Surface properties of 1,2-dipalmitoyl-3-acyl-sn-glycerols

Stereospecific 1,2-dipalmitoyl-sn-glycerol and a series of 1,2-dipalmitoyl-3-acyl-sn-glycerols (TGs) with 3-acyl chains of two through six and eight carbons in length were synthesized. Pressure-area isotherms at 27 degrees C, surface melting temperatures (Ts), and equilibrium spreading pressures (esp) measured at the bulk melting temperature (Tf) were obtained for each TG and for dipalmitin. Whereas dipalmitin and the 3-acetyl-TG condense directly to an expanded mesomorphous state (30-33 A2/palmitoyl chain at the vapor pressure, pi v), the 3-propionyl- through 3-octanoyl-TGs show an area per molecule (in the liquid at pi v) that increases linearly from 105 to 130 A2/molecule (slope = 5 A2/CH2 group). This slope suggests that the 3-acyl chains are lying flat on the water at the end of the gas-liquid transition. Before solidification at 42-47 A2/molecule, the 3-propionyl- through 3-hexanoyl-TGs show a transition corresponding to the immersion of the 3-acyl chain. The pressure at this transition, pi tr, vs. 3-acyl carbon number is linear and indicates a chain immersion energy of 497 cal mol-1 per CH2. In contrast, the 3-octanoyl chain is not forced into the water but rather is pushed into the monolayer to lie parallel to the palmitoyl chains. As the sn-3 chain is lengthened, Ts decreases from 68 to 25 degrees C, but the 3-octanoyl monolayer does not solidify even at 5 degrees C because the short upright octanoyl chains fluidize the palmitoyl chains. The esp (at Tf) drops from 31.7 mN m-1 for dipalmitin to 20.6 mN m-1 for the 3-acetyl-TG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyanidioschyzon merolae ferredoxin: a high resolution crystal structure analysis and its thermal stability

Cyanidioschyzon merolae (Cm) is a single-cell red alga that grows under moderately thermophilic (40-50°C), acidic (pH 1-3) conditions. We purified a Cm ferredoxin (Fd) that was characterized as a plant-type [2Fe-2S] Fd by physicochemical techniques. X-ray crystallography revealed that the overall three-dimensional structure of CmFd was highly similar to, but slightly different from, the [2Fe-2S] Fd from Spinacia oleracea, whose growth temperature is 15-20°C. Therefore, slight structural differences, including non-covalent-bond number and amino acid sequence, may underlie the differential thermostabilities of the plant-type Fds.     

X-ray crystal structure of canine myeloperoxidase at 3 A resolution.

The three-dimensional structure of the enzyme myeloperoxidase has been determined by X-ray crystallography to 3 A resolution. Two heavy atom derivatives were used to phase an initial multiple isomorphous replacement map that was subsequently improved by solvent flattening and non-crystallographic symmetry averaging. Crystallographic refinement gave a final model with an R-factor of 0.257. The root-mean-square deviations from ideality for bond lengths and angles were 0.011 A and 3.8 degrees. Two, apparently identical, halves of the molecule are related by local dyad and covalently linked by a single disulfide bridge. Each half-molecule consists of two polypeptide chains of 108 and 466 amino acid residues, a heme prosthetic group, a bound calcium ion and at least three sites of asparagine-linked glycosylation. There are six additional intra-chain disulfide bonds, five in the large polypeptide and one in the small. A central core region that includes the heme binding site is composed of five alpha-helices. Regions of the larger polypeptide surrounding this core are organized into locally folded domains in which the secondary structure is predominantly alpha-helical with very little organized beta-sheet. A proximal ligand to the heme iron atom has been identified as histidine 336, which is in turn hydrogen-bonded to asparagine 421. On the distal side of the heme, histidine 95 and arginine 239 are likely to participate directly in the catalytic mechanism, in a manner analogous to the distal histidine and arginine of the non-homologous enzyme cytochrome c peroxidase. The site of the covalent linkage to the heme has been tentatively identified as glutamate 242, although the chemical nature of the link remains uncertain. The calcium binding site has been located in a loop comprising residues 168 to 174 together with aspartate 96. Myeloperoxidase is a member of a family of homologous mammalian peroxidases that includes thyroid peroxidase, eosinophil peroxidase and lactoperoxidase. The heme environment, defined by our model for myeloperoxidase, appears to be highly conserved in these four mammalian peroxidases. Furthermore, the conservation of all 12 cysteine residues involved in the six intra-chain disulfide bonds and the calcium binding loop suggests that the three-dimensional structures of members of this gene family are likely to be quite similar.     

Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile

Hydrophobins are proteins specific to filamentous fungi. Hydrophobins have several important roles in fungal physiology, for example, adhesion, formation of protective surface coatings, and the reduction of the surface tension of water, which allows growth of aerial structures. Hydrophobins show remarkable biophysical properties, for example, they are the most powerful surface-active proteins known. To this point the molecular basis of the function of this group of proteins has been largely unknown. We have now determined the crystal structure of the hydrophobin HFBII from Trichoderma reesei at 1.0 A resolution. HFBII has a novel, compact single domain structure containing one alpha-helix and four antiparallel beta-strands that completely envelop two disulfide bridges. The protein surface is mainly hydrophilic, but two beta-hairpin loops contain several conserved aliphatic side chains that form a flat hydrophobic patch that makes the molecule amphiphilic. The amphiphilicity of the HFBII molecule is expected to be a source for surface activity, and we suggest that the behavior of this surfactant is greatly enhanced by the self-assembly that is favored by the combination of size and rigidity. This mechanism of function is supported by atomic force micrographs that show highly ordered arrays of HFBII at the air water interface. The data presented show that much of the current views on structure function relations in hydrophobins must be re-evaluated.     

Atomic resolution structure of prokaryotic phospholipase A2: analysis of internal motion and implication for a catalytic mechanism

We have found a secreted phospholipase A(2) (PLA(2), EC 3.1.1.4) from Streptomyces violaceoruber A-2688, which is the first PLA(2) identified in prokaryote, and determined its tertiary structure by NMR and X-ray analyses. In this study, we collected the X-ray diffraction data of the bacterial PLA(2) at room temperature (297 K) using conventional MoK(alpha) radiation and refined the structure at a 1.05 A resolution. The atomic resolution analysis led us to introduce disordered conformations and hydrogen atoms into a full anisotropic model. The molecular motion, which is expressed as the sum of rigid-body motion and internal motion of protein, is roughly estimated as the thermal motion when the X-ray diffraction data are collected at room temperature. In this study, we applied a TLS (rigid-body motion in terms of translation, libration, and screw motions) model to analyze the rigid-body motion of the bacterial PLA(2) and calculated the internal motion by subtracting the estimate of the rigid-body motion from the observed anisotropic temperature factor. We also subjected the TLS model to estimate the internal motion of the bovine pancreatic PLA(2) using the anisotropic temperature factor deposited in the Protein Data Bank. Both results indicate that the localization of regions exhibiting larger internal motion in the bacterial PLA(2) is almost the same as that in the bovine pancreatic PLA(2), suggesting that although the tertiary structure of the bacterial PLA(2) is strikingly different from that of the bovine pancreatic PLA(2), the internal motion, which is associated with the calcium(II) ion-binding, phospholipid-binding, and allosteric interfacial activation, is commonly observed in both PLA(2)s.     

Water structure in a protein crystal: rubredoxin at 1.2 A resolution

The model for rubredoxin based on X-ray diffraction data has been extensively refined with a 1.2 Å resolution data set. Water oxygen atoms were deleted from the model if B exceeded 50 Ų and occupancy was less than 0.3 eÅ^?3. The final water model consists of 127 sites with B values ranging from 15 to $\text{6}\text{?}$0 Ų and occupancies from unity down to 0.3, the most tightly bound water oxygen atoms being hydrogen bonded to two or more main-chain nitrogen or oxygen atoms. The water forms extensive hydrogen bond networks bridging the crevices on the molecular surfaces and between adjacent molecules. The minimum distances of the water sites from the protein surface are distributed about two distinct maxima, the major one at 2.5 to 3 Å and a minor one at 4 to 4.5 Å. Beyond $\text{5}\text{?}$ to 6 Å from the protein surface, the discrete water merges into the aqueous continuum.     

Ultra-high resolution crystal structure of a dimeric defensin SPE10

Defensins are key players of the innate immune system known to act against bacteria, fungi and viruses. Here we report the 0.98-Å crystal structure of SPE10, a dimeric plant defensin. SPE10 associates as a dimer through a unique amino acid triplet involving residues R36–W42–R40. The helix from one subunit interacts with arginines R36 and R40 from the other subunit, forming a sheet-like dimer with a highly extended molecular surface. A conserved hydrophobic patch on the molecular head largely overlaps with the putative receptor-binding site previously reported for another defensin. Structural analysis and mutational studies indicate that the dimeric association of SPE10 is relevant to its function, and that the hydrophobic patch on the molecular head is required for its antifungal activity.

Structured summary

SPE10binds to SPE10 by X-ray crystallography(View interaction)     

The crystal structure of erabutoxin a at 2.0-A resolution

The three-dimensional structure of erabutoxin a, a single-chain, 62-residue protein neurotoxin from snake venom, has been determined to 2.0-A resolution by x-ray crystal structure analysis. Molecular replacement methods were used, and the structure refined to a residual R = 0.17. The sites of 62 water molecules and 1 sulfate ion have been located and refined. The structure of erabutoxin a is very similar to that established earlier for erabutoxin b. These toxins from venom of the same snake differ in sequence only at residue 26, which is Asn in erabutoxin a and His in erabutoxin b. The substitution leads to only minor variations in intramolecular hydrogen bonding. Furthermore, the distribution of thermal parameters and the implied regional mobilities are similar in the two structures. In particular, the highly mobile character of the peripheral segment Pro44-Gly49 in both structures supports the specific role proposed for this segment in neurotoxin binding to the acetylcholine receptor. Forty-eight of the solvent sites determined are first surface positions; approximately one-half of these are equivalent to solvent sites in erabutoxin b.     

Preliminary crystal structure analysis of a microbial, guanine-specific ribonuclease St at 2.5 A resolution

The three-dimensional structure of Ribonuclease St (RNase St), the extracellular ribonuclease from Streptomyces erythreus, has been deduced based on a preliminary electron density map at 2.5 A resolution. RNase St has a substrate specificity similar to ribonuclease T1 which catalyzes the splitting of the phosphodiester bond of guanylic acid. Crystals grown as diamond plates have space group C2 with unit cell parameters a=88.4, b=33.0, c=69.0 A, beta = 98.4 degrees having two enzyme molecules per asymmetric unit. Phases were obtained by use of KAu(CN)4, phenylmercuric acetate and UO2 (CH3COO)2. The overall dimensions of the molecule are 40 X 30 X 25 A. The most prominent secondary structural features are two turns of alpha-helix and a three strand stretch of antiparallel beta-sheet. The alpha-carbon backbone of RNase St seems to have no apparent correlation with that of ribonuclease A.     

The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited

The crystal structure of the monoclinic form of yeast phenylalanine tRNA has been redetermined at a resolution of 1.93 A. The structure of yeast tRNAphe described here is more accurate than its predecessors not only because it incorporates higher resolution data, but also because it has been refined using techniques that had not been developed when its predecessors were determined more than 20 years ago. The 1.93 A resolution version of this structure differs interestingly from its predecessors in its details. In loop regions particularly, the backbone torsion angles in the new structure are not the same as those reported earlier. Several new divalent cation binding sites have been identified, and the water structure that has emerged is also different.     

Atomic resolution structure of a succinimide intermediate in E.coli CheY

Isomerization of aspartate to isoaspartate occurs spontaneously in proteins, causes changes in protein structures, and correlates positively with the aging processes of many organisms, including Alzheimer disease in humans. Aspartate isomerization proceeds through an unstable cyclic succinimide intermediate. There are few protein structure determinations that have characterized the intermediates and products of this isomerization reaction. Here we report the discovery of an unusually stabilized succinimide ring in the 1.1A structure of the Escherichia coli CheY protein, as determined from a crystal eight years old. The ring is formed by the side-chain of aspartate 75 and the backbone nitrogen of glycine 76 in an exposed loop of the molecule. Stabilization of the succinimide is through interaction of a sulfate ion oxygen atom with the imide nitrogen atom. Formation of the ring caused conformational changes in the loop, but did not alter the overall structure of the protein.     

Salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans: crystal structure of a peculiar ring-cleaving dioxygenase

The crystallographic structure of salicylate 1,2-dioxygenase (SDO), a new ring fission dioxygenase from the naphthalenesulfonate-degrading strain Pseudaminobacter salicylatoxidans BN12, which oxidizes salicylate to 2-oxohepta-3,5-dienedioic acid by a novel ring fission mechanism, has been solved by molecular replacement techniques and refined at 2.9 Å resolution (R_free 26.1%; R-factor 19.3%). SDO is a homo-tetramer member of type III extradiol-type dioxygenases with a subunit topology characteristic of the bicupin β-barrel folds. The catalytic center contains a mononuclear iron(II) ion coordinated to three histidine residues (His119, His121, and His160), located within the N-terminal domain in a solvent-accessible pocket. SDO is markedly different from the known gentisate 1,2-dioxygenases (GDO) or 1-hydroxy-2-naphthoate dioxygenase because of its unique ability to oxidatively cleave numerous salicylates, gentisates and 1-hydroxy-2-naphthoate with high catalytic efficiency. The comparison of the structure and substrate specificity for a series of different substrates with the corresponding data for several GDOs and the docking of salicylates/gentisates in the active site of SDO, allowed the identification of several active site residues responsible for differences of substrate specificity. In particular, a more defined electron density of the N-terminal region allowed the discovery of a novel structure fragment in SDO previously unobserved in GDO. This region contributes several residues to the active site that influence substrate specificity for both of these enzymes. Implications on the catalytic mechanism are discussed.     

Polymorphic behavior of 1,2-dipalmitoyl-3-lauroyl(PP12)- and 3-myristoyl(PP14)-sn-glycerols

The polymorphic behavior and molecular packing in different polymorphic forms of stereospecific triacylglycerols, 1,2-dipalmitoyl-3-lauroyl-sn-glycerol (PP12) and 1,2-dipalmitoyl-3-myristoyl-sn-glycerol (PP14) were examined by X-ray diffraction, differential scanning calorimetry, infrared and Raman spectroscopy techniques. The molecular packing and the polymorphic behavior of the metastable forms of these two compounds are very similar. In both compounds the isotropic liquid, on quenching, crystallizes into a hexagonally packed alpha-phase. The long spacing periodicity of the alpha-phase indicates a tilted bilayer structure to compensate the voids created by the short acyl chains. Upon heating, the alpha-phase converts into an orthorhombic perpendicular (O perpendicular) beta'2-phase. The beta' 2-phase, on further heating, exothermally converts to beta' 1-phase with slightly different O perpendicular subcell. On incubation of PP12 near the melting temperature of beta' 1-phase, there is a slow (hours) conversion to a beta-phase with triclinic parallel (T//) packing. However the beta' 1-phase of PP14 is the most stable structure and the beta-phase is absent. The thermodynamic parameters of the O perpendicular packings of these compounds compared to those of the higher homologue, tripalmitoylglycerol, suggest that the O perpendicular subcell is more stable in PP12 and PP14. The X-ray diffraction long spacings indicate that all the polymorphic forms of these compounds pack in a bilayer structure. The vibrational spectra confirm the lateral chain packing and inter- and intra-molecular order/disorder in the various polymorphic forms. The Raman and infrared spectra further indicate perturbation in the carbonyl and the end methyl plane regions of the beta'-phases.     

Atomic resolution (0.98 A) structure of eosinophil-derived neurotoxin

Human eosinophil-derived neurotoxin (EDN) is a small, basic protein that belongs to the ribonuclease A superfamily. EDN displays antiviral activity and causes the neurotoxic Gordon phenomenon when injected into rabbits. Although EDN and ribonuclease A have appreciable structural similarity and a conserved catalytic triad, their peripheral substrate-binding sites are not conserved. The crystal structure of recombinant EDN (rEDN) has been determined at 0.98 A resolution from data collected at a low temperature (100 K). We have refined the crystallographic model of the structure using anisotropic displacement parameters to a conventional R-factor of 0.116. This represents the highest resolution structure of rEDN determined to date and is only the second ribonuclease structure to be determined at a resolution greater than 1.0 A. The structure provides a detailed picture of the conformational freedom at the various subsites of rEDN, and the water structure accounts for more than 50% of the total solvent content of the unit cell. This information will be crucial for the design of tight-binding inhibitors to restrain the ribonucleolytic activity of rEDN.     

Atomic resolution structure of the major endoglucanase from Thermoascus aurantiacus

Breast cancer is a common disease in females but very rare in males, in whom it shows a more metastatic behavior, and a worse prognosis. Matrix metalloprotease-2 (MMP-2) and MMP-9 are proteolytic enzymes balanced by tissue inhibitor of MMP-2 (TIMP-2), commonly involved in cancer metastasis. This is the first study on gelatinolytic activity in male breast cancer patients, compared to that in female patients. In cancer tissues, both gelatinases were more expressed than in normal samples, being and more concentrated in male than in female patients. TIMP-2 levels were slightly increased in normal compared to those in cancer tissues and more concentrated in males than in females. Immunostaining showed that in male cancer tissues MMP-2 and MMP-9 staining was more intense and diffuse than in female cancer tissues, while no differences were observed regarding TIMP-2. In conclusion, the increased expression of gelatinases in male breast cancer patients together with anatomical features might explain the high tendency toward metastasis and the worse prognosis.     

Atomic resolution analysis of a 2:1 complex of CpG and acridine orange.

Cytidylyl-3', 5'-guanosine and acridine orange crystallize in a highly-ordered triclinic lattice which diffracts X-rays to 0.85 angstrom resolution. The crystal structure has been solved and refined to a residual factor of 9.5%. The two dinucleoside phosphate molecules form an antiparallel double helix with the acridine orange intercalated between them. The two base pairs of the double helical fragment have a twist angle of 10 degrees and it is found to have a C3' endo-(3', 5')-C2' endo mixed sugar puckering along the nucleotide backbone as has been observed for other simple intercalator complexes. Twenty-five water molecules have been located in the lattice together with a sodium ion. The intercalator double helical fragments form sheets which are held together by van der Waals interactions in one direction and hydrogen bonding interactions in the other. The crystal lattice contains aqueous channels in which sixteen water molecules are hydrogen bonded to the nucleotide, none to the intercalator, five water molecules are coordinated about the sodium ion and four water molecules bind solely to other water molecules. The bases in the base pairs have a dihedral angle of 7 to 8 degrees between them.     

High resolution crystal structure of human Rab9 GTPase: a novel antiviral drug target

Rab GTPases and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome to trans-Golgi transport and has recently been found to be a key cellular component for human immunodeficiency virus-1, Ebola, Marburg, and measles virus replication, suggesting that it may be a novel target in the development of broad spectrum antiviral drugs. As part of our structure-based drug design program, we have determined the crystal structure of a C-terminally truncated human Rab9 (residues 1-177) to 1.25-A resolution. The overall structure shows a characteristic nucleotide binding fold consisting of a six-stranded beta-sheet surrounded by five alpha-helices with a tightly bound GDP molecule in the active site. Structure-based sequence alignment of Rab9 with other Rab proteins reveals that its active site consists of residues highly conserved in the Rab GTPase family, implying a common catalytic mechanism. However, Rab9 contains seven regions that are significantly different in conformation from other Rab proteins. Some of those regions coincide with putative effector-binding sites and switch I and switch II regions identified by structure/sequence alignments. The Rab9 structure at near atomic resolution provides an excellent model for structure-based antiviral drug design.     

X-ray analysis of pepsin. VI. Atomic structure of the enzyme at 2-angstrom resolution

The crystallographic refinement of pepsin structure at 2 A resolution is described. Real space refinement and Jack and Levitt methods were used. As a result, the refined atomic coordinates of 2436 nonhydrogen atoms were obtained. Values of crystallographic R-factor and conformational energy are 29.2% and -1347 kcal/mol correspondingly. The most important and interesting features of pepsin structure are discussed.     

High resolution crystal structure of Rubrivivax gelatinosus cytochrome c'

The structure of the cytochrome c′ from the purple non-sulfur phototrophic bacterium Rubrivivax gelatinosus was determined using two crystals grown independently at pH 6.3 and pH 8. The resolution attained for the two structures (1.29 Å and 1.50 Å for the crystals at high and low pH, respectively) is the highest to date for this class of proteins. The two structures were compared in detail in an attempt to investigate the influence of pH on the geometry of the haem and of the coordination environment of the Fe(III) ion. However, while the results suggest some small propensity for the movement of the metal atom out of the plane of the haem ring upon pH increase, the accuracy of the measurements at these two pH below the pK of the axial histidine is not sufficient to provide hard evidence of a shift in the iron position and associated changes.