as in the model system and the geometry optimized structure helps explain part of the enzyme reaction.

We report the results of a series of density functional theory (DFT) calculations of the Mössbauer quadrupole splittings and isomer shifts in NO heme model compounds, together with the results of calculations of the Mössbauer quadrupole splittings, isomer shifts, and electron paramagnetic resonance hyperfine coupling constants in a model Fe(II)(NO)(imidazole) complex as a function of Fe-NO bond length and Fe-N-O bond angle. The results of the Mössbauer quadrupole splitting and isomer shift calculations on the NO heme model compounds show good accord between theory and experiment, with the largest errors being observed for structures having the largest crystallographic R(1) values. The results of the property surface calculations were then used to calculate Fe-NO bond length and Fe-N-O bond angle probability surfaces (Z-surfaces) for a nitrosyl hemoglobin, using, in addition, an energy filter. The results obtained yielded a most probable Fe-NO bond length (r) of 1.79 A and an Fe-N-O bond angle (beta) of 136 degrees -137 degrees. This bond length is somewhat longer than those observed in most model compounds but may be due, at least in part, to hydrogen bond formation with the distal His residue. Bond elongation was also observed in a geometry optimized Fe(II)(NO)(imidazole) complex hydrogen bonded to an imidazole residue, in which we find r = 1.76-1.78 A and beta = 137 degrees -138 degrees. The computed bond angles are close to the canonical approximately 140 degrees value found in most model systems. Highly bent Fe-N-O bond angles or very long Fe-NO bond lengths seem unlikely to occur in proteins, due to their high energies. We also investigated the molecular orbitals and spin densities in each of the six coordinate systems investigated and found the orbitals and spin densities to be generally similar those described previously for five coordinate systems. Taken together, these results show that Mössbauer quadrupole splittings and isomer shifts, in addition to electron paramagnetic resonance hyperfine coupling constants, can now be calculated for nitrosyl heme systems with relatively good accuracy and that the results so obtained can be used to determine Fe-N-O geometries in metalloproteins. The Z-surface approach is thus applicable to both diamagnetic (CO) and paramagnetic (NO) heme proteins with in both cases the metal-ligand binding geometries found in the proteins being very close to those seen in model systems.

We have obtained the three-dimensional X-ray crystallographic structure of a C67A mutant Escherichia coli isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase (EC 5.3.3.2) complexed with the bromohydrin of isopentenylpyrophosphate, at 1.93 Å resolution. The overall backbone fold is very similar to that obtained previously for the wild-type enzyme in the presence of a divalent metal cation (Mn2+ or Mg2+). However, in the new structure, there are two metal binding sites, not just one. The first metal binding site is occupied by Mn2+, coordinated to three histidine and two glutamate residues, while the second is occupied by Mg2+, coordinated to two bromohydrin-ligand phosphate oxygens, the carbonyl oxygen of A67, a carboxyl oxygen of E87, and two water molecules. The C3 hydroxyl group of the bromohydrin inhibitor is involved in a short hydrogen bond to the carboxyl group of E116, one of the two Mn-bound glutamates. The structure obtained is consistent with a mechanism of action of the enzyme in which the carboxyl group of E116 protonates the double bond in isopentenylpyrophosphate, forming a carbocation, followed by removal of a C2 proton by the thiolate of C67, in the wild-type enzyme. The inhibition of the enzyme by a wide variety of other potent inhibitors is also readily explained on the basis of the bromohydrin inhibitor structure.