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Using different 1D and 2D NMR methods including Nuclear Overhauser effects in the rotating frame the structural features of 1-(3-n-butoxypropyl)-4-benzoyloxypiperidine hydrochloride and its inclusion complex with β-cyclodextrin were investigated in aqueous solution. 1-(3-n-butoxypropyl)-4-benzoyloxypiperidine hydrochloride exists as a mixture of two conformational isomers with axial and equatorial orientation of the benzoyloxy group (ca. 2:1). Both these isomers effectively form inclusion complexes with β-cyclodextrin as host molecule.

The interaction of the analgesic prosidol [1-(2-ethoxyethyl)-4-phenyl-4-propionyl-oxypiperidine] and the anaesthetic kazcaine [1-(2-ethoxyethyl)-4-ethynyl-4-benzoyloxypiperidine] with β-cyclodextrin (β-CD) in aqueous solutions has been studied by nuclear magnetic resonance (NMR), ultraviolet (UV) and infrared (IR) spectroscopy. The composition and structure of the formed guest:β-CD inclusion complexes have been determined and were found to have a molar ratio of 1:2, with the guest molecule located in the cavity formed by two β-CD molecules in head-to-head orientation, with the O(2), O(3) rims interacting. The phenyl and ethoxyethyl substituents of the guests are in contact with the β-CD molecules. In contrast to prosidol–base and kazcaine–base the complexes with β-CD show a higher analgesic and local anaesthetic activity.

The base form of the local anaesthetic kazcaine (BFK, [1-(2-ethoxyethyl)-4-ethynyl-4-benzoyloxypiperidine, C18H23NO3]) and β-cyclodextrin (β-CD) co-crystallized as BFK:β-CD inclusion complex in 1:2 M ratio from a mixture of water and ethanol while the filtered mother liquor yielded crystals of free BFK. X-ray diffraction showed that the crystals of BFK and its inclusion complex with β-CD belong to monoclinic (P21/c) and triclinic (P1) space groups, respectively. The crystals of free BFK are stabilized by pairs of C–H⋯O, C–H⋯π and ≡C–H⋯O type interactions and van der Waals contacts. In the 1:2 BFK:β-CD complex the two β-CD molecules are in hydrogen-bonding contact with their primary hydroxyl groups, the 1-(2-ethoxyethyl)-4-ethynyl-piperidine moiety being located in one and the benzoyloxy group of BFK in the other β-CD. This crystal structure is of the channel-type, the β-CD molecules of the 1:2 BFK:β-CD complex interacting with their secondary hydroxyl groups. The pharmacological activities of the 1:2 BFK/β-CD inclusion complex have been determined in mice, rats, porpoises and rabbits and compare favourably with those of kazcaine, procaine, dicaine, lidocaine and trimecaine. The methods used include terminal (superficial), infiltration, conduction anaesthesia, and acute toxicity.

The photosynthetic oxygen-evolving photosystem II (PSII) is the only known biochemical system that is able to oxidize water molecules and thereby generates almost all oxygen in the Earth's atmosphere. The elucidation of the structural and mechanistic aspects of PSII keeps scientists all over the world engaged since several decades. In this Minireview, we outline the progress in understanding PSII based on the most recent crystal structure at 2.9 A resolution. A likely position of the chloride ion, which is known to be required for the fast turnover of water oxidation, could be determined in native PSII and is compared with work on bromide and iodide substituted PSII. Moreover, eleven new integral lipids could be assigned, emphasizing the importance of lipids for the perfect function of PSII. A third plastoquinone molecule (Q(C)) and a second quinone transfer channel are revealed, making it possible to consider different mechanisms for the exchange of plastoquinone/plastoquinol molecules. In addition, possible transport channels for water, dioxygen and protons are identified.

SummaryUsing the 2.9 Å resolution structure of the membrane-intrinsic protein-cofactor complex photosystem II (PSII) from the cyanobacterium Thermosynechococcus elongatus, we calculated and characterized nine possible substrate/product channels leading to/away from the Mn4Ca cluster, where water is oxidized to dioxygen, protons, and electrons. Five narrow channels could function in proton transport, assuming that no large structural changes are associated with water oxidation. Four wider channels could serve to supply water to or remove oxygen from the Mn4Ca cluster. One of them might be regulated by conformational changes of Lys134 in subunit PsbU. Data analyses of Kr derivatized crystals and complexes with dimethyl sulfoxide (DMSO) confirm the accessibility of the proposed dioxygen channels to other molecules. Results from Xe derivatization suggest that the lipid clusters within PSII could serve as a drain for oxygen because of their predominant hydrophobic character and mediate dioxygen release from the lumen.

Water oxidation generating atmospheric oxygen occurs in photosystem II (PSII), a large protein-pigment complex located in the thylakoid membrane. The recent crystal structures at 3.2 and 3.5 A resolutions provide novel details on amino acid side chains, especially in the D1/D2 subunits. We calculated the redox potentials for one-electron oxidation of the chlorophyll a (Chla) molecules in PSII, considering the protein environment in atomic detail. The calculated redox potentials for the dimer Chla (P(D1/D2)) and accessory Chla (Chl(D1/D2)) were 1.11-1.30 V relative to the normal hydrogen electrode at pH 7, which is high enough for water oxidation. The D1/D2 proteins and their cofactors contribute approximately 390 mV to the enormous upshift of 470 mV compared to the redox potential of monomeric Chla in dimethylformamide. The other subunits are responsible for the remaining 80 mV. The high redox potentials of the two accessory Chla Chl(D1/D2) suggests that they also participate in the charge separation process.

The crystal structure of a decameric HNA/RNA (HNA = 2',3'-dideoxy-1',5'-anhydro-d-arabinohexitol nucleic acid) hybrid with the RNA sequence 5'-GGCAUUACGG-3' is the first crystal structure of a hybrid duplex between a naturally occurring nucleic acid and a strand, which is fully modified to contain a six-membered ring instead of ribose. The presence of four duplex helices in the asymmetric unit allows for a detailed discussion of hydration, which revealed a tighter spinelike backbone hydration for the HNA- than for the RNA-strands. The reinforced backbone hydration is suggested to contribute significantly to the exceptional stability of HNA-containing duplexes and might be one of the causes for the evolutionary preference for ribose-derived nucleic acids.

Crystal structures are reported of cycloamylose containing 26 D-glucose residues (CA26, cyclohexaicosaose, C156H260O130) in complexes with undecanoic acid (CA26 x 2C10H21COOH x 34.95 H2O, orthorhombic P2(1)2(1)2(1), one CA26 and two bound undecanoic acids F1 and F2 in the asymmetric unit, resolution 0.95 angstroms) and with dodecanol ((CA26)(0.5) x C12H25OH x 32.0H2O, monoclinic C2, half a CA26 binding one dodecanol, A, in the asymmetric unit, resolution 1.0 angstroms). The macrocycle of CA26 is folded like the figure '8' into two 10 D-glucoses long left-handed V-amylose helices forming approximately 5A wide V-channels that are occupied by undecanoic acid (F1, F2) or dodecanol (A) as guest molecules. The functional head groups of the guests near the O(6) ends of the V-channels are hydrogen bonded with d-glucose O(6)n-H; the aliphatic termini beyond C(9) protrude from the O(2), O(3) ends. Parts of the aliphatic chains enclosed in the V-channels are all-trans except for one torsion angle each (approximately 130 degrees ) in undecanoic acid molecules F1 and F2. There are several (guest)C-H...O hydrogen bonds to O(4) and O(6) of CA26 in both complexes, and H...H van der Waals interactions with d-glucose C(3)-H and C(5)-H dominate. C(5)-H determine the position of the aliphatic chains of undecanoic acid F1 and of dodecanol A in contrast to F2 where both C(3)-H and C(5)-H contribute equally, probably because the V-channel is narrower than in F1 and in dodecanol. Complexes of polymeric V-amylose with fatty acids and alcohols studied by X-ray fiber diffraction could not provide the here described high resolution.

Cyclomaltohexaicosaose (CA26) is folded into two 1(2)/(3) turns long V-helices that are oriented antiparallel. Crystals of complexes of CA26 with NH(4)I(3) and Ba(I(3))(2) are brown and X-ray analyses show that I(3)(-) units are located in the approximately 5 A wide central channels of the V-helices. In the complex with NH(4)I(3), two CA26 molecules are stacked to form 2 x 1(2)/(3) turns long channels harbouring 3 I(3)(-) at 3.66-3.85 A inter I(3)(-) distance (shorter than van der Waals distance, 4.3 A), whereas in the Ba(I(3))(2) complex, CA26 are not stacked and only one I(3)(-) each fills the V-helices. Glucose...I contacts are formed with C5-H, C3-H, C6-H and (at the ends of the V-helices) with O6 in (+) gauche orientation. By contrast, O2, O3, O4 and O6 in the preferred (-) gauche orientation do not interact with I because these distances are/=4.01 A and exceed the van der Waals I...O sum of radii by about 0.5 A except for one O2...I distance of 3.68 A near the end of one V-helix. Raman spectra indicate that the complexes share the presence of I(3)(-) with blue amylose-iodine.

The calf spleen purine nucleoside phosphorylase (PNP) ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate ion has been crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 94.11 Angstrom and one monomer per asymmetric unit. X-ray diffraction data were collected using synchrotron radiation (Station X31, EMBL Outstation, DESY, Hamburg). The crystal structure was refined to a resolution of 2.2 Angstrom and R and R-free values of 17.5 and 24.5%, respectively. The acyclonucleoside inhibitor is bound in the active site in an inverted ('upside- down') orientation of the purine base compared with natural substrates. The side chain of Asp243 forms two hydrogen bonds with the base ring: N-delta donates a hydrogen to N(3) and O-delta accepts a hydrogen from the guanine N(2)-amino group. N(1)-H of the base is hydrogen bonded to O-delta of Glu201, while N(9) accepts a hydrogen bond from Thr242 O-gamma. In addition, a water molecule (W417) bridges the N(2)-amino group of the base and O-epsilon of Glu201. In the phosphate-binding site, a phosphate ion is bound to Ser33, His64, Arg84, His86, Ala116 and Ser220. The acyclic chain of the N(7)-acycloguanosine inhibitor is in a folded conformation and together with a water molecule (W388) occupies the pentose- binding site, with possible hydrogen bonds to Tyr88 O-eta and His257 N-delta1. This new binding mode fully accounts for the previously observed substrate properties of 7-beta -D- ribofuranosides of hypoxanthine and guanine. It also provides a new starting point for the design of inhibitors of PNP for therapeutic and other applications.

Octakis(2,3,6-tri-O-methyl)-gamma-CD (TRIMEG) cocrystallized at 18 degrees C with 4.5 water molecules in the orthorhombic space group P2(1)2(1)2(1), unit cell dimensions a = 10.7879(3), b = 29.0580(9), c = 32.2909(11) A. The TRIMEG macrocycle is in a 'round' form with all glucose units oriented syn, and one O-6-CH3 methoxy group points 'toward' the molecular cavity. The TRIMEG x 4.5 H2O molecules are stacked to form infinite cylinders with the central cavities aligned into channels filled for each TRIMEG by 4.5 water molecules distributed over 15 partially occupied sites. This structure differs from the two known structures of TRIMEG in which two diametrically opposed glucoses are oriented anti to yield an 'elliptical' form, and their O-6-CH3 groups are directed 'toward' the cavity and close it at this side to form a bowl-shaped molecule.

To understand the rapid diffusion mechanism of water molecules in the crystal lattice of hydrated beta-cyclodextrin (beta-CD), molecular dynamics (MD) simulations of crystalline beta-CD were performed at five different relative humidities corresponding to hydration states ranging from beta-CD-9.4H2O to beta-CD-12.3H2O, and in aqueous solution. The trajectories for the crystalline beta-CD hydrates had lengths of 4 ns each, whereas the simulation in aqueous solution extended to 2 ns. Transport of water molecules in the crystal was characterized in terms of a spatially varying diffusion constant and the main direction of diffusion, which were evaluated using newly developed algorithms. The main diffusion pathway winds through the cavities of adjacent doughnut shaped beta-CD molecules and is slightly slanted with respect to the crystallographic b-axis. Water molecules outside the beta-CD cavities have access to the main diffusion pathway. The diffusion constant for transport of water molecules along the main pathway calculated from the MD simulation data adopts 1/30 of the value in bulk water at room temperature. This is in agreement with estimates that can be made from experimental data on the adjustment of a beta-CD crystal to changes in relative humidity.

Hexakis(2,6-di-O-methyl)-alpha-cyclodextrin (DIMEA) crystallizes from 1:1 water-acetonitrile as DIMEA-acetonetril-dihydrate in the orthorhombic space group P2(1)2(1)2(1), unit cell dimensions a = 14.2775(5), b = 15.7312(5), c = 31.1494(11) A. Refinement of the structure against 5540 X-ray diffraction data converged at an R factor of 0.083. The macrocycle exhibits a 'round' conformation and is stabilized by intramolecular, interglucose O-3-H(n)...O-2(n + 1) and C-6-H(n)...O-5(n + 1) hydrogen bonds. Acetonitrile is included in the central cavity of DIMEA and held in position by C-5-H...N interactions. The two water molecules in the asymmetric units are distributed over six sites. One is fully occupied due to hydrogen bonding to O-3 groups of two symmetry-related DIMEA molecules, whereas the five remaining sites show occupancies between 0.15 and 0.25. These sites are in hydrogen bonding contact with O...O distances between 2.59 and 3.50 A and are located in infinite, hydrophobic channels parallel to the alpha-axis, which are coated with methyl groups of symmetry-related DIMEA.

The amylose fraction of starch occurs in double-helical A- and B-amyloses and the single-helical V-amylose. The latter contains a channel-like central cavity that is able to include molecules, “iodine’s blue” being the best-known representative. Molecular models of these amylose forms have been deduced by solid state 13 C cross-polarization/magic angle spinning NMR and by x-ray fiber and electron diffraction combined with computer-aided modeling. They remain uncertain, however, as no structure at atomic resolution is available. We report here the crystal structure of a hydrated cycloamylose containing 26 glucose residues (cyclomaltohexaicosaose, CA26), which has been determined by real/reciprocal space recycling starting from randomly positioned atoms or from an oriented diglucose fragment. This structure provides conclusive evidence for the structure of V-amylose, as the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by twofold rotational pseudosymmetry. In the V-helices, all glucose residues are in syn orientation, forming systematic interglucose O(3) n ⋅⋅⋅O(2) n +l and O(6) n ⋅⋅⋅O(2) n +6 /O(3) n +6 hydrogen bonds; the central cavities of the V-helices are filled by disordered water molecules. The folding of the CA26 macrocycle is characterized by typical “band-flips” in which diametrically opposed glucose residues are in anti rather than in the common syn orientation, this conformation being stabilized by interglucose three-center hydrogen bonds with O(3) n as donor and O(5) n +l , O(6) n +l as acceptors. The structure of CA26 permitted construction of an idealized V-amylose helix, and the band-flip motif explains why V-amylose crystallizes readily and may be packed tightly in seeds.

Octakis(2,3,6-tri-O-methyl)-γ-cyclodextrin (TRIMEG) crystallized from saturated aqueous solution at 18 °C in the monoclinic space group P21 with 4 TRIMEG and a total of 19.3 water molecules in the ...

An improved electron density map of photosystem I from Synechococcus elongatus calculated at 4-A resolution for the first time reveals a second phylloquinone molecule and thereby completes the set of cofactors constituting the electron transfer system of this iron-sulfur type photosynthetic reaction center: six chlorophyll a, two phylloquinones, and three Fe4S4 clusters. The location of the newly identified phylloquinone pair, the individual plane orientations of these molecules, and the resulting distances to other cofactors of the electron transfer system are discussed and compared with those determined by magnetic resonance techniques.

Heptakis(2,6-di-O-methyl)-β-cyclodextrin crystallized from water at 18 °C as dihydrate (DIMEB·2 H2O) in the monoclinic space group P21 with unit cell dimensions a=15.2415(5), b=10.6391(4), c=23.3239 (8) A, β=101.798(1)°. X-ray analysis reveals a torus-like macrocycle stabilized by systematic hydrogen bonds O-3H··O-2′ and C-6H··O-5′ between adjacent glucose residues. The O-6CH3 methoxy groups of three glucose units are directed toward but do not close the cavity. Both ends of the cavity are closed by adjacent molecules which are related by the characteristics of ‘cage type’ crystal packing. One water molecule is included in the apolar cavity of the torus and has very high anisotropic thermal displacement factor due to lack of hydrogen bonding interactions to DIMEB, while the other one is located in an intermolecular void and forms hydrogen bonds to O-3 and O-6 oxygen atoms.

Glucose-flips in a small cycloamylose: the molecular structure of permethylated γ-cyclodextrin, which crystallizes as the dihydrate, is characterized by 180° flips of two diametrically opposed glucose residues. This leads to an elliptical distortion of the molecule and closure of the molecular cavity on one side so that it is no longer doughnut-shaped but is bowl-shaped. The hydrate water molecules are not located in the cavities!

X-ray diffraction analysis of the crystalline inclusion complex β -cyclodextrin-but-2-yne-1,4-diol heptahydrate has shown that a number of C–H…O and C–H… π interactions occur between the cavity wall and the guest molecules. The interaction energies of these contacts are estimated by ab initio quantum chemical calculations. They are in the range 0.7 to 1.1 kcal mol −1 , which is far below values of conventional hydrogen bonding (4 to 6 kcal mol −1 for O–H…O hydrogen bonds in carbohydrates), but appreciably above energies of van der Waals contacts.

Crystals of the ternary α-cyclodextrin-acetonitrile hexahydrate inclusion complex belong to the orthorhombic space group P212121 with unit cell dimensions a=9.479(2), b=14.323(4), c=37.397(9) A. Refinement against 4202 X-ray diffraction data converged at R=0.059. The α-cyclodextrin macrocycle forms a regular torus which is stabilized by intramolecular O-3–O-2 hydrogen bonds between neighboring glucose units. Within the cavity, one acetonitrile and one water molecule are located with occupancy factors of 0.8 and 0.7, respectively. They are hydrogen bonded to disordered O65B, and acetonitrile is stabilized in position by C-5–H⋯N interactions. The remaining five water molecules link symmetry related cyclodextrin molecules.

Helicases are ATP-driven enzymes essential for DNA unwinding. The broad host range plasmid RSFI010 harbours a gene (repA) encoding for one of the smallest known oligomeric helicases, RepA, a homo-hexamer with 30 kDa subunits. Electron micrographs indicate that the overall shape of RepA resembles a hexagon with globular monomers at the corners, diameter 140 A, and a central channel. Below pH 6, the molecules aggregate into tubular structures. The enzyme has been purified and crystallized using the hanging-drop vapour-diffusion method with polyethyleneglycol monomethylether as precipitating agent. The crystals exhibit the monoclinic space group P2(1) with unit-cell parameters a = 105.8, b = 180.3, c = 115.4 A, beta = 95.2 degrees, and diffract to 3.5 A resolution using rotating-anode Cu Kalpha radiation. Assuming two 180 kDa molecules per asymmetric unit, the volume per unit weight is V(m) = 3.06 A Da(-1), equivalent to a solvent content of 60%. A self-rotation search indicates that the sixfold axis of the hexamer is parallel to the ac plane and inclined at about 2 degrees to the c axis. The two hexamers are oriented head-to-head with point-group symmetry D(6).

X-ray diffraction studies were carried out on hexakis-(2,6-di- O -methyl)-cyclomaltohexaose (dimethyl-α-cyclodextrin, DIMEA) crystallized at room temperature from solutions in pure acetone as the 1:1 inclusion complex, and crystallized from aqueous solution at 80 °C without water of hydration. In both crystal structures, the DIMEA molecules adopt a round shape stabilized by systematic intramolecular O-3H … O-2 hydrogen bonds between neighbouring glucose units, and the DIMEA-cavities are closed at the O-6Me rims by methoxy groups forming van der Waals contacts across the molecular opening. In the DIMEA-acetone complex, the guest molecule is fully included and exhibits excessive thermal motions. In anhydrous DIMEA, the remaining cavity volume is occupied by a methoxy group of a neighbouring DIMEA molecule.

An X-ray diffraction study was carried out on dimethyl-β-cyclodextrin which crystallized from aqueous solution at 60°C without water of hydration. The molecule adopts a round shape which is stabilized by systematic intramolecular O-3H z O-2 hydrogen bonds between neighboring glucose units. The O-6C-8 groups of three glucoses are oriented towards the molecular axis so that the cyclodextrin cavity is closed at one end. The remaining volume of the bowl-shaped cavity is occupied by part of the C-6O-6C-8 rim of a neighboring molecule (self-inclusion).

N-(n- Octyl )-6- deoxy - d - gluconamide crystallizes in the orthorhombic space group P 2 1 2 1 2 1 , with a = 5.4524(5), b = 16.662(3), and c = 36.897(5) A. The structure was determined by X-ray diffraction and refined to R = 9.2%. The asymmetric crystal unit contains two molecules (A,B) with significantly different conformations. In contrast to the crystal structures of the N-(n- alkyl )- d - gluconamide family, in which the molecules were found arranged in head-to-tail monolayers, molecules A and B form a complex motif with pairwise alternating orientations and interdigitating antiparallel aliphatic chains. The two symmetry independent molecules form considerably different hydrogen bond patterns.

Single-crystal X-ray diffraction studies were carried out for the title compounds at room temperature. The crystal packings are of the cage-type and isomorphous to that of beta-cyclodextrin (beta CD) hydrate. In both crystal structures, disorder and extensive thermal vibrations of the complexed guest molecules are observed. In beta CD-ethylene glycol.8H2O, one ethylene glycol molecule (disordered over two discrete sites) and three water molecules (four discrete sites) are included in the beta CD cavity. Within the beta CD cavity, all oxygen sites (ordered and disordered) are in positions occupied by water molecules in beta CD.12H2O; this is only possible because the ethylene glycol molecule adopts the low-energy conformation with the O-C-C-O torsion angle approximately 60 degrees and an O...O separation of 2.9 A, in which its hydroxyl groups can directly substitute for two hydrogen-bonding water molecules. In beta CD-glycerol.7.2H2O, one glycerol molecule (disordered over two discrete sites) and two water molecules (two fully occupied sites) are included in the beta CD cavity. The general situation in both compounds parallels that found earlier in beta CD-ethanol.8H2O. It is assumed that the disorder is dynamic, i.e., associated with jumps between the partially occupied molecular sites.

(±)-3,4-Di-O-acetyl-,2,5,6-tetra-O-benzyl-myo-inositol, C 38 H 40 O 8 , M r =624.7 («jumping crystal»). Three monoclinic crystal forms. Form I, T= 18 o C, P2 1 /n, a=28.883 (3), b=15.189 (2), c= 15.566 (2) A, β=100.37 (1) o , V=6717 (1) A 3 , Z= 8, R=0.090 for 5788 reflections [F>σ(F)]. Form II, T=18 o C [T=60 o C], P2 1 /c, a=14.269 (2) [14.464 (6)], b=14.862 (2) [14.923 (5)], c= 16.506 (3) A [16.385 (6) A], β=103.21 (1) o [102.73 (3) o ], V=3407 (1) A 3 [3450 (2) A 3 ], Z=4, R=0.086 [0.093] for 3505 [2338] reflections [F>σ(F)]

The crystal structure of a transition state/product complex formed by the interaction between proteinase K and the substrate analogue N-Ac-L-Pro-L-Ala-L-Pro-L-Phe-D-Ala-L-Ala-NH2 has been determined at a resolution of 2.2 A and refined to an R-factor of 0.165 for 12,725 reflections. The inhibitor forms a stable complex through a series of hydrogen bonds with protein atoms and water molecules. The inhibitor is hydrolyzed between Phe 4I and D-Ala5I (I indicates inhibitor). The two fragments are separated by a distance of 3.07 A between the carbonyl carbon and the main chain nitrogen. Both fragments remain bound to the protein. The N-terminal fragment occupies subsites S5 to S1, whereas the C-terminal part is bound in S1' and S2', the first time that electron density for a substrate analogue has been observed in the P1' and P2' sites of a subtilisin-like enzyme. The flexible segments of the substrate recognition sites Gly100-Tyr104 and Ser132-Gly136 move appreciably to accommodate the inhibitor. Biochemical results indicate an inhibition by this specifically designed peptide of 95%.

The crystal structure of the title compound [ 5 ; space group P 2 1 ; a = 8.375(5), b = 51.52(2), c = 5.6473(3) A; β = 89.64(4)°] was determined by X-ray diffraction methods and refined to R = 0.107. There are two molecules in the asymmetric unit, which adopt nearly identical conformations. Each molecule has two parts that differ only in the torsion angle N-C-7-C-8-C-9, which can be synclinal (−64°) or anti (168°). The molecules are arranged in sheets and the packing is stabilised by strong intra- and inter-molecular hydrogen bonds, some of which form a homodromic arrangement.

X-ray structure analysis shows that the monomer of trimeric photosystem I (PS I) of Synechococcus sp. consists of a catalytic domain and a smaller domain that connects the monomers. The 4Fe–4S clusters FX,FA and FB, 28 α-helices and 45 chlorophyll a molecules were located. The two large subunits of PS I are represented by nine a-helices each; they are related by a local 2-fold rotation axis passing through Fx. Electron densities close to this axis are interpreted as carriers of the electron transfer chain.

The ternary complex formed between RNase T1, guanosine 3',5'-bisphosphate (3',5'-pGp) and Pi crystallizes in the cubic space group I23 with a = 8.706(1) nm. In a previous publication [Lenz, A., Heinemann, U., Maslowska, M.Saenger, W. (1991) Acta Crystallogr. B47, 521-527], the structure of the complex (in which Pi was not located) was described at a resolution of 0.32 nm. This is now extended to 0.19 nm with newly grown, larger crystals. Refinement with restrained least-squares converged at R = 17.8% for 8027 reflections with [Fo[or = 1 sigma ([Fo[); the final model comprises 120 water molecules. 3',5'-pGp is bound to RNase T1 in the anti form, with guanine in the specific recognition site; the 3'-phosphate protrudes into the solvent, and the 5'-phosphate hydrogen bonds with Lys41 O and Asn43 N4. A tetrahedral anion assigned as Pi occupies the catalytic site and hydrogen bonds to the side chains of Tyr38, Glu58, Arg77 and His92. The overall polypeptide fold of RNase T1 in the cubic space group does not differ significantly from that in the orthorhombic space group P2(1)2(1)2(1) except for changesor = 0.2 nm in loop regions 69-72 and 95-98.

X-ray structure analysis shows that the monomer of trimeric photosystem I (PS I) of Synechococcus sp. consists of a catalytic domain and a smaller domain that connects the monomers. The 4Fe–4S clusters FX,FA and FB, 28 α-helices and 45 chlorophyll a molecules were located. The two large subunits of PS I are represented by nine a-helices each; they are related by a local 2-fold rotation axis passing through Fx. Electron densities close to this axis are interpreted as carriers of the electron transfer chain.

The binding of six herbicides of the triazine family to the photosynthetic reaction center of Rhodopseudomonas viridis was investigated with energy-minimization techniques, in order to correlate experimental with calculated data. The inhibitors were modeled in the active site according to the X-ray structure analysis of the complex formed between the triazine terbutryn (2-ethylamino-4-t-butylamino-6-methylthio-s-triazine) and the reaction center of R. viridis [Michel, H., Epp. O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. 40 different energy minimizations were carried out with varying cutoff radii, partial charges on inhibitor atoms and dielectric constants, i.e. 10 different combinations of these were tested. The impact of these parameters on the calculated binding and interaction energy was either examined for all protein/triazine complexes or, in the case of the dielectric constant, a smaller sample was used. The calculated energies are dominated by van der Waals interactions, which change by up to 20% when extending the cutoff radius from 0.8 nm to 1.5 nm. The use of uniform or distance-dependent dielectric constant or partial charges on the inhibitor atoms does not severely influence the resulting structures, but shows a great impact on the calculated energies. In the two groups of triazines, each containing three inhibitors with methoxy or methylthio substituents, correlations of biological and calculated data were found quite often, but only once with all six triazines. The energy-minimized structures were compared and analysed. A third hydrogen bond, not seen in the X-ray analysis of the reaction center/tertubryn complex, was found between the t-butylamino moiety of terbutryn (and equivalent moieties in the other triazines) and the carbonyl oxygen of TyrL222.

A single crystal X-ray diffraction study of the title complex carried out at room temperature revealed space group P2(1), a = 21.199(12), b = 9.973(3), c = 15.271(8) A, beta = 110.87(3) degrees, V = 3017(3) A3, 4681 unique reflections with Fo greater than 1 sigma (Fo). The structure was refined to R = 0.069, resolution lambda/2sin theta max = 0.89 A. The crystal packing is of the cage type and is isomorphous to that of beta-cyclodextrin (beta CD) dodecahydrate. One 1,4-butanediol and approximately 1.25 water molecules are enclosed in each beta CD cavity. The hydroxyl groups of the 1,4-butanediol molecule are located at each end of the cavity and form hydrogen bonds with neighboring water and beta CD molecules. The flexible (CH2)4 moiety vibrates extensively in the central part of the cavity. Water molecules and hydroxyl groups are chelated between O-6 and O-5 of at least five glucose residues.

The distribution of 4-nitrophenyl-α- d -malto-oligosaccharides produced by phosphorolytic synthesis was analyzed by an amine-modified and a C 18 -bonded silica gel. With both systems high resolution of components up to d.p.40 can be achieved by gradient elution using acetonitrile-water and water-methanol, respectively. The use of water-acetonitrile in reversed-phase (C 18 ) chromatography allows only a separation up to d.p. 9. The elution behaviour of oligomers in the d.p. range of 1–10 glucosyl residues per molecule eluted by water-methanol is discussed. The observation of rearrangement products of 4-nitrophenyl-α- d -malto-oligomers was successful by normal-phase chromatography.

The complex of fatty-acid-binding protein (FABP) from bovine heart (cFABP, pI4.9) with endogenous lipid was crystallized in the presence of ammonium sulfate as precipitant. The needle-shaped crystals belong to the monoclinic space group C2, with unit-cell constants a = 5.262(6) nm, b = 7.631(8) nm, c = 3.945(5) nm and beta = 94.47(9) degrees. A native data set to 0.35 nm resolution was collected using synchrotron radiation and film methods. An initial model for the three-dimensional structure of the protein was constructed based on the crystal structure of the related bovine P2 myelin protein [Jones, T.A., Bergfors, T., Sedzik, J.Unge, T. (1988) EMBO J. 7, 1597-1604] to which the amino acid sequence of bovine cFABP was adapted. Energy minimizations were carried out under different conditions using both an all-atom and a united-atom force field. The optimized models were used to determine the crystal structure of cFABP by molecular-replacement techniques. The structure was refined by simulated annealing to R = 0.267. As the bound lipid is heterogeneous, it could not be located in the electron-density map and/or the attained resolution was not sufficient. Bovine cFABP is composed of ten antiparallel beta strands forming a beta barrel, and by two alpha helices. The structural features are similar to those of other members of the superfamily of hydrophobic molecule transporters.

The crystalline complex between the ring-shaped β-cyclodextrin (β-CD) and 11 water molecules (β-CD · 11H2O) shows extensive orientational disorder of β-CD hydroxyl groups and water molecules and in addition positional disorder of six water molecules which are enclosed in the β-CD molecular cavity. A quasielastic incoherent neutron scattering (QINS) study was performed employing three different energy resolutions. The quasielastic lineshape can be approximated by a sum of two Lorentzian curves of constant width and intensity ratio and intensities decreasing with the scattering angle φ and extrapolating to zero for φ = 0 consistently for all three energy resolutions. This is indicative of local diffusion of hydrogen atoms in the sample. A previous high resolution (0.6A) neutron crystallographic study provides the knowledge of the number and jump distances of the disordered hydrogen atoms. This gives a sound basis for model calculations which aim to describe the experimental spectra in terms of theoretical s...

The title compound. 9-(1,3-dihydroxy-2-propoxymethyl)adenine (DHP-Ade). an analogue of the antiviral acyclonucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), crystallizes in the monoclinic space group P21. with unit cell dimensions of a = 10.848(4), b = 8.765(3), c = 11.432(4) Å. β = 102.14(3)° . with two independent molecules in the asymmetric unit. The crystal structure of DHP-Ade was determined and compared with that for DHPG. The solution conformations of both acyclonucleosides were also determined with the aid of 1H and ,13C NMR spectroscopy. In the solid state the acyclic chain may adopt a “folded” form, i.e. gauche about the C(1′)-O(1′) bond (as in DHP-Ade), or an “extended” form (as in DHPG), results which corre- spond to the rotations about this bond in solution. A general discussion is presented of the conformations of the acyclic chains of various acyclonucleosides, from the antiviral 9-(2-hydroxyethoxymethyl)guanine (Acyclovir, ACV) through to 2′,3′-seco-nucleosides, both in the solid state and in solution, and the relevance of these to biological activities.

Tetracyclines coordinate metal(II) ions under physiological conditions forming chelate complexes with their ketoenolate moiety at rings B and C. These metal(II) complexes are the biologically relevant molecules conferring the antibiotic character of the drug by inhibiting ribosomal protein biosynthesis in prokaryotes. The Tet repressor, TetR, is the molecular switch for tetracycline resistance determinants in gram-negative bacteria. TetR controls transcription of a gene encoding the integral membrane protein TetA, which mediates active efflux of a tetracycline-metal(II) cation, [MeTc](+), by equimolar antiport with a proton. We evaluated distinct characteristics of the metal binding by crystal structure determination of TetR/[MeTc](+) complexes and of association equilibrium constants of [MeTc](+) and TetR/[MeTc](+) complexes. Various divalent metal ions bind to the same octahedral coordination site, defined by a histidine side chain of TetR, the tetracycline, and three water molecules. Whereas association constants for [MeTc](+) vary within 3 orders of magnitude, association of the [MeTc](+) cation to TetR is very similar for all measured divalent metals. Taking intracellular cation concentrations into account, it is evident that no other metal ion can compete with Mg(2+) for TetR/[MeTc](+) complex formation.

The enzyme arylsulfatase A (ASA) occurs in solution as dimer (alpha(2)) above pH 6 and associates to octamers (alpha(2))(4) below pH 6. The crystal structure of ASA suggests that the (alpha(2))-(alpha(2))(4) equilibrium is regulated by protonation/deprotonation of Glu-424 located at the interface between (alpha(2)) dimers in the octamer. The reason for this assumption is that Glu-424 can be in two different conformers where it forms an intra or intermolecular hydrogen bond, respectively. In the present study we investigate this protein association process theoretically. The electrostatic energies are evaluated by solving the Poisson-Boltzmann equation for the inhomogeneous dielectric of the protein-water system for the dimer and octamer configurations. If a conventional surface energy term is used for the nonelectrostatic interactions, the absolute value of free energy of association fails to agree with experiment. A more detailed treatment that explicitly accounts for hydrophilic and hydrophobic character of the amino acids in the dimer-dimer interface of the octamer can explain this discrepancy qualitatively. The pH dependence of the computed association energy clearly demonstrates that the octamer is more stable at low pH if Glu-424 becomes protonated and forms an intermolecular hydrogen bond. We found a slight preference of Glu-424 to be in a conformation where its acidic group is fully solvent-exposed in the dimer state to form hydrogen bonds with water molecules. Application of the proton linkage model to calculate the association energy from the simulated data yielded results identical to the one obtained from the corresponding direct method.

The structure of a new crystal form (space group C2), grown at pH 8.0 and diffracting to 1.95 A resolution, of the replicative homo-hexameric DNA helicase RepA encoded by plasmid RSF1010 is reported. In contrast to previous crystals grown at pH 6.0 in space group P2(1) (Niedenzu et al., 2001), only one half (a trimer) of the RepA hexamer occupies the asymmetric unit of the space-group C2 crystals. The new crystal packing explains the pH-dependent hexamer-hexamer association mechanism of RepA. The C-terminus (264)VLERQRKSKGVPRGEA(279), which could not be modelled in the previous structure, is clearly defined in the present electron density except for the last four amino acids. Sulfate anions occupy the six ATPase active sites of RepA at positions where the product phosphates are supposed to bind. Binding of sulfate anions induces conformational changes both at the ATPase active sites and throughout the whole molecular structure. In agreement with electron microscopy, the above studies implicate structural changes to an "open" form that may occur upon binding and hydrolysis of nucleotide 5'-triphosphates and could be essential for DNA duplex-unwinding activity.

The reasons for the association of the human major histocompatibility complex protein HLA-B27 with spondyloarthropathies are unknown. To uncover the underlying molecular causes, we determined the crystal structures of the disease-associated B*2705 and the nonassociated B*2709 subtypes complexed with the same nonapeptide (GRFAAAIAK). Both differ in only one residue (Asp(116) and His(116), respectively) in the F-pocket that accommodates the peptide C terminus. Several different effects of the Asp(116) --His replacement are observed. The bulkier His(116) induces a movement of peptide C-terminal pLys(9), allowing the formation of a novel salt bridge to Asp(77), whereas the salt bridge between pLys(9) and Asp(116) is converted into a hydrogen bond with His(116). His(116) but not Asp(116) adopts two alternative conformations, one of which leads to breakage of hydrogen bonds. Water molecules near residue 116 differ with regard to number, position, and contacts made. Furthermore, F-pocket atoms exhibit higher B-factors in B*2709 than in B*2705, indicating an increased flexibility of the entire region in the former subtype. These changes induce subtle peptide conformational alterations that may be responsible for the immunobiological differences between these HLA-B27 subtypes.

The molecular structures of peracylated beta-cyclodextrins (CDs)--heptakis(2,3,6-tri-O-acetyl)-beta-CD (TA), heptakis(2,3,6-tri-O-propanoyl)-beta-CD (TP), and heptakis(2,3,6-tri-O-butanoyl)-beta-CD (TB)--have been determined by single crystal X-ray structure analysis. Due to the lack of O2...O3' hydrogen bonds between adjacent glucose units of the peracylated CDs, the macrocycles are elliptically distorted into nonplanar boat-shaped structures. The glucose units are tilted with respect to the O4 plane to relieve steric hindrance between adjacent acyl chains. In TB, all glucose units adopt the common (4)C(1)-chair conformation and one butanoyl chain intramolecularly penetrates the cavity, whereas, in TA and TP, one glucose unit each occurs in (O)S(2)-skew-boat conformation and one acyl chain closes the O6 side like a lid. In each of the three homologous molecules the intramolecular self-inclusion and lidlike orientation of acyl chains forces the associated O5-C5-C6-O6 torsion angle into a trans-conformation never observed before for unsubstituted CD; the inclusion behavior of TA, TP, and TB in solution has been studied by circular dichroism spectroscopy with the drug molsidomine and several organic compounds. No inclusion complexes are formed, which is attributed to the intramolecular closure of the molecular cavity by one of the acyl chains.

Cycloamylose containing 26 glucose residues (cyclohexaicosaose, CA26) crystallized from water and 30% (v/v) polyethyleneglycol 400 in the orthorhombic space group P 2 1 2 1 2 1 in the highly hydrated form CA26·32.59 H 2 O. X-ray analysis of the crystals at 0.85 A resolution shows that the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by a twofold rotational pseudosymmetry as reported recently for the (CA26) 2 ·76.75 H 2 O triclinic crystal form [Gesler, K. et al. Proc. Natl. Acad. Sci. USA 1999 , 96 , 4246–4251]. In the orthorhombic crystal form, CA26 molecules are packed in motifs reminiscent of V-amylose in hydrated and anhydrous forms. The intramolecular interface between the V-helices in CA26 is dictated by formation of an extended network of interhelical CH⋯O hydrogen bonds; a comparable molecular arrangement is also evident for the intermolecular packing, suggesting that it is a characteristic feature of V-amylose interaction. The hydrophobic channels of CA26 are filled with disordered water molecules arranged in chains and held in position by multiple CH⋯O hydrogen bonds. In the orthorhombic and triclinic crystal forms, the structures of CA26 molecules are equivalent but the positions of the individual water molecules are different, suggesting that the patterns of water chains are perturbed even by small structural changes associated with differences in packing arrangements in the two crystal lattices rather than with differences in the CA26 geometry.

The inclusion complex beta-cyclodextrin.2,7-dihydroxynaphthalene.4.6 H(2)O crystallized in the monoclinic space group P2(1), with a=14.082(3), b=19.079(4), c=12.417(3) A, beta=109.28(3) degrees, V=3149.0(11) A(3), and Z=2. An X-ray study performed at room temperature shows that the crystal packing is of the herringbone type with one 2,7-dihydroxynaphthalene included completely in the beta-CD cavity, its long axis being oriented along the beta-CD molecular axis, and 4.6 water molecules are placed in the interstitial space. The beta-CD macrocycle is elliptically distorted, and the guest molecule is held in the hydrophobic beta-CD cavity by C-H...O and C-H...pi interactions.

A single-crystal neutron diffraction study of cyclodecaamylose (CA10) was carried out at 20 K. CA10 crystallizes with 27.18 water molecules [(C6H10O5)10·27.18H2O] in space group C2 with unit-cell constants a = 29.31 (5), b = 9.976 (10), c = 19.34 (2) Å, β = 121.07 (2)°. The asymmetric unit contains a half molecule of CA10 and 13.59 water molecules, the other half being related by a crystallographic twofold rotation axis. All H atoms except two water H atoms could be located from difference neutron-density maps; structure refinement converged at R = 0.635. Two of the five CH2—O6 groups and one of the 15 O2, O3 hydroxyl groups of CA10 are twofold orientationally disordered. A total of 13.59 water molecules in the asymmetric unit are distributed over 23 positions; 20 of which are in the CA10 cavity, and the other three occupy intermolecular interstices. Of the 123 symmetry-independent hydrogen bonds, 25 (= 20%) are three-centered and 7 (= 6%) are four-centered. Water molecules and O—H groups of CA10 form an extended network with cooperative O—H...O—H...O—H hydrogen bonds. They are arranged in 11 polygons with three, four, five, six and eight O—H bonds and in homodromic, antidromic and heterodromic arrangements. Nine polygons are located within the cavity and the others are outside.

Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5 ˛ 2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)6. At pH 5.8, (RepA)6 associates to form a dimer with a molecular mass of 366.2 ˛ 4.1 kDa. Analytical ultracentrifuga- tion was also applied to characterize the interaction of single- stranded DNA (ssDNA) with the two different oligomeric states of (RepA)6 at pH 5.8 and 7.6. The dissociation constants, Kd, for the equilibrium binding of (dA)30 to the (RepA)6 dimer at pH 5.8 and to (RepA)6 at pH 7.6 were determined at 10‡C in the presence of 0.5 mM ATPQS, 10 mM MgCl2 and 60 mM NaCl as Kd5:8 = 0.94 ˛ 0.13 W WM at pH 5.8 and Kd7:6 = 25.4 ˛ 6.4 W Ma t pH 7.6. The stoichiometries, n, for the two complexes (dA)30/ (RepA)6 dimer and (dA)30/(RepA)6 at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA)30 molecule was bound per (RepA)6 dimer, while at pH 7.6 one (dA)30 molecule was bound to one (RepA)6. Binding curves were compatible with a single ssDNA binding site present on the (RepA)6 dimer and on (RepA)6, respectively, with no indication of cooperativity. (RepA)6 tends to form larger aggregates under acidic conditions (pH6 6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)6 dimer was observed both in the absence and presence of (dA)30. fl 2000 Federation of European Biochem- ical Societies. Published by Elsevier Science B.V. All rights reserved.