Atomic Resolution Structure of a Protein Prepared by Non-Enzymatic His-Tag Removal. Crystallographic and NMR Study of GmSPI-2 Inhibitor

Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and R _free of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β₂αβ fold characteristic for Kazal-family serine proteinase inhibitors.     

A dihydrocinnamoyl neoflavanoid from pityrogramma calomelanos

The structure of a neoflavanoid isolated from Pityrogramma calomelanos was confirmed by an X-ray diffraction study.     

Letter to the editor: The polyuridylic acid complex with polyamines: an x-ray fiber diffraction observation.

The poly(U)-spermine complex has been studied by X-ray fiber diffraction techniques. The X-ray pattern is generally similar to that of A RNA or of the A form of DNA, suggesting a double-helical structure with strands of opposite polarity. Models with 9 to 11 residues per turn of the helix are considered.     

Lasianthin,a neo-clerodane diterpenoid from Salvia lasiantha

From the aerial part of Salvia lasiantha a neo-clerodane diterpenoid, lasianthin, was isolated. The structure of this compound was established by spectroscopic means and X-ray diffraction analysis.     

Phospholipid arrangements in multilayers and artificial membranes: quantitative analysis of the X-ray diffraction data from a multilayer of 1,2-dimyristoyl-DL-phosphatidylethanolamine.

The X-ray diffraction data from a multilayer of 1,2-dimyristoyl-dl-phosphatidylethanolamine is interpreted quantitatively (R₁ = 0.20) from a single crystal analysis of the molecular structure of 1,2-dilauroyl-dl-phosphatidylethanolamine.     

The fine structure of silk fibroin

The fine structure of Bombyx mori silk fibroin was investigated by electron microscopy and X-ray diffraction techniques. Examination of silk fibers fragmented with ultrasonic radiation and negatively stained revealed the presence of ribbon-like filaments of well-defined lateral dimensions. Analysis of the breadths of the equatorial reflections in the X-ray diffraction pattern of fibroin yielded similar dimensions for the lateral extent of the crystallites. It is concluded that the crystalline material in B. mori silk fibroin is in the form of ribbon-like filaments of considerable length parallel to the fiber axis and of lateral dimensions approximately 20 x 60 A.     

Synthesis, crystal structure, magnetic properties and EPR studies of Cu/Hg bimetallic thiocyanato-bridged coordination polymer

The new Cu/Hg bimetallic thiocyanato-bridged coordination polymer, [Cu(2-bzpy)Hg(SCN)₄]_n, has been synthesized and characterized by elemental analysis, magnetic measurements, EPR, and X-ray crystallography. X-ray diffraction analysis reveals that the title complex structure creates 3D network.     

Synthesis and aggregation phenomena of multifunctional Schiff bases and Ni(II) complexes: an X-ray investigation

Multifunctional Schiff base ligands Lⁿ, namely the tetradentate N,N^′-bis[2-hydroxy-5-(azopyridine)benzylidene]propylendiamine and the bidentate N-dodecyl-5-(azopyridine)salicylaldimine, both containing a flexible azo spacer, a metallation site and a terminal pyridine group, were synthetised and fully characterised. Mesogenic structures, analysed by polarised optical microscopy, DSC and powder X-ray diffraction, were obtained from self-assembly of the mono or bifunctional hydrogen-bond acceptors Lⁿ with carboxylic acid donors. Ni(II) mono and bis-chelate, four- and six-coordinated, Lⁿ derivatives were synthetised. The octahedral structure of the [Ni(py)₂(L²)₂] complex was confirmed by single crystal X-ray analysis. H-bonded self-assembly of Ni(II) complexes and carboxylic acids results in the formation of supramolecular networks whose structure and thermal stability were studied by DSC and powder X-ray diffraction analysis at variable temperatures.     

Mayoside,an oxanthrone from Picramnia hirsuta

A new oxanthrone named mayoside was isolated from the roots of Picramnia hirsuta and the structure established by X-ray diffraction.     

The agarose double helix and its function in agarose gel structure

Agarose and eight different derivatives carrying O-methyl, O-sulphate, O-hydroxyethyl or O-carboxyethylidene substituents in various positions were studied by optical rotation, X-ray diffraction and computerised molecular model building methods. All samples showed essentially the same order-disorder transition during gel-sol interconversion. In addition, all the samples that could be made into oriented films or fibres gave X-ray diffraction diagrams corresponding to a common molecular structure. A double helix model for this structure is proposed that has the 0.95 nm axial periodicity observed and a calculated cylindrically averaged Fourier transform in good agreement with the observed (continuous) layer line intensities. Each chain in the double helix forms a lefthanded 3-fold helix of pitch 1.90 nm and is translated axially relative to its partner by exactly half this distance. This model accounts for the sign and magnitude of the optical rotation shift that accompanies the sol-gel transitions and is sterically accessible to each of the various substituted forms. The relationship between agarose gel properties and the double helix is discussed and the structure compared with i-carrageenan.     

Salvifarin,x-ray structure determination of a cis neo-clerodane diterpenoid from salvia farinacea

The structure and absolute configuration of salvifarin, a neo-clerodane diterpenoid isolated from Salvia farinacea, have been established by X-ray diffraction analysis. This result modifies the structure previously assigned to this compound.     

Fibre and molecular structure of thymidylyl-3′,5′-deoxyadenosine

X-ray diffraction and molecular model building studies of an ordered structure of thymidylyl-3′,5′-deoxyadenosine which gives fibre-type diffraction patterns, are consistent with a seven-residues per turn, left-handed structure in which the adenine of one molecule and the thymine of the next are linked together by Hoogsteen type of hydrogen bonds. The structure thus resembles a macromolecule in which units are linked together by hydrogen bonds and stabilized by base stocking. Both nucleosides in the basic molecule are in the anti conformation and both sugar rings have C3′-endo puckers. The C5′-05′ bond of the deoxyadenosine is trans relative to C4′-C3′ and the conformations about the P-03′ and P-05′ bond are gauche^?, trans.     

X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.     

Sesquiterpene lactones from Montanoa gigas and the crystal structure of gigantanolide a

Chemical analysis of Montanoa gigas afforded in addition to known kaurane-derivatives three new 12,6-cis-germacrolides named gigantanolides, A, B and C. The structures of the new compounds were established by spectral methods mainly ¹H NMR. The structure of gigantanolide A was confirmed by single crystal X-ray diffraction.     

A revised structure for the diterpene rosmanol

The diterpene rosmanol, previously isolated from Rosmarinus officinalis, has been isolated from the flowers of Salvia canariensis and its structure revised as 7α,11,12-trihydroxyabieta-8,11,13-trien-20-oic acid 20,6-lactone, on the basis of chemical evidence and an X-ray diffraction analysis.     

Novel method to observe subtle structural modulation of stratum corneum on applying chemical agents

In the development of functional chemicals such as percutaneous penetration enhancers and cosmetics, the structural evidence at the molecular level in stratum corneum (SC) is highly desirable. We developed a method to observe a minute structural change of intercellular lipid matrix and corneocytes on applying the chemicals to the SC using synchrotron X-ray diffraction technique. The performance of the present method was demonstrated by applying typical chemicals, chloroform/methanol mixture, hydrophilic ethanol and hydrophobic d-limonene. From the small- and wide-angle X-ray diffraction we obtained the following results: on applying chloroform/methanol mixture the intercellular lipids were extracted markedly, on applying ethanol the intercellular lipid structure was slightly disrupted, ethanol molecules were taken into the corneocytes and in addition the pools of ethanol seem to be formed in the hydrophilic region of the intercellular lipid matrix in the SC, and on applying d-limonene the repeat distance of the long lamellar structure increased by incorporating d-limonene molecules, the intercellular lipid structure was slightly disrupted, and the pools of d-limonene were formed in the hydrophobic region of the intercellular lipid matrix in the SC.     

Structure of Lamprey Haemoglobin

The crystal structure of haemoglobin V from the sea lamprey Petromyzon marinushas been analysed by X-ray diffraction. This report describes the structure of the cyanide complex of methaemo-globin crystals, type D₂.     

Synthesis,Structural and Antioxidant Studies of Some Novel N-Ethyl Phthalimide Esters

A series of N-ethyl phthalimide esters 4(a-n) were synthesized and characterized by spectroscopic studies. Further, the molecular structure of majority of compounds were analysed by single crystal X-ray diffraction studies. The X-ray analysis revealed the importance of substituents on the crystal stability and molecular packing. All the synthesized compounds were tested for in vitro antioxidant activity by DPPH radical scavenging, FRAP and CUPRAC methods. Few of them have shown good antioxidant activity.     

Synthetic versus analytic approaches to protein and DNA structure determination

The structures of protein and DNA were discovered primarily by means of synthesizing component-level information about bond types, lengths, and angles, rather than analyzing X-ray diffraction photographs of these molecules. In this paper, I consider the synthetic and analytic approaches to exemplify alternative heuristics for approaching mid-twentieth-century macromolecular structure determination. I argue that the former was, all else being equal, likeliest to generate the correct structure in the shortest period of time. I begin by characterizing problem solving in these cases as proceeding via the elimination of candidate structures through the successive application of component-level information and interpretations of X-ray diffraction photographs, each of which serves as a kind of constraint on structure. Then, I argue that although each kind of constraint enables the elimination of a considerable proportion of candidate structures, component-level constraints are significantly more likely to do so correctly. Thus, considering them before X-ray diffraction photographs is a better heuristic than one that reverses this order. Because the synthetic approach that resulted in the determination of the protein and DNA structures exemplifies such a heuristic, its use can help account for these discoveries.     

Structure and phase behavior of O-stearoylethanolamine: A combined calorimetric, spectroscopic and X-ray diffraction study

Recent studies show that O-acylethanolamines (OAEs), structural isomers of the putative stress-fighting lipids, namely N-acylethanolamines (NAEs), can be derived from NAEs and are present in biological membranes under physiological conditions. In view of this, we have synthesized O-stearoylethanolamine (OSEA) as a representative OAE and investigated its phase behavior and crystal structure. The thermotropic phase transitions of OSEA dispersed in water and in 150 mM NaCl were characterized using calorimetric, spectroscopic, turbidimetric and X-ray diffraction studies. These studies have revealed that when dispersed in water OSEA undergoes a cooperative phase transition centered at 53.8 °C from an ordered gel phase to a micellar structure whereas in presence of 150 mM NaCl the transition temperature increases to 55.8 °C and most likely the bilayer structure is retained above the phase transition. O-Stearoylethanolamine crystallized in the orthorhombic space group P2₁2₁2₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that OSEA molecules adopt a linear structure with all-trans conformation in the acyl chain region. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. These studies are relevant to understanding the role of salt on the phase properties of this new class of lipids.