Morphology and crystal structure of a recombinant silk-like molecule,SLP4

Morphology and crystal structure of a recombinant silk-like molecule, SLP4, were studied. Wide angle x-ray scattering (WAXS) and electron diffraction revealed that SLP4 lyophilized powder and thin films were isomorphic with the silk I crystal structure. Transmission electron microscopy of SLP4 thin films demonstrated a morphology of flat, variable width, crystallites that may aggregate in an epitaxial manner. Theoretical diffraction patterns from silk I crystal structure models were critically compared with SLP4 WAXS data. The analysis concluded that while the crankshaft model is capable of describing details of the SLP4 structural data well, the out-of-register model does not explain the experimental results. In particular, the predicted intensities of the crystallographic reflections for the out-of-register model are inconsistent with the SLP4 WAXS data. © 1998 John Wiley & Sons, Inc. Biopoly 45: 307–321, 1998     

The glucan-chitin complex in Saccharomyces cerevisiae

Scar rings (SR) from scarless cells at the early stages of budding and mature bud scars from Saccharomyces cerevisiae isolated by both chemical and enzymic treatment of cell walls were observed by selected-area electron diffraction (SAED), X-ray diffraction and electron microscopy with simultaneous physico-chemical characterization (including molar mass, intrinsic viscosity and crystallite size) of -chitin and glucan. The SR, composed of glucan with strong 0.608; 0.397 and 0.293 nm X-ray reflections, was formed at the start of budding. The SAED patterns of -chitin both in the adjacent circular zone and in the parts of newly formed primary septum (PS), observed when the development of the PS started, did not differ from those of -chitin in the single mature bud scar. The bud scar consisted of -chitin, glucan and mannan and their content, as well as the crystallite size of chitin, depended on the mode of preparation.Non-Standard Abbreviations ACZ Adjacent circular zone - BS Birth scar - CFW Calcofluor white M2R new: Disodium salt of 4,4-bis-(4-anilino-bis-diethyl-amino-S-triazine-2-yl-amino)-2,2-stilbene-disulfonic acid - EDI Electron dense istmus - PS Primary septum - SAED Selected area electron diffraction - SEM Scanning electron microscopy - SR Scar ring - TEM Transmission electron microscopy - XRD X-ray diffraction     

Green synthesis of silver nanoparticles using Rhodobacter Sphaeroides

The use of microorganisms in the synthesis of nanoparticles emerges as an eco-friendly and exciting approach. In this study, silver nanoparticles were successfully synthesized from AgNO₃ by reduction of aqueous Ag⁺ ions with the cell filtrate of Rhodobacter sphaeroides. Nanoparticles were characterized by means of UV–vis absorption spectroscopy, X-Ray Diffraction (XRD), transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). Crystalline nature of the nanoparticles in the fcc structure are confirmed by the peaks in the XRD pattern corresponding to (111), (200), (220) and (311) planes, bright circular spots in the selected are a electron diffraction (SAED) and clear lattice fringes in the high-resolution TEM image. Also, the size of silver nanoparticles was controlled by the specific activity of nitrate reductase in the cell filtrate.     

The crystal structure of Bombyx mori silk fibroin at the air–water interface

A new crystalline polymorph of Bombyx mori silk, which forms at the air–water interface, has been characterized. A previous study found this structure to be trigonal, and to be distinctly different than the two previously observed silk crystal structures, silk I and silk II. This new structure was named silk III. Identification of this new silk polymorph was based on evidence from transmission electron microscopy and electron diffraction, coupled with molecular modeling. In the current paper, additional data enables us to refine our model of the silk III structure. Some single crystal electron diffraction patterns indicate a deviation in symmetry away from a perfect trigonal unit cell to monoclinic unit cell. The detailed shape of the powder diffraction peaks also supports a monoclinic cell. The monoclinic crystal structure has an nonprimitive unit cell incorporating a slightly distorted hexagonal packing of silk molecular helices. The chains each assume a threefold helical conformation, resulting in a crystal structure similar to that observed for polyglycine II, but with some additional sheet-like packing features common to the threefold helical crystalline forms of many glycine-rich polypeptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 705–717, 1997     

The Characterization of Intracellular and Extracellular Biomineralization Induced by Synechocystis sp. PCC6803 Cultured under Low Mg/Ca Ratios Conditions

In order to simulate the precipitation process of microbial limestone at the offshore of the ancient ocean, different calcites induced by Synechocystis sp. PCC6803 in culture media with low Mg/Ca ratios (0.01 M Ca²⁺, Mg/Ca = 0, 0.2, 0.4, 0.6) were investigated, and the characteristics of intracellular and extracellular biomineralization were described. Carbonic anhydrase activity of Synechocystis sp. PCC6803 in different culture medium was further detected. The ultrathin slices of Synechocystis sp. PCC6803 cells were analyzed by transmission electron microscope (TEM) and selected area electron diffraction (SAED). Then the precipitates were analyzed by polarizing microscope, scanning electron microscope (SEM), X-ray diffraction (XRD) and energy dispersive spectrometer (EDS). The results showed that the biomineralization precipitates of Synechocystis sp. PCC6803 under low Mg/Ca ratios were mainly calcites with different morphologies. The CA accelerated the pivotal rate limiting step of the calcite precipitation. It was also found that the morphology, microstructure, particle size, preferred orientation, crystallinity and cell volume of calcites changed gradually with the increasing Mg²⁺ concentrations. What is more important, it was found that Synechocystis sp. PCC6803 had the ability of intracellular biomineralization without crystal structure. The intracellular biomineralization product could be divided into two types. This study can provide some useful information for further understanding the characteristics and mechanisms of biomineralization and even the diagenetic environment research of microbial limestone.     

Biogenic synthesis of floral-shaped gold nanoparticles using a novel strain,Talaromyces flavus

A biogenic route was adopted towards the synthesis of gold nanoparticles using the extract of a novel strain, Talaromyces flavus. Reduction of chloroauric acid by the fungal extract resulted in the production of gold nanoparticle, which was further confirmed by the concordant results obtained from UV–visible spectroscopy, energy dispersive spectroscopy (EDS), and dynamic light scattering (DLS) analysis. Morphology and the crystal nature of the synthesized nanoparticles were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD) and selected area electron diffraction (SAED). A direct correlation was observed between nanoparticle formation and the concentration of reducing agent present in the fungal extract. The time-dependent kinetic study revealed that the bioreduction process follows an autocatalytic reaction. Crystalline, irregular, and mostly flower-shaped gold nanoparticles with a mean hydrodynamic radius of 38.54?±?10.34 nm were obtained. pH played a significant role on production of mono-dispersed nanoparticle. FTIR analysis partially deciphered the involvement of –NH₂, ?SH, and –CO groups as the probable molecules in the bio-reduction and stabilization process. Compared to the conventional methods, a time-resolved, green, and economically viable method for floral-shaped nanoparticle synthesis was developed.     

Impact of DAG stimulation on mineral synthesis, mineral structure and osteogenic differentiation of human cord blood stem cells

It remains unexplored in what way osteogenic stimulation with dexamethasone, ascorbic acid and β-glycerol phosphate (DAG) influences the process of mineralization, the composition and structure of the assembled mineral. Therefore, we analyzed and characterized biomineralization in DAG-stimulated and unstimulated 3D human unrestricted somatic stem cell (USSC) cultures. Microspheres were analyzed by histological staining, scanning electron microscopy (SEM), semi-quantitative energy-dispersive X-ray spectroscopy (EDX), quantitative wavelength-dispersive X-ray spectroscopy (WDX), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and Raman spectroscopy.Mineral material was detected by SEM and histological staining in both groups, and showed structural differences. DAG influenced the differentiation of USSCs and the formation, structure and composition of the assembled mineral. SEM showed that cells of the + DAG spheres exhibited morphological signs of osteoblast-like cells. EDX and WDX confirmed a Ca-P mineral in both groups. Overall, the mineral material found showed structural similarities to the mineral substance of bony material.     

Fruit extract capped colloidal silver nanoparticles and their application in reduction of methylene blue dye

Green synthesis of silver nanoparticles (AgNPs) has become a promising environmentally benign synthetic route in nanoscience and nanotechnology during recent years. In the present work, we have developed an environment-friendly and low-cost method for synthesis of silver nanoparticles from silver nitrate using aqueous fruit extract of Dillenia indica. The as-synthesized nanoparticles were characterized by UV-Vis spectrophotometer, transmission electron microscopy (TEM) and X-ray diffraction (XRD). FTIR study was performed to know the interaction of bio-molecules present in the fruit extract with AgNPs. The catalytic application of the as-synthesized AgNPs was demonstrated against degradation of methylene blue (MB) in aqueous system. The absorption spectra of colloidal suspension of AgNPs showed characteristic surface plasmon resonance (SPR) band centred at a wavelength of 416?nm. TEM image showed that the AgNPs were almost spherical in shape having an average diameter of 10.78?±?.48?nm. XRD pattern and selected area electron diffraction (SAED) pattern with bright spots signify the crystalline nature of nanoparticles. The fruit extract-capped AgNPs was highly stable and have showed the effective catalytic activity in reduction of MB dye.     

Validating Solution Ensembles from Molecular Dynamics Simulation by Wide-Angle X-ray Scattering Data

Wide-angle x-ray scattering (WAXS) experiments of biomolecules in solution have become increasingly popular because of technical advances in light sources and detectors. However, the structural interpretation of WAXS profiles is problematic, partly because accurate calculations of WAXS profiles from structural models have remained challenging. In this work, we present the calculation of WAXS profiles from explicit-solvent molecular dynamics (MD) simulations of five different proteins. Using only a single fitting parameter that accounts for experimental uncertainties because of the buffer subtraction and dark currents, we find excellent agreement to experimental profiles both at small and wide angles. Because explicit solvation eliminates free parameters associated with the solvation layer or the excluded solvent, which would require fitting to experimental data, we minimize the risk of overfitting. We further find that the influence from water models and protein force fields on calculated profiles are insignificant up to q ≈ 15 nm^?1. Using a series of simulations that allow increasing flexibility of the proteins, we show that incorporating thermal fluctuations into the calculations significantly improves agreement with experimental data, demonstrating the importance of protein dynamics in the interpretation of WAXS profiles. In addition, free MD simulations up to one microsecond suggest that the calculated profiles are highly sensitive with respect to minor conformational rearrangements of proteins, such as an increased flexibility of a loop or an increase of the radius of gyration by  <  1%. The present study suggests that quantitative comparison between MD simulations and experimental WAXS profiles emerges as an accurate tool to validate solution ensembles of biomolecules.     

Studies on Xylanase from Microorganisms

X-Ray diffraction analysis of the α-cyclodextrin complexes with a number of organic guest molecules were carried out. Several different kinds of the X-ray diffraction patterns were obtained. It was found that different guest molecules enclosed within the void of the dextrin cause large changes in the diffraction patterns of the complexes. However, most of the diffraction patterns could be reasonably interpreted in terms of the hexagonal unit cells with minor differences in the unit cell dimensions ranging a = b = 27.0 ~ 27.8 Å and c = 14.7 ~ 16.7 Å. The crystal structure of the complexes could be accounted for by a closest packing of channel cylinders that are made by coaxial alignments of the dextrin molecules and the cage structure in the crystal, in which the dextrin molecules align non-coaxially, may not be plausible.     

Nucleic Acid Conformation: Crystal Structure of a Naturally Occurring Dinucleoside Phosphate (UpA)

THEORIES of the molecular structure of nucleic acids have so far been based on evidence from the crystal structures of monomeric units such as nucleosides and mononucleotides, the interpretation of diffraction patterns of oriented nucleic acid fibres and molecular model building^1–6. Such approaches can help to suggest structures of periodic molecules such as helices, but they are insufficient for predicting and understanding nonrepetitive structures such as the loops in transfer RNA (tRNA), presumably associated with many of the functions of tRNA. To understand the geometry of nucleic acids and possible constraints on their conformation, it is therefore essential to know the detailed conformation of the sugar residues and the conformational relationship between the sugar residue, the base and the phosphate group^7–9. The simplest molecule which contains this information is a 3´5´-dinucleoside phosphate. We now report the structure of uridine-3´,5´-adenosine phosphate (UpA). This is the first naturally occurring dinucleoside phosphate whose crystal structure has been determined by X-ray diffraction. The only other dinucleoside phosphate with known crystal structure is adenosine-2´,5´-uridine phosphate¹⁰, but it does not have the naturally occurring 3´5´ sugar phosphate linkage.     

Equivalence of the projected structure of thin catalase crystals preserved for electron microscopy by negative stain,glucose or embedding in the presence of tannic acid

Thin crystals of beef liver catalase have been examined by electron microscopy following various preservation procedures. In the first part of this investigation, micrographs of three principal projections were obtained from thin sections of micro-crystals embedded in the presence of tannic acid. Computer reconstructions confirmed the space group assignment of P2₁2₁2₁ and permitted the packing arrangement of the catalase tetramers to be deduced to a resolution of about 20 Å. These results corroborate the packing model for this crystal form proposed by Unwin (1975) on the basis of molecular modeling of one projection. In the second part of this investigation, the projected structures of the thin crystals in various preserving media were compared. The negative contrasting of crystals embedded in the presence of tannic acid was confirmed by direct comparison with nonembedded, negatively stained thin platelet crystals. In addition, good agreement at 20 Å resolution was observed between the structure of negatively stained crystals and the structure of crystal platelets preserved in glucose and examined by lowdose methods, while moderate agreement was established with the published data of Taylor (1978) for crystals embedded in thin ice films. Tannic acid alone was also found to serve as a suitable medium for preserving catalase crystals to a resolution of 3.7 Å as judged by electron diffraction. Overall, we demonstrate that projections obtained from thin sections of catalase crystals embedded in the presence of tannic acid can provide a reliable, negatively contrasted representation of the protein structure to 20 Å resolution. Examination of sectioned crystals could thus provide a useful adjunct to X-ray crystallographic studies of protein crystals and three-dimensional reconstruction of crystal thin sections should ultimately be possible.     

WAXS studies of the structural diversity of hemoglobin in solution

Specific ligation states of hemoglobin are, when crystallized, capable of taking on multiple quaternary structures. The relationship between these structures, captured in crystal lattices, and hemoglobin structure in solution remains uncertain. Wide-angle X-ray solution scattering (WAXS) is a sensitive probe of protein structure in solution that can distinguish among similar structures and has the potential to contribute to these issues. We used WAXS to assess the relationships among the structures of human and bovine hemoglobins in different liganded forms in solution. WAXS data readily distinguished among the various forms of hemoglobins. WAXS patterns confirm some of the relationships among hemoglobin structures that have been defined through crystallography and NMR and extend others. For instance, methemoglobin A in solution is, as expected, nearly indistinguishable from HbCO A. Interestingly, for bovine hemoglobin, the differences between deoxy-Hb, methemoglobin and HbCO are smaller than the corresponding differences in human hemoglobin. WAXS data were also used to assess the spatial extent of structural fluctuations of various hemoglobins in solution. Dynamics has been implicated in allosteric control of hemoglobin, and increased dynamics has been associated with lowered oxygen affinity. Consistent with that notion, WAXS patterns indicate that deoxy-Hb A exhibits substantially larger structural fluctuations than HbCO A. Comparisons between the observed WAXS patterns and those predicted on the basis of atomic coordinate sets suggest that the structures of Hb in different liganded forms exhibit clear differences from known crystal structures.     

Biogenic Synthesis of Selenium Nanoparticles and Their Effect on As(III)-Induced Toxicity on Human Lymphocytes

A bioreductive capacity of a plant, Terminalia arjuna leaf extract, was utilized for preparation of selenium nanoparticles. The leaf extract worked as good capping as well as stabilizing agent and facilitated the formation of stable colloidal nanoparticles. Resulting nanoparticles were characterized using UV–Vis spectrophotometer, transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDAX), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction analysis (XRD), respectively. The colloidal solution showed the absorption maximum at 390 nm while TEM and selected area electron diffraction (SAED) indicated the formation of polydispersed, crystalline selenium nanoparticles of size raging from 10 to 80 nm. FT-IR analysis suggested the involvement of O–H, N–H, C=O, and C–O functional group of the leaf extract in particle formation while EDAX analysis indicated the presence of selenium in synthesized nanoparticles. The effect of nanoparticles on human lymphocytes treated with arsenite, As(III), has been studied. Studies on cell viability using MTT assay and DNA damage using comet assay revealed that synthesized selenium nanoparticles showed protective effect against As(III)-induced cell death and DNA damage. Chronic ingestion of arsenic infested groundwater, and prevalence of arsenicosis is a serious public health issue. The synthesized benign nanoselenium can be a promising agent to check the chronic toxicity caused due to arsenic exposure.     

Structural characterization of ordered domains in a hydrophobic membrane protein

A delipidized proteolipid protein fraction was purified from organic solvent extracts of bovine cerebral cortex and studied by means of diffraction, electron microscopic, and ir techniques. Special use was made of an electron diffraction procedure which minimized the electron damage to the biological specimens. The ir spectroscopy of the apoprotein fraction indicated the presence of polypeptides in extended β-conformation, possibly in the antiparallel mode of packing. Electron microscopy of the fraction, negatively stained in organic media, made apparent the presence of both ordered and amorphous material. Only the former, characterized by repeating units of about 40–45 Å in diameter and varying length, produced diffraction patterns in the selected area mode exhibiting a highly undistorted lattice. The two-dimensional cell parameters of the protein fraction were a = 4.79 Å, b = 7.20 Å, and γ = 90°. The plane group symmetry, corresponding to the systematic absences, was p 2gg, consistent with the β-pleated sheet structure of simple polypeptides.     

Characterization of protein fold by wide-angle X-ray solution scattering

Wide-angle X-ray solution scattering (WAXS) patterns contain substantial information about the three-dimensional structure of a protein. Although WAXS data have far less information than is required for determination of a full three-dimensional structure, the actual amount of information contained in a WAXS pattern has not been carefully quantified. Here we carry out an analysis of the amount of information that can be extracted from a WAXS pattern and demonstrate that it is adequate to estimate the secondary-structure content of a protein and to strongly limit its possible tertiary structures. WAXS patterns computed from the atomic coordinates of a set of 498 protein domains representing all of known fold space were used as the basis for constructing a multidimensional space of all corresponding WAXS patterns (‘WAXS space’). Within WAXS space, each scattering pattern is represented by a single vector. A principal components analysis was carried out to identify those directions in WAXS space that provide the greatest discrimination among patterns. The number of dimensions that provide significant discrimination among protein folds agrees well with the number of independent parameters estimated from a naïve Shannon sampling theorem approach. Estimates of the relative abundances of secondary structures were made using training/test sets derived from this data set. The average error in the estimate of α-helical content was 11%, and of β-sheet content was 9%. The distribution of proteins that are members of the four structure classes, α, β, α/β and α+β, are well separated in WAXS space when data extending to a spacing of 2.2 Å are used. Quantification of the information embedded within a WAXS pattern indicates that these data can be used as a powerful constraint in homology modeling of protein structures.     

The effect of temperature on antibacterial activity of biosynthesized silver nanoparticles

The purpose of this study was the evaluation of two different temperatures on antibacterial activity of the biosynthesized silver nanoparticles. 38 silver nanoparticles-producing bacteria were isolated from soil and identified. Biosynthesis of silver nanoparticles by these bacteria was verified through visible light spectrophotometry. Two strains were relatively active for production of silver nanoparticles. These strains were subjected for molecular identification and recognized as Bacillus sp. and Acinetobacter schindleri. In the present study, the effect of temperatures was evaluated on structure and antimicrobial properties of the silver nanoparrticles by transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis and antimicrobial Agar well diffusion methods. The silver nanoparticles showed antibacterial activity against all the pathogenic bacteria; however, this property was lost after treatment of the silver nanoparticles by high temperatures (100 and 300 °C). TEM images showed that the average sizes of heated silver nanoparticles were >100 nm. However, these were <100 nm for non-heated silver nanoparticles. Although, XRD patterns showed the crystalline structure of heated silver nanoparticles, their antibacterial activities were less. This was possible because of the sizes and accordingly less penetration of the particles into the bacterial cells. In addition, elimination of the capping agents by heat might be considered another reason.     

The dynamics of camphor in the cytochrome P450 CYP101D2

The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate‐free) and camphor‐soaked forms have open conformations. Furthermore, two other potential camphor‐binding sites were also identified from electron densities in the camphor‐soaked structure, one being located in the access channel and the other in a cavity on the surface near the F‐helix side of the F‐G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor‐bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor‐bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit.     

Electron diffraction of Valonia cellulose. A quantitative interpretation

The crystal structure of native cellulose (Valonia) has been analyzed by electron diffraction. Possible models for the structure were refined by rigid-body least squares methods, which incorporated parameters defining the preferred orientation of the fibrils around their long axes in the cell wall lamellae. The structure was found to consist of an array of chains having the same sense (i.e., parallel), with packing parameters similar to those recently determined by X-ray diffraction. The eight-chain unit cell could be approximated adequately by a two-chain monoclinic unit cell with dimensions a = 8.18 Å, b = 7.84 Å, c = 10.38 Å (fiber axis), and γ = 97.04°; the space group is P2₁.     

Crystal structure of Escherichia coli TEM1 β-lactamase at 1.8 Å resolution

The X-ray structure of Escherichia coli TEM1β-lactamase has been refined to a crystallorgphic R-factor of 16.4% for 22,510 reflections between 5.0 and 1.8 Å resolution; 199 water molecules and 1 sulphate ion were included in refinement. Except for the tips of a few solvent-exposed side chains, all protein atoms have clear electron density and refined to an average atomic temperature factor of 11 Ų. The estimated coordinates error is 0.17 Å. The substrate binding site is located at the interface of the two domains of the protein and contains 4 water molecules and the sulphate anion. One of these solvent molecules is found at hydrogen bond distance from S70 and E166. S70 and S130 are hydrogen bonded to K73 and K234, respectively. It was found that the E. coli TEM1 and Staphylococcus aureus PC1 β-lactamases crystal structures differ in the relative orientations of the two domains composing the enzymes, which result in a narrowed substrate binding cavity in the TEM1 enzyme. Local but significant differences in the vicinity of this site may explain the occurrence of TEM1 natural mutants with extended substrate specificities. © 1993 Wiley-Liss, Inc.