Refined X-ray Crystal Structure Research Articles

New building blocks in the 2/1 crystalline approximant of a Bergman-type icosahedral quasicrystal.

The refined x-ray crystal structure of the phase Mg(27)Al(10.7(2))Zn(47.3(2)) (Pa3) establishes it as the new 2/1 Bergman-type approximant of the icosahedral quasicrystal. The primitive cubic lattice consists of condensed triacontahedral and novel prolate rhombohedral (PR) clusters. Each triacontahedron encapsulates the traditional, multiply endohedral Bergman-type clusters, and each PR encapsulates an Al(2) dimer. This phase exhibits the same long-range order as recently established for the Tsai-type Sc-Mg-Zn 2/1 approximant crystal, with substantial geometric and atomic distribution differences between the two only in the short range orders. This common feature suggests that Bergman- and Tsai-type quasicrystals may be more similar than earlier conceived. Factors germane to the formation of, and the differences between, Bergman- vs. Tsai-type 1/1 and 2/1 approximate structures are considered, including notably different distributions of the more electropositive elements.

Experimental support for the "E pathway hypothesis" of coupled transmembrane e- and H+ transfer in dihemic quinol:fumarate reductase.

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called "E pathway hypothesis." According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display approximately 1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, K(m). The refined x-ray crystal structures at 2.19 A and 2.76 A resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 --> Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180.

Cation Substitutional Chemistry in the R7X12Z‐Type Structure for Four Lanthanum Iodide Examples.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Cation substitutional chemistry in the r(7)x(12)z-type structure for four lanthanum iodide examples.

A series of quaternary lanthanide halide cluster compounds ALa(6)I(12)Z with transition metal interstitials (principally Os) and alkali or alkaline-earth metal cations (A = Na, Mg, Ca, Sr) have been synthesized by high temperature solid state techniques. The compounds were structurally characterized by single crystal and powder X-ray diffraction methods. The new compounds are isotypic (R3, Z = 3) with rhombohedral R(7)X(12)Z (R = Sc, Y, La-Lu; X = Cl, Br, I; Z = transition or main group element) and contain nominally octahedral R(6)X(12) units centered by interstitial Z. Here, the cation (A) rather than the seventh R occupies the isolated position between clusters along c as A(x)R(1-x) with 0 < x <or= 1. The balance of the structure remains essentially unchanged. The refined X-ray crystal structures of NaLa(6)I(12)Os, (Ca(0.906)La(0.094))La(6)I(12)Os, (Ca(0.621)La(0.379))La(6)I(12)Os, SrLa(6)I(12)Os, and (Sr(0.526)La(0.474))La(6)I(12)Os are reported, along with results of electrical resistivity and magnetic susceptibility measurements on these and some analogous phases. The phases are generally semiconductors, but the complex paramagnetic properties are not those of simple spin-only systems.

Structural properties and heat-induced oxidation-dehydrogenation of manganoan ilvaite from Perda Niedda mine, Sardinia, Italy

An unusually Mn-rich ilvaite sample from the Perda Niedda mine in Sardinia, Italy, was studied in order to clarify the Mn 2 + distribution among the different structural sites, and to observe the structural response of the mineral upon thermally induced oxidation-dehydrogenation. The crystal structure and the chemical composition of one crystal [a = 13.014(5), b = 8.867(3), c = 5.838(4) A, β = 90.02(4)°] were investigated. X-ray crystal-structure refinement, performed in the Pnam space group, and electron microprobe analyses yielded the formula (Ca 0 . 9 8 Mn 2 + 0 . 0 2 )(Fe 3 + Fe 2 + )(Mn 0 . 7 2 Fe 2 + 0 . 2 8 ) (Si 2 O 2 )O(OH). Crystal chemical details, compared to structural data from literature, led to the assumption that Mn 2 + replaces Fe 2 + , mainly at the M2 site. Annealing experiments and structure refinements were performed in the temperature range 400-690 °C. No phase transition was observed over the entire temperature range. Oxidation of Fe 2 + at the M1 site, with concomitant dehydrogenation, was deduced from examination of the structural adjustments occurring as the temperature was increased. A useful model to evaluate a possible OH - ↔ O 2 - substitution in ilvaite was obtained.

Magnetic properties of the Fe 2 SiO 4 -Fe 3 O 4 spinel solid solutions

Magnetic measurement of Fe3− x Si x O4 spinel solid solutions indicates that their Curie temperatures decrease gradually, but not linearly, from 851 to 12 K with increasing content of nonmagnetic ions Si4+. Magnetic hysteresis becomes more noticeable in solid solutions having a larger content of Fe2SiO4. Saturation magnetizations of Fe3− x Si x O4 samples increase up to x=0.357 and they are easily saturated in the field of H=0.1 T. However, magnetization of the sample of x=0.794 does not approach saturation even at high field of H=7.0 T and has a large coercive force. The Si4+ disordered distribution is confirmed to be tetr[Fe3+1− x + x t Si4+ x (1− t )] octa[Fe2+1+ x Fe3+1− x − x t Si4+ x t ] O4 by the spin moment, which is consistent with site occupancy obtained from X-ray crystal structure refinement. Their molecular magnetizations would be expressed as MB={4(1+x)+10xt}μB as functions of composition parameter x and Si4+ ordering parameter t of the solid solution. The sample of x=0.794 is antiferromagnetic below the Neel temperature, mainly due to the octahedral cation interaction MO–MO, while both MT–MO and MO–MO interactions induce a ferrimagnetic property. Concerning magnetic spin configuration, in the case of x>0.42, the lowest dɛ level becomes a singlet, resulting in no orbital angular momentum.

Influence of niobium on the charge-density-wave transition ofNi(Ta1−xNbx)2Se7

The influence of doping by niobium on the low-temperature charge-density-wave (CDW) state of ${\mathrm{NiTa}}_{2}{\mathrm{Se}}_{7}$ has been investigated by x-ray diffraction and measurements of the electrical conductivity. Doping concentrations in $\mathrm{Ni}({\mathrm{Ta}}_{1\ensuremath{-}x}{\mathrm{Nb}}_{x}{)}_{2}{\mathrm{Se}}_{7}$ varied between 0.5% and 30%. X-ray crystal structure refinements showed that the niobium atoms statistically replace the tantalum atoms, but with a ratio of 2:1 for the two crystallographically independent tantalum sites Ta1 and Ta2. Niobium was not found on the Ni sites nor intercalated between the layers. Previous investigations have established that the primary CDW is located on the Ni and Se2 atoms, and only a small influence on the CDW was expected from Nb doping of the Ta chains. Nevertheless, we found that the CDW was very sensitive to the level of doping x with ${\mathrm{dt}}_{c}/dx=\ensuremath{-}16,$ where ${t}_{c}{=T}_{c}{(x)/T}_{c}(0)$ is the reduced transition temperature. Similar values for ${\mathrm{dt}}_{c}/dx$ were calculated for different isoelectronically doped compounds with transition temperatures varying between ${T}_{c}=59\mathrm{K}$ ${(\mathrm{N}\mathrm{b}\mathrm{S}\mathrm{e}}_{3})$ and 263 K $[{(\mathrm{T}\mathrm{a}\mathrm{S}\mathrm{e}}_{4}{)}_{2}\mathrm{I}].$ It is suggested that this can be explained by a common mechanism of weak pinning of the CDW to the impurities. The results are rationalized by considering the recently found ${4k}_{F}$ component of the CDW, which is located on the Ta2 atoms. The large effects of doping on the CDW transition can then be understood in terms of the effect of doping on the ${4k}_{F}$ CDW and the interactions between the primary CDW and the ${4k}_{F}$ CDW.

1 Å crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium

The 1 Å resolution X-ray crystal structures of Mg2+ and Ca2+ salts of the B-DNA decamers CCAACGTTGG and CCAGCGCTGG reveal sequence-specific binding of Mg2+ and Ca2+ to the major and minor grooves of DNA, as well as non-specific binding to backbone phosphate oxygen atoms. Minor groove binding involves H-bond interactions between cross-strand DNA base atoms of adjacent base-pairs and the cations’ water ligands. In the major groove the cations’ water ligands can interact through H-bonds with O and N atoms from either one base or adjacent bases, and in addition the softer Ca2+ can form polar covalent bonds bridging adjacent N7 and O6 atoms at GG bases. For reasons outlined earlier, localized monovalent cations are neither expected nor found.Ultra-high atomic resolution gives an unprecedented view of hydration in both grooves of DNA, permits an analysis of individual anisotropic displacement parameters, and reveals up to 22 divalent cations per DNA duplex. Each DNA helix is quite anisotropic, and alternate conformations, with motion in the direction of opening and closing the minor groove, are observed for the sugar-phosphate backbone. Taking into consideration the variability of experimental parameters and crystal packing environments among these four helices, and 24 other Mg2+ and Ca2+ bound B-DNA structures, we conclude that sequence-specific and strand-specific binding of Mg2+ and Ca2+ to the major groove causes DNA bending by base-roll compression towards the major groove, while sequence-specific binding of Mg2+ and Ca2+ in the minor groove has a negligible effect on helix curvature. The minor groove opens and closes to accommodate Mg2+ and Ca2+ without the necessity for significant bending of the overall helix.The program Shelxdna was written to facilitate refinement and analysis of X-ray crystal structures by Shelxl-97 and to plot and analyze one or more Curves and Freehelix output files.

Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: gated substrate entry and proton relays support the proposed catalytic mechanism

The degradation of the toxic phenol p-cresol by Pseudomonas bacteria occurs by way of the protocatechuate metabolic pathway. The first enzyme in this pathway, p-cresol methylhydroxylase (PCMH), is a flavocytochrome c. The enzyme first catalyzes the oxidation of p-cresol to p-hydroxybenzyl alcohol, utilizing one atom of oxygen derived from water, and yielding one molecule of reduced FAD. The reducing electron equivalents are then passed one at a time from the flavin cofactor to the heme cofactor by intramolecular electron transfer, and subsequently to cytochrome oxidase within the periplasmic membrane via one or more soluble electron carrier proteins. The product, p-hydroxybenzyl alcohol, can also be oxidized by PCMH to yield p-hydroxybenzaldehyde. The fully refined X-ray crystal structure of PCMH in the native state has been obtained at 2.5 Å resolution on the basis of the gene sequence. The structure of the enzyme-substrate complex has also been refined, at 2.75 Å resolution, and reveals significant conformational changes in the active site upon substrate binding. The active site for substrate oxidation is deeply buried in the interior of the PCMH molecule. A route for substrate access to the site has been identified and is shown to be governed by a swinging-gate mechanism. Two possible proton transfer pathways, that may assist in activating the substrate for nucleophilic attack and in removal of protons generated during the reaction, have been revealed. Hydrogen bonding interactions between the flavoprotein and cytochrome subunits that stabilize the intramolecular complex and may contribute to the electron transfer process have been identified.

Neutron and X-Ray Structure Refinements between 15 and 1073 K of Piezoelectric Gallium Arsenate, GaAsO4: Temperature and Pressure Behavior Compared with Other α-Quartz Materials

Previous X-ray crystal structure refinement of GaAsO4 gave spurious As–O (1.72 Å) and Ga–O (1.77 Å) bond distances by comparison with theoretical values and with experimental values from AlAsO4 and GaPO4 ([As–O]≅1.67 Å and [Ga–O]≅1.82 Å). Therefore, a complete structural reinvestigation of gallium arsenate was carried out between 15 and 1073 K by both neutron and X-ray diffraction. All new analyses led to experimental bond values in agreement with those predicted, i.e., close to 1.67 and 1.82 Å. This new structural investigation of GaAsO4 over a wide range of temperatures emphasizes the high thermal stability of the α-quartz packing in this compound, the best amongst quartz-like materials. The experimental relation between structural deformation and piezoelectric properties of these quartz-like materials also allows us to predict that GaAsO4 would have the best piezoelectric characteristics for its AT-cut. Conversely, its higher covalent character should imply a greater pressure sensitivity, with a crystal structure transition around 9 GPa. Thus, GaAsO4 would be the best piezoelectric candidate for high temperature applications but not for high pressure devices.

Current Methods and Optimization Algorithms for the Refinement of X-Ray Crystal Structures

Optimization algorithms and other techniques used for the crystallographic refinement of both small molecule and macromolecular X-ray crystal structures are reviewed in this work. Emphasis is made on the advantages and disadvantages of every method in its application to the refinement process of different problems, as well as on their actual implementation in current program packages. A description of the most basic (first-level) algorithms based on physical grounds, such as maximum-likelihood, least-squares, molecular dynamics, maximum-entropy, Fourier and graphical methods, and holographic methods, is followed by a second-level approach, including Newton methods, simulated annealing, and gradient methods, among others, and finally by a third-level study which includes matrix decompositions and iterative methods for the solution of the resulting linear systems of equations. In addition, by descending to the most basic mathematical and physical level, connections with other disciplines that use almost the same tools are easily made, showing not only the interdisciplinarity of crystallography but also the flow of information between different sciences.

Commensurately modulated structure of4Hb−TaSe2determined by x-ray crystal-structure refinement

The $(\sqrt{13}a\ifmmode\times\else\texttimes\fi{}\sqrt{13}a\ifmmode\times\else\texttimes\fi{}c)$ superstructure of ${4H}_{b}\ensuremath{-}{\mathrm{TaSe}}_{2}$ at room temperature has been determined by x-ray crystal-structure refinement using (3+2)-dimensional superspace. The resulting modulation has its largest amplitudes for a longitudinal displacement of the Ta atoms in the octahedrally coordinated (T) layers, thus leading to a superstructure of hexagram-shaped clusters containing thirteen Ta atoms each. The atoms in the corresponding planes of Se atoms show a transversal modulation, which can be interpreted as the relaxation of these layers to the modulation of the Ta layers. Forming a similar pattern, modulations were found of the atoms in the layers with Ta in trigonal prismatic coordination $(H$ layers), with amplitudes of about 1/10 of the sizes of the modulations in the T layers. These results confirm the presence of a charge-density wave in the T layers, while the modulations of the H layer can be assigned to elastic coupling between the two types of layers. The observation of nonzero displacements of the atoms of the H layers provides an interpretation for recent observations by scanning tunneling microscope (STM): the superstructure features observed by STM on the H layers is a direct consequence of the atomic displacements in this layer.

1H, 15N, 13C, and 13CO Assignments and Secondary Structure Determinationof Basic Fibroblast Growth Factor using 3D Heteronuclear NMR Spectroscopy

Summary The assignments of the ~H, 15N, 13CO and ~3C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ~SN- and ~SN-/13C-labeled FGF-2 with an isotope incorporation > 95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C ~, C ~ and H a to the backbone amide ~H and ~SN of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C ~, C ~ and H ~ to the backbone amide ~H and ~SN in the CBCANH and HNHA experiments. In addition, C a and C ~ chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C ~ correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the atiphatic ~H and ~3C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H ~ and H ~ protons as well as 3JHNHC~ coupling constants, amide exchange and ~3C~ and 13C1~ secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel 13-sheets (residues 30-34, 39-44, 48-53, 62-67, 71-76, 81-85, 91-94, 103-108, 113-118, 123-125 and 148-152) and a helix-like structure (residues 131-136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131-136 were defined as 13-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128-138) instead of the 13-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9-28. This is consistent with the Xray structures of FGF-2, where the first 17-19 residues were ill defined.

An investigation of microporous cetineite-type phases A 6[B 12O 18][CX 3] 2[D x(H 2O,OH,O) 6−y] I. The cetineite structure field

Hydrothermal syntheses between 120 and 200 °C have been performed to determine the chemical variability of semiconducting microporous materials with cetineite structure. The syntheses were based on the general formula A 6[B 12O 18][CX 3] 2[D x(H 2O,OH,O) 6−y], (0≤×≤2; 0≤y≤6), which was derived from X-ray crystal structure refinements. A = Li +, Na +, K +, Rb +, Cs +, Tl +, NH 4 +, Ca 2+, Sr 2+, and Ba 2+ were introduced as hydroxides, in some cases as carbonates, B = C = As 3+, Sb 3+, and Bi 3+, and X = S 2−, Se 2−, and Te 2− as elements. Only syntheses with B = C = Sb 3+ and X = S 2− and Se 2− were successfull. Known cetineite-type phases now include the element combinations A/Sb 3+/S 2− with A = Na + and K +, and A/Sb 3+/Se 2− with A = Na +, K +, Rb +, Sr 2+, Ba 2+, and probably Tl +. Variable amounts of Na +, Sb 3+ and C 4+ were found to occupy the D position of the cetineite-type structure. The chemical variability can be described by the coupled substitutions A + + H 2OA 2+ + OH − mH 2O D m+ + mOH, and nOH −D n+ + nO 2−. The crystals obtained are orange to dark red, in agreement with their semiconducting properties.

Structure and morphology of synthetic magnesium calcite

The X-ray crystal structure refinements of two synthetic calcite single crystals containing 3.1 and 5.7 mol% magnesium were carried out. The structure of the second crystal was also refined at –80 °C. No significant structural difference from biogenic magnesium calcite was found. Magnesium within the crystals of calcite forms a solid solution over the compositional range investigated. The principal axes of vibration ellipsoids at –80 °C resemble very closely those of calcite suggesting that the large thermal parameters in magnesium calcite reflect increased thermal vibrations more than positional disorder. The crystals of magnesium calcite express new rhombohedral faces due to the interaction of magnesium with growing crystals. The role of magnesium in the morphology of single crystal and globular aggregates of magnesium calcite is discussed in view of its possible relevance in both biomineralization and crystal growth.

Osmium Complexes of 1,4,7-Triazacyclononane (tacn) and 1,4,7-Trimethyl-1,4,7-triazacyclononane (Me(3)tacn) and the X-ray Crystal Structure of [(Me(3)tacn)Os(eta(6)-C(6)H(5)BPh(3))]BPh(4).CH(3)CN.

The complexes of osmium with tacn (1,4,7-triazacyclononane) and Me(3)tacn (1,4,7-trimethyl-1,4,7-triazacyclononane), [LOs (eta(6)-C(6)H(6))](PF(6))(2) (L = tacn) and LOsCl(3) (L = tacn, Me(3)tacn), have been prepared by substitution of L on [Os(eta(6)-C(6)H(6))Cl(2)](2) or [Os(2)Cl(8)](2)(-), respectively. Reaction of LOsCl(3) with neat triflic acid leads to partial replacement of chloride and formation of the binuclear Os(III)-Os(III) complexes [LOs(&mgr;-Cl(3))OsL](PF(6))(3) (L = tacn, Me(3)tacn). The binuclear nature was established by NMR spectroscopy and elemental analysis and, for L = tacn, a partially refined X-ray crystal structure which shows the Os-Os separation to be 2.667 Å, indicative of significant metal-metal bonding. Reduction of [LOs(&mgr;-Cl(3))OsL](3+) over zinc amalgam in either aqueous or non-aqueous solution yields the intensely colored Os(II)-Os(III) mixed-valence ions [LOs(&mgr;-Cl(3))OsL](2+). Electrochemical measurements on [LOs(&mgr;-Cl(3))OsL](3+) in CH(3)CN reveal the reversible formation of the mixed valence ions. These are further reduced at lower potential to the Os(II)-Os(II) binuclear species, reversibly for L = Me(3)tacn. (Me(3)tacn)OsCl(3) is oxidized by persulfate ion to give [(Me(3)tacn)OsCl(3)](+); zinc amalgam reduction in an aqueous solution at high concentration produces the binuclear complex [(Me(3)tacn)Os(&mgr;-Cl(3))Os(Me(3)tacn)](3+) or, at low concentration, a solution containing an air sensitive osmium(II) species. Addition of BPh(4)(-) results in the eta(6)-arene zwitterion [(Me(3)tacn)Os(eta(6)-C(6)H(5)BPh(3))](+), which was characterized by X-ray diffraction on the BPh(4)(-) salt. The compound crystallizes in the triclinic space group P1 with a = 11.829(2) Å, b = 12.480(3) Å, c = 17.155(4) Å, alpha = 84.42(2) degrees, beta = 83.52(2) degrees, gamma = 71.45(2) degrees, V = 2380(2) Å(3), Z = 2, and R = 7.62%, and R(w) = 7.39%.

Synthesis and use of iodinated nonsteroidal antiinflammatory drug analogs as crystallographic probes of the prostaglandin H2 synthase cyclooxygenase active site.

The cyclooxygenase activity of the membrane protein prostaglandin H2 synthase isoform 1 (PGHS-1) is the target of the nonsteroidal antiinflammatory drugs (NSAIDs). The X-ray crystal structures of PGHS-1 in complex with the NSAIDs flurbiprofen and bromoaspirin have been determined previously [Picot, D., et al. (1994) Nature 367, 243-249; Loll, P. J., et al. (1995) Nat. Struct. Biol. 2, 637-643]. We report here the preparation and characterization of novel potent iodinated analogs of the NSAIDs indomethacin and suprofen, as well as the refined X-ray crystal structures of their complexes with PGHS-1. The PGHS-iodosuprofen complex structure has been refined at 3.5 A to an R-value of 0.189 and shows the suprofen analog to share a common mode of binding with flurbiprofen. The PGHS-iodoindomethacin complex structure has been refined at 4.5 A to an R-value of 0.254. The low resolution of the iodoindomethacin complex structure precludes detailed modeling of drug-enzyme interactions, but the electron-dense iodine atom of the inhibitor has been unambiguously located, allowing for the placement and approximate orientation of the inhibitor in the enzyme's active site. We have modeled two equally likely binding modes for iodoindomethacin, corresponding to the two principal conformers of the inhibitor. Like flurbiprofen, iodosuprofen and iodoindomethacin bind at the end of the long channel which leads into the enzyme active site. Binding at this site presumably blocks access of substrate to Tyr-385, a residue essential for catalysis. No evidence is seen for significant protein conformational differences between the iodoindomethacin and iodosuprofen of flurbiprofen complex structures.

The Refined X-ray Structure of Muconate Lactonizing Enzyme from Pseudomonas putidaPRS2000 at 1.85 Å Resolution

We report here the refined X-ray crystal structure of muconate lactoniz- ing enzyme (MLE) from Pseudomonas putidaPRS2000 at a resolution of 1.85 Å with an R-factor of 16.8%. An enzyme from the β-ketoadipate path- way, MLE catalyses the conversion of cis,cis-muconate to muconolactone. It is a homo-octamer, one monomer consisting of 373 amino acid residues. MLE has two large domains and a C-terminal subdomain: an α + β domain, an αβ-barrel domain and a C-terminal meandering subdomain. The αβ-barrel domain is highly irregular. Its structure is (β/α) 7β, with the structural role of the last α-helix being replaced by both the C-terminal subdomain and part of the N-terminal domain. The fifth, seventh and eighth barrel strands are unusual because they have left-handed twist about their axes. The strand crossing angles also vary enormously, from +9° to −69°; the first and last strands, which close the barrel, cross at an angle of −69°, making extensive strand – strand hydrogen bonding impossible. The first barrel strand is also unusual because it starts in the N-terminal domain and forms hydrogen bonds to the C-terminal subdomain β-sheet as well as to its neighbouring strands in the barrel. It thus cements the whole protein together. As in other αβ-barrel proteins, the active site of MLE, present in each subunit is at the C-terminal ends of the barrel β-strands. The active site cleft contains an essential manganese ion, is lined with charged and other polar residues, and contains many of the crystallographic water molecules. The manganese ion is octahedrally co-ordinated to three side-chain carboxylate groups and three water molecules, and is at the centre of a radiating web of ionic and hydrogen-bonding interactions. Additionally, two water molecules are buried in the centre of the barrel and two hydrophilic side-chains (Lys167 and Arg196) make both hydrophobic and hydrophilic packing interactions with much of the barrel interior. The barrel interior is thus also unusual because it is so hydrophilic; the dominating force appears to be the need to solvate the metal ion effectively. This might account for the irregularity of the barrel. The catalytic mechanism has been investigated by docking both substrate and product in the active site with the C-COO −of muconolactone superimposed on the corresponding atoms of cis,cis-muconate. In agreement with earlier kinetic and spectroscopic results, the manganese ion does not interact directly with substrate or product. Our model thus confirms that Glu327 is the general acid, that Lys169 is the general base, and that the manganese ion and Lys167 provide electrophilic stabilisation of the enol/enolate intermediate that forms during the course of the reaction.

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy.

The assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising of 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation > 95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, C alpha and C beta chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C alpha correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H alpha and H beta protons as well as 3JHNH alpha coupling constants, amide exchange and 13C alpha and 13C beta secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel beta-sheets (residues 30-34, 39-44, 48-53, 62-67, 71-76, 81-85, 91-94, 103-108, 113-118, 123-125 and 148-152) and a helix-like structure (residues 131-136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131-136 were defined as beta-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128-138) instead of the beta-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9-28. This is consistent with the X-ray structures of FGF-2, where the first 17-19 residues were ill defined.

Three-dimensional structure of chemotactic Che Y protein in aqueous solution by nuclear magnetic resonance methods.

The three-dimensional structure of chemotactic Che Y protein from Escherichia coli in aqueous solution has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. A total of 20 converged structures were computed from 1545 conformationally relevant distance restraints derived from 1858 unambiguously assigned NOE cross-correlations. The resulting average pairwise root-mean-square deviation is 1.03 A for the backbone atoms and 1.69 A for all heavy atoms. If residues in the regions structurally least defined (1 to 5, 47 to 50, 76 to 79, 88 to 91 and 124 to 129) are excluded from the analysis, the root-mean-square deviations are reduced to 0.53 A and 1.23 A, respectively. The solution structure is closely similar to the refined X-ray crystal structure, except in the regions found to be less defined by NMR spectroscopy. The root-mean-square deviation between the average solution structure and the X-ray crystal structure is 0.92 A for the backbone residues (2 to 129). The highly refined solution structure determined herewith provides an essential background to delineate functionally important conformational changes brought about by different effectors.