Nonstoichiometric complex of gramicidin D with KI at 0.80 A resolution.

The crystal structure of a nonstoichiometric complex of gramicidin D (gD) with KI has been determined at 100 K using synchrotron radiation. The final R factor was 0.106 for 83 988 observed reflections (Friedel pairs were not merged) collected to 0.80 A. The structure consists of four independent pentadecapeptides and numerous solvent molecules and salt ions. The general architecture of the antiparallel double-stranded gramicidin dimers in the crystal (a right-handed antiparallel DSbetaH(R) form) closely resembles that of previously published cation complexes of gD. However, a significantly different mixture of gramicidin isomers is found in the crystal of the KI complex, including partial occupancy of phenylalanine at position 11. Only three sites in each of the two crystallographically independent channels are partially occupied by potassium cations instead of the commonly observed seven sites. The sum of the partial occupancies of K(+) (1.10 per two dimers) is consistent with the sum of the iodide occupancies (1.095 over eight sites), which is also confirmed by the anomalous signal of the iodide. There was a significant asymmetry of the distribution and occupancies of cations in the crystallographically independent gramicidin channels, in contrast to the distribution found in the rubidium chloride complex with gD.

Structure of gramicidin D-RbCl complex at atomic resolution from low-temperature synchrotron data: interactions of double-stranded gramicidin channel contents and cations with channel wall.

Gramicidin D (gD) is a naturally occurring ionophoric antibiotic that forms membrane channels specific for monovalent cations. The crystal structure of the RbCl complex of gD has been determined at 1.14 A resolution from low-temperature (100 K) synchrotron-radiation data with a final R of 16%. The structure was refined with anisotropic temperature factors for all non-H atoms and with partial occupancies for many of them. The asymmetric unit in the crystal contains four crystallographically independent molecules that form two right-handed antiparallel double-stranded dimers. There are seven distinct rubidium-binding sites in each dimeric channel. The occupancy factors of Rb cations are between 0.11 and 0.35 and the total ion contents of the two crystallographically independent channels are 1.59 and 1.22 ions, respectively. Although each channel is 'chemically symmetrical', the side-chain conformations, the distributions of rubidium cations and their binding sites in the two independent channels are not. Cations are 'coordinated' by delocalized pi-electrons of three to five carbonyl groups that together with peptide backbone chains form the gramicidin channel walls. The water:cation ratio in the channel interior is four or five:one, and five or six waters separate Rb cations during their passage through the channel.

Solvent dependence of the structure and magnetic ordering of ferrimagnetic manganese(III) meso-tetraphenylporphyrin tetracyanoethenide, [MnTPP]+[TCNE]*-.

Tetraphenylporphinatomanganate(III) tetracyanoethenide, [MnTPP][TCNE], is the prototype of a growing family of linear chain (1-D) coordination polymers that magnetically order as ferrimagnets. [MnTPP][TCNE].xS [S = PhMe (x = 2), 1,2-C(6)H(4)Me(2) (x = 1), 1,2-C(6)H(4)Cl(2) (x = 3), 1,2,4-C(6)H(3)Cl(3) (x = 2), and 1,3-C(6)H(4)Cl(2) (x = 2)] have been prepared and structurally and magnetically characterized. All form 1-D chain structures with intrachain Mn.Mn separations ranging from 9.202 to 10.218 A. The 173 K crystal structure of [MnTPP][TCNE].2PhMe has been rerefined, revealing that the [TCNE](*)(-) is 2-fold-disordered and coordinated to Mn(III) by a pair of trans cyano nitrogen atoms to form parallel one-dimensional chains. The two orientations of [TCNE](*)(-) are related by a 180 degrees rotation about the diagonal axis joining the trans nitrogen atoms bound to Mn(III). The major form has an occupancy of 83.3(4)% with a Mn-N(TCNE) distance of 2.328(3) A and a MnNC angle of 146.8(8) degrees. The minor form, with 16.7(4)% occupancy, has a Mn-N(TCNE) distance of 2.176(15) A and a MnNC angle of 152.3(39) degrees. Lattice packing and molecular bonding imply static as opposed to dynamic disorder. The magnetic properties depend on the type and quantity of the solvent present in the structure. Desolvation via heating in n-octane (127 degrees C), n-dodecane (216 degrees C), and/or vacuum thermolysis (175 degrees C) leads to numerous different desolvated materials with differing magnetic properties. At higher temperatures the magnetic susceptibility can be fit by the Curie-Weiss expression, chi varies with (T - theta)(-1), with theta = 44, 52, 72, 55, and 77 K for the toluene, 1,2-xylene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, and 1,2,4-trichlorobenzene solvates, respectively. The T(c)'s were taken as the maximum in 10 Hz chi'(T) and are 7.8, 9.2, 11.3, 10.8, and 8.2 K for the PhMe, 1,2-xylene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, and 1,2,4-trichlorobenzene solvates, respectively. Upon desolvation the T(c)'s increase for the PhMe, 1,2-xylene, 1,2,4-trichlorobenzene solvates and decrease for the 1,2- and 1,3-dichlorobenezene solvates. The compounds show one-dimensional ferrimagnetic exchange behavior at high temperatures with intrachain exchange of J/k(b) = -63, -99, -234, -100, and -200 K for toluene, 1,2-xylene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, and 1,2,4-trichlorobenzene solvates, respectively, as determined from fits to the Seiden expression, which models isolated 1-D interactions among alternating S = 2 classical and S = (1)/(2) quantum spins. For variation in the temperature at which the peak occurs per decade of frequency, phi, (DeltaT(f)/T(f))/Delta(log omega) is 0.167, 0.168, 0.066, 0.171, and 0.024 for toluene, 1,2-xylene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, and 1,2,4-trichlorobenzene, respectively, typical of spin glass behavior. Since [TCNE](*)(-) is only disordered for the PhMe solvate and all five solvates exhibit spin glass behavior, the spin glass behavior cannot be attributed to this disorder.

Structure of an activity suppressing Fab fragment to cytochrome P450 aromatase: insights into the antibody-antigen interactions.

The crystal structure of a Fab fragment (Fab3-2C2) of a monoclonal antibody raised against aromatase cytochrome P450 P450arom) has been determined at 3.0 A resolution. P450arom is a membrane bound enzyme responsible for the catalysis of indrogens to estrogens, the process of aromatization, and hence has been implicated in hormone-dependent breast cancer. The Fab fragment of MAb3-2C2 IgG suppresses P450arom activity in a dose dependent manner. The Fab3-2C2 molecule crystallizes n the space group P2(1)2(1)2(1) with a unit cell of a= 154.89 A, b = 73.51 A, and c= 36.90 A. The crystal structure consists of a light and a heavy chain in the asymmetric unit, each characterized by the greek-key antiparallel beta barrel folding seen in all Fab structures. The average elbow angle between the two domains is 143 degrees. Modeling of the interactions between the variable domains of the antibody and a known model of P450arom maps the epitope to a region of the enzyme that is consistent with the available biochemical data and the activity-suppressing function of the antibody. The epitope mapping result is further supported by the inability of MAb3-2C2 IgG to suppress the activity of, or to interact with placental porcine P450arom, which is 81% identical (86% similar) to human P450arom but has a few key substitutions in the putative epitope region.

Gramicidin D conformation, dynamics and membrane ion transport.

The linear pentadecapeptide antibiotic, gramicidin D, a heterogeneous mixture of six components, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The conformation of gramicidin A in the solid state, in organic solvents, and in planar lipid bilayers and the relationship between the composition and the conformation of gramicidin and its selective transport of ions across membranes has been the subject of intense investigation for over 50 years. The x-ray crystal structure and nmr solution spectroscopy agree fully with one another and reveal that entirely different conformations of gramicidin are present in uncomplexed and ion complexed forms. Precise refinements of the three-dimensional structures of naturally occurring gramicidin D in crystals obtained from methanol, ethanol, and n-propanol demonstrate the unexpected presence of stable left-handed antiparallel double-helical heterodimers that vary with the crystallization solvent. The side chains of Trp residues in the three structures exhibit sequence-specific patterns of conformational preference. Tyr substitution for Trp at position 11 appears to favor beta ribbon formation and stabilization of the antiparallel double helix. This conformation acts as a template for gramicidin folding and nucleation of the different crystal forms. The fact that a minor component in a heterogeneous mixture influences aggregation and crystal nucleation has potential applications to other systems in which anomalous behavior is exhibited by aggregation of apparently homogeneous materials, such as the enigmatic behavior of prion proteins. The crystallographically determined structures of cesium, potassium, rubidium, and hydronium ion complexes of gramicidin A are in excellent agreement with the nmr structure determination of the cesium ion gramicidin complex in a methanol chloroform mixture (50 : 50). The right-handed antiparallel double stranded double helical structures (DSDHR) also exhibit geometric features compatible with the solid-state 15N and 2H nmr data recorded for gramicidin in planar lipid bilayers and attributed to the active form of gramicidin A. The DSDHR crystal structures reveal an ion channel with a single partially solvated cation distributed over three ion binding sites. The channel lumen is relatively smooth and electrostatically negative as required for cation passage, while the exterior is electrostatically neutral, a requirement for membrane insertion. The "coordination" of the Cs+ ion is achieved by interaction with the pi orbitals of the carbonyls which do not point toward the ions. The K+ binding sites, which are similar in position to Cs+ binding sites, are shifted off center slightly toward the wall of the channel.

Heterodimer formation and crystal nucleation of gramicidin D.

The linear pentadecapeptide antibiotic gramicidin D is a heterogeneous mixture of six components. Precise refinements of three-dimensional structures of naturally occurring gramicidin D in crystals obtained from methanol, ethanol, and n-propanol demonstrate the unexpected presence of stable left-handed antiparallel double-helical heterodimers that vary with the crystallization solvent. The side chains of Trp residues in the three structures exhibit sequence-specific patterns of conformational preference. Tyr substitution for Trp at position 11 appears to favor beta ribbon formation and stabilization of the antiparallel double helix that acts as a template for gramicidin folding and nucleation of different crystal forms. The fact that a minor component in a heterogeneous mixture influences aggregation and crystal nucleation has potential applications to other systems in which anomalous behavior is exhibited by aggregation of apparently homogeneous materials, such as the enigmatic behavior of prion proteins.

The conducting form of gramicidin A is a right-handed double-stranded double helix.

The linear pentadecapeptide antibiotic, gramicidin D, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The x-ray crystal structures of the right-handed double-stranded double-helical dimers (DSDH) reported here agree with 15N-NMR and CD data on the functional gramicidin D channel in lipid bilayers. These structures demonstrate single-file ion transfer through the channels. The results also indicate that previous crystal structure reports of a left-handed double-stranded double-helical dimer in complex with Cs+ and K+ salts may be in error and that our evidence points to the DSDH as the major conformer responsible for ion transport in membranes.

The X-ray structure of the monoclinic crystal form of [D-Hyi2, L-Hyi4] meso-valinomycin.

The conformation and intermolecular association of [D-Hyi2, L-Hyi4] meso-valinomycin [cyclo[-D-Val-D-Hyi-L-Val-L-Hyi-(D-Val-L-Hyi-L-Val-D-+ ++Hyi)2-], C60H102N6O18] in a crystal form obtained from ethanol solution has been determined by x-ray crystallography. Two depsipeptides and one ethanol molecule per asymmetric unit crystallize in space group P2(1) (Z = 4); a = 14.579, b = 39.795, c = 13.928 A, beta = 116.90, Rl = 0.0757. The molecular conformation is very similar to that observed in the trigonal P3(2) crystal form obtained from acetone solution [V. Z. Pletnev et al. (1991) Biopolymers, Vol. 31, pp. 409-415]. Both independent molecules in the crystal adopt a similar distorted bracelet structure with a sterically inaccessible, partially formed, ion-binding center that is stabilized by six 4-->1 type H bonds. The observed conformation accounts for the inability of the molecule to complex ions. Close examination of the three crystallographically independent molecules reveals that differences in the backbone conformation associated with solvent interaction are significantly larger than those associated with hydrophobic van der Waals interactions of crystal packing.

Characterization of crystals of Penicillium purpurogenum acetyl xylan esterase from high-resolution x-ray diffraction.

Acetyl xylan esterase from Penicillium purpurogenum, a single-chain 23 kDa member of a newly characterized family of esterases that cleaves side chain ester linkages in xylan, has been crystallized. The crystals diffract to better than 1 A resolution at the Cornell High Energy Synchrotron Source (CHESS) and are highly stable in the synchrotron radiation. The space group is P2(1)2(1)2(1) and cell dimensions are a = 34.9 A, b = 61.0 A, C = 72.5 A.

2',3'-Dideoxy-3'-C,2'-N-[(3R,5R)-5-ethoxy-carbonyl-2-methyl-1, 2-isoxazolidine]-ribothymidine.

The title compound, C15H21N3O7, is a thymidine derivative with a tetrahydroisoxazole (THI) ring fused to the sugar ring at the 2'- and 3'-C atoms. The thymine base is in an anti conformation [chi = -122.5 (3) degrees] while the ribose moiety has C2'-exo,C3'-endo (3T2) conformation with a highly flattened sugar ring [P = -5.8, tau m = 13.1 degrees]. The molecule contains a pseudo-twofold axis on the C2'-C3' bond with the ethyl ester group acting as the base and the N-methyl group acting as the 5'-hydroxyl group in a nucleoside. The THI ring fused to the furanose has a C3'-endo,C4'-exo (3T4) conformation [P = 4.18, tau m = 19.3 degrees]. The ethyl ester moiety is disordered with respect to two conformations, the population of the major form being 87.4 (8)% and that of the minor form 12.6 (8)%.

Structure of the trigonal form of recombinant oxidized flavodoxin from Anabaena 7120 at 1.40 A resolution.

The oxidized recombinant flavodoxin from the cyanobacterium Anabaena 7120 has been crystallized in a trigonal form. The recombinant protein has an identical primary structure to that purified directly from Anabaena, which functions as a substitute for ferredoxin in an iron-deficient environment for electron transfer from photosystem I to ferredoxin-NADP(+) reductase. X-ray data to 1.40 A were collected on a Siemens area detector. Of the 311 379 reflections collected, 36069 reflections were unique in space group P3(1)21 (a = 55.36, c = 102.59 A) with an R(merge) of 3.8%. The structure was solved by molecular replacement using coordinates from the wild-type monoclinic structure previously solved in this laboratory [Rao, Shaffie, Yu, Satyshur, Stockman & Markley (1992). Protein Sci. 1, 1413-1427]. The structure was refined with X-PLOR and SHELXL93 to a crystallographic R-factor of 13.9% for 32963 reflections with I> 2sigma(I). The final structure contains 2767 atoms including 31 flavin mononucleotide (FMN) atoms, 299 water molecules, and one sulfate ion. The protein is comprised of a central five-stranded beta-sheet surrounded by five helices and binds a single molecule of FMN at the C-terminus of the sheet. The trigonal protein structure and the crystal packing are compared with the monoclinic wild-type protein. Helix alpha3 in this structure is less distorted than in the monoclinic structure and shows additional hydrogen bonds in the N-terminal portion of the helix. The trigonal structure is extensively hydrogen bonded in three major areas with neighboring molecules compared with five regions in the monoclinic structure, but using significantly fewer hydrogen bonds to stabilize the lattice. There are several hydrogen bonds to the amide groups from water molecules several of which stabilize and extend the ends of the beta-sheet.