A method to correlate optical properties and structures of metallic nanoparticles.

The optical response of individual nanoparticles is strongly influenced by their structures. In this report, we present a quick and simple pattern-matching based approach in which optical images of nanoparticles from localized surface plasmon resonance and single-molecule surface-enhanced Raman spectroscopy were used in conjunction with transmission electron microscopy for correlation of optical responses and the nanostructures of exactly the same nanoparticles or clusters of nanoparticles.

Structure of a yeast hypothetical protein selected by a structural genomics approach.

Yeast hypothetical protein YBL036C (SWISS-PROT P38197), initially thought to be a member of an 11-protein family, was selected for crystal structure determination since no structural or functional information was available. The structure has been determined independently by MIR and MAD methods to 2.0 A resolution. The MAD structure was determined largely through automated model building. The protein folds as a TIM barrel beginning with a long N-terminal helix, in contrast to the classic triose phosphate isomerase (TIM) structure, which begins with a beta-strand. A cofactor, pyridoxal 5'-phosphate, is covalently bound near the C-terminal end of the barrel, the usual active site in TIM-barrel folds. A single-domain monomeric molecule, this yeast protein resembles the N-terminal domain of alanine racemase or ornithine decarboxylase, both of which are two-domain dimeric proteins. The yeast protein has been shown to have amino-acid racemase activity. Although selected as a member of a protein family having no obvious relationship to proteins of known structure, the protein fold turned out to be a well known and widely distributed fold. This points to the need for a more comprehensive base of structural information and better structure-modeling tools before the goal of structure prediction from amino-acid sequences can be realised. In this case, similarity to a known structure allowed inferences to be made about the structure and function of a widely distributed protein family.

Crystallographic evidence for doxorubicin binding to the receptor-binding site in Clostridium botulinum neurotoxin B.

The neurotoxins of Clostridium botulinum and tetanus bind to gangliosides as a first step of their toxin activity. Identifying suitable receptors that compete with gangliosides could prevent toxin binding to the neuronal cells. A possible ganglioside-binding site of the botulinum neurotoxin B (BoNT/B) has already been proposed and evidence is now presented for a drug binding to botulinum neurotoxin B from structural studies. Doxorubicin, a well known DNA intercalator, binds to the neurotoxin at the receptor-binding site proposed earlier. The structure of the BoNT/B-doxorubicin complex reveals that doxorubicin has interactions with the neurotoxin similar to those of sialyllactose. The aglycone moiety of the doxorubicin stacks with tryptophan 1261 and interacts with histidine 1240 of the binding domain. Here, the possibility is presented of designing a potential antagonist for these neurotoxins from crystallographic analysis of the neurotoxin-doxorubicin complex, which will be an excellent lead compound.

Structure of staphylococcal enterotoxin C2 at various pH levels.

The three-dimensional structure of staphylococcal enterotoxin C2 (SEC2), a toxin as well as a superantigen, has been determined at various pH levels from two different crystal forms, tetragonal (pH 5.0, 5.5, 6.0 and 6.5) and monoclinic (pH 8.0) at 100 and 293 K, respectively, by the molecular-replacement method. Tetragonal crystals belong to space group P4(3)2(1)2, with unit-cell parameters a = b = 42.68, c = 289.15 A (at pH 5.0), and monoclinic crystals to space group P2(1), with unit-cell parameters a = 43.3, b = 70.6, c = 42.2 A, beta = 90.3 degrees. SEC2 contains a zinc-binding motif, D+HExxH, and accordingly a Zn atom has been identified. The coordination of the zinc ion suggests that it may be catalytic zinc rather than structural, but there is so far no biological evidence that it possesses catalytic activity. However, superantigen staphylococcal exfoliative toxins A and B have been shown to have enzymatic activity after their fold was identified to be similar to that of serine protease. The structure and its conformation are similar to the previously reported structures of SEC2. Though it was expected that the zinc ion may be leached out, as the histidines coordinating the zinc ion are expected to be protonated below pH 6.0, zinc is present at all pH values. The coordination distances to zinc increase with decreasing pH, with the distances being the least at pH 8.0. The results of automated model building using the ARP/wARP program for different data sets collected at various pH values are discussed.

Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi.

Outer surface protein C (OspC) is a major antigen on the surface of the Lyme disease spirochete, Borrelia burgdorferi, when it is being transmitted to humans. Crystal structures of OspC have been determined for strains HB19 and B31 to 1.8 and 2.5 A resolution, respectively. The three-dimensional structure is predominantly helical. This is in contrast to the structure of OspA, a major surface protein mainly present when spirochetes are residing in the midgut of unfed ticks, which is mostly beta-sheet. The surface of OspC that would project away from the spirochete's membrane has a region of strong negative electrostatic potential which may be involved in binding to positively charged host ligands. This feature is present only on OspCs from strains known to cause invasive human disease.

Crystallization and preliminary X-ray analysis of Borrelia burgdorferi outer surface protein C (OspC).

Single crystals of the outer surface protein C (OspC) from Borrelia burgdorferi HB19 have been obtained by the vapor-diffusion method. These crystals belong to space group P2(1), with unit-cell parameters a = 66.218, b = 46.113, c = 112.079 A, beta = 99.30 degrees, and diffract to at least 2.2 A resolution. Native data have been collected from flash-frozen crystals at the National Synchrotron facility of Brookhaven National Laboratory. There are two dimers per asymmetric unit, related by a non-crystallographic twofold axis and a pseudo-translational symmetry.

Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B.

Clostridium botulinum neurotoxins are among the most potent toxins to humans. The crystal structures of intact C. botulinum neurotoxin type B (BoNT/B) and its complex with sialyllactose, determined at 1. 8 and 2.6 A resolution, respectively, provide insight into its catalytic and binding sites. The position of the belt region in BoNT/B is different from that in BoNT/A; this observation presents interesting possibilities for designing specific inhibitors that could be used to block the activity of this neurotoxin. The structures of BoNT/B and its complex with sialyllactose provide a detailed description of the active site and a model for interactions between the toxin and its cell surface receptor. The latter may provide valuable information for recombinant vaccine development.

Crystallization and preliminary X-ray analysis of Clostridium botulinum neurotoxin type B.

Single crystals of Clostridium botulinum neurotoxin type B have been obtained by the vapor-diffusion method. These crystals belong to space group P2(1), with unit-cell parameters a = 76.08, b = 123.11, c = 95.86 A, beta = 113.03 degrees and diffract to at least 1.8 A resolution. Native data have been collected from flash-frozen crystals at the National Synchrotron facility of Brookhaven National Laboratory. These crystals often tend to be non-isomorphic.

The tertiary structure at 1.59 A resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti bainier.

We report the crystal structure at 1.59 A and the proposed amino acid sequence of an endo-1,4-beta-xylanase (PVX) from the thermophilic fungus Paecilomyces varioti Bainier (PvB), stable up to 75 degrees C. This fungus is attracting clinical attention as a pathogen causing post-surgical infections. Its xylanase, known as a skin-contact allergen, is the first protein from this fungus whose three-dimensional structure has been elucidated. The crystals of PVX conform to the space group P2(1)2(1)2(1 )with a=38.76 A, b=54.06 A and c=90.06 A. The structure was solved by molecular replacement techniques using polyalanine coordinates of the Thermomyces lanuginosus xylanase (PDB code 1YNA) and a careful model building based on the amino acid sequence known for two trypsin-digested peptide fragments (17 residues), the sequence and structural alignment of family-11 xylanases and electron density maps. The final refined model has 194 amino acid residues and 128 water molecules, with a crystallographic R-factor of 19.07 % and a free R-factor of 21.94 %. The structure belongs to an all-beta fold, with two curved beta-sheets, forming the cylindrical active-site cleft, and a lone alpha-helix, as present in other family-11 xylanases. We have carried out a quantitative comparison of the structure and sequence of the present thermophilic xylanase (PVX) with other available native structures of mesophiles and thermophiles, the first such detailed analysis to be carried out on family-11 xylanases. The analysis provides a basis for the rationalisation of the idea that the "hinge" region is made more compact in thermophiles by the addition of a disulphide bridge between Cys110 and Cys154 and a N-H.O hydrogen bond between Trp159 near the extremity of the lone alpha-helix and Trp138 on beta-strand B8. This work brings out explicitly the presence of the C-H.O and the C-H.pi type interactions in these enzymes. A complete description of structural stability of these enzymes needs to take account of these weaker interactions.

Crystal structure of the complex of bovine pancreatic phospholipase A2 with the inhibitor 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol,.

The structure of recombinant bovine pancreatic phospholipase A2 (PLA2) complexed with the competitive inhibitor 1-hexadecyl-3-(trifluoroethyl)-sn-glycero -2-phosphomethanol (hereafter MJ33), a phospholipid analogue without the sn-3 phosphodiester group, has been determined. The crystals are trigonal, space group P3121, a = b = 46.36 A and c = 102.56 A, and isomorphous to the recombinant PLA2 with one molecule in the asymmetric unit. The structure was refined using 8082 reflections between 8.0 and 1.91 A resolution to a final R-value of 18.4% [Rfree = 28.0%]. The model includes 957 protein atoms, 86 water molecules, one calcium ion, and 26 non-hydrogen atoms of the inhibitor MJ33. The overall tertiary fold of the complex is very similar to that of the inhibitor-free recombinant PLA2 with a root mean square deviation of 0.32 A for all the backbone atoms. The electron density of the surface loop residues 62-66 is clear and ordered, unlike the other trigonal bovine PLA2 structures done to date. This structural change could be responsible for the interfacial allosteric activation, which thermodynamically relates the enhanced binding of the substrate mimic to the active site of the enzyme. MJ33 is tightly bound in the active-site cleft, dislodging the equatorial coordinated calcium water (W5), the putative catalytic water W6, and the neighboring water W7. The axial coordinated calcium water is missing; thus the hexacoordinated calcium is a monocapped pentagonal pyramid. Although MJ33 is a sn-2 tetrahedral mimic, its phosphate binds to PLA2 differently from the sn-2 phosphonate analogue of phospholipids, another tetrahedral mimic. The knowledge of the active-site geometry of MJ33 would be useful in the design of more useful therapeutic agents for PLA2.

Crystallization and preliminary X-ray crystallographic studies of thermostable xylanase crystals isolated from Paecilomyces varioti.

A highly thermostable xylanase isolated from the thermophilic fungus Paecilomyces varioti has been crystallized by the vapour diffusion method. The isolation of this enzyme by crystallization directly from the culture filtrate projects this fungus as an important source for large-scale production of pure xylanase. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with the unit cell dimensions a = 38.48 A, b = 53.87 A and c = 90.23 A. Four molecules occupy a volume of 187,039.4 A3 along with 34% of solvent. The data collected with an area detector to the resolution of 2.7 A were used to calculate the unit cell parameters and Matthews' constant. The optical behaviour of the crystal was studied at different temperatures to understand its thermal stability.