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Chemical cross-linking coupled to mass spectrometry analysis has become a realistic alternative to the study of proteins structure and interactions, especially when these systems are not amenable to high-resolution techniques such as protein crystallography or nuclear magnetic resonance. One of the main bottlenecks of this approach relies on the detection of cross-linked peptides, as they are usually present in substoichiometric amounts in complex samples. It was shown that one of the main fragmentation pathways of disuccinimidyl suberate (DSS) cross-linked peptides yields diagnostic ions, whose structure is composed of a rearranged lysine side chain and the spacer arm of the linker. In this report, we demonstrate the feasibility of detecting these modified peptides based on a precursor ion scan in a quadrupole time-offlight (Q-TOF) instrument. It was shown that the fragmentation of nonmodified tryptic peptides hardly generates ions with the same nominal mass of the diagnostic ions, making the precursor ion scan very specific to N-hydroxysuccinimide (NHS)-based cross-linkers. Moreover, the experimental setup is the same as in the case of a regular cross-linking experiment, not demanding any additional experimental steps that would increase sample handling. The results obtained with protein samples allowed us to propose an algorithm that could be implemented in a software to process data from cross-linking experiments in an automated and high-thrnughput way. 10.1021/ac902051q

Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix. hydrophobic cavity | molecular movie | protein dynamics | time-resolved crystallography

Cold rolled Cu-24 wt% Ag composite and the effects of crystallographic structure and defect anisotropy and microstructure refinement on properties of the composite are analyzed. The curved lamellae seen in the rolling direction (RD) are attributed to the development of shear bands during rolling, where both the texture and shear bands are related to the anisotropy of the properties.

A new fragment-based approach, fragment hopping, is described for de novo inhibitor design by focusing on ligand diversity and isozyme selectivity. This new approach has incorporated the concept of early 'ADME/Tox' considerations and has provided a basic platform for medical chemistry-driven efforts.

The first crystallographic data for a ternary type II R67 DHFR.DHF.[NADP.sup.} (DHFR = dihyrofolate reductase) catalytic complex is presented. The structure provides insight into function of DHFR enzyme that lacks the active site carboxyl residue ubiquitous in type I DHFRs.

The crystallographic studies of human and certain bacterial forms of manganese superoxide dimutase (MnSOD) are reported. The studies demonstrate the structural differences in catalytic properties and contribution of residue 66 in catalytic and inhibition activity.

Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. Connexin26 (Cx26) is the second smallest member of the gap junction protein family, and mutations in Cx26 cause certain hereditary human diseases such as skin disorders and hearing loss. Here, we report the electron crystallographic structure of a human Cx26 mutant (M34A). Although crystallization trials used hemichannel preparations, the density map revealed that two hemichannels redocked at their extracellular surfaces into full intercellular channels. These orthorhombic crystals contained two sets of symmetry-related intercellular channels within three lipid bilayers. The 3D map shows a prominent density in the pore of each hemichannel. This density contacts the innermost helices of the surrounding connexin subunits at the bottom of the vestibule. The density map suggests that physical blocking may play an important role that underlies gap junction channel regulation. Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule. connexin channels | electron crystallography | intercellular communication | membrane protein structure | two-dimensional crystals

A simple general route of obtaining very stable octacoordinated non-oxovanadium(IV) complexes of the general formula V[L.sub.2] where [H.sub.2]L is a tetradentate ONNO donor showing crystallographic 4 symmetry and having a dodecahedral structure existing in a tetragonal space group P4n2 is presented.

The spectroscopic properties and high-power continuous-wave laser performance of Yb:Y[Al.sub.3][(B[O.sub.3]).sub.4] crystals using various samples cut along the crystallographic axes are analyzed. A very strong anisotropy in the spectroscopy is observed in the polarized absorption and emission cross section derived in the whole wavelength range of interest for laser operation.

Apoptosis is held in check by prosurvival proteins of the Bcl-2 family. The distantly related BH3-only proteins bind to and antagonize them, thereby promoting apoptosis. Whereas binding of the BH3-only protein Noxa to prosurvival Mcl-1 induces Mcl-1 degradation by the proteasome, binding of another BH3-only ligand, Bim, elevates Mcl-1 protein levels. We compared the three-dimensional structures of the complexes formed between BH3 peptides of both Bim and Noxa, and we show that a discrete C-terminal sequence of the Noxa BH3 is necessary to instigate Mcl-1 degradation. apoptosis | Bim | Noxa | crystallography

Transmission electron microscopy has shown extensive twinning in the low-temperature [(AgSb[Te.sub.2]).sub.15] [(GeTe).sub.85] thermoelectric material that has resulted from lattice strain during the dilation along the (111) crystallographic direction. The results of electrical conductivity measurements as a function of temperature on as-cast samples are discussed in terms of the twinning.

A study is conducted to find out the composition width in which the relaxor to ferroelectric transition occurs and the presence of any intermediate low symmetry phase formation during the crossover. No symmetry phase formation is found across the phase boundary and the phase change is described in terms of growth of short range to long range rhombohedral symmetry without consideration of any polarization rotation.

The enzymatic and crystallographic properties of the P168A variant of trypanosomal triosephosphate isomerase (TIM) are analyzed to investigate the importance of the fully conserved active site proline, Pro168, for the reaction mechanism of TIM. The results have shown that the presence of the proline side chain is required for transmitting the signal of ligand binding and loop 6 closure to the Glu167 side chain.

Monte Carlo simulations were performed within a model based on a set of distance-dependent effective potentials, which are used to describe the interactions between a pair of distinct amino acids. The model has a reduced number of variables and its main is the use of effective pair potentials (EPPs) extracted from experimental correlation functions that were obtained from the crystallographic data of the Protein Data Bank (PDB).

Hot-wall epitaxy and molecular-beam epitaxy are used for growing quaterthiophene thin films on the (010) cleavage face of potassium hydrogen phthalate, and the results are compared in terms of film properties and growth mode. A clear correlation between the morphology of the thin films and the crystallographic orientation is observed.

D-alanine:D-alanine ligase (DDI) is an essential enzyme in bacterial cell wall biosynthesis and an important target for developing new antibiotics. It catalyzes the formation of D-alanine:D-alanine dipeptide, sequentially by using one D-alanine and one ATP as substrates for the first-half reaction, and a second D-alanine substrate to complete the reaction. Some gain of function DDI mutants can use an alternate second substrate, causing resistance to vancomycin, one of the last lines of defense against life-threatening Gram-positive infections, Here, we report the crystal structure of Staphylococcus aureus DDI (StaDDI) and its cocrystal structures with 3-chloro-2,2-dimethyl-N-[4(trifluoromethyl)phenyl]propanamide (inhibitor 1) ([K.sub.i] = 4 [micro]M against StaDDI) and with ADP, one of the reaction products, at resolutions of 2.0, 2.2, and 2.6 [angstrom], respectively. The overall structure of StaDDI can be divided into three distinct domains. The inhibitor binds to a hydrophobic pocket at the interface of the first and the third domain. This inhibitor-binding pocket is adjacent to the first D-alanine substrate site but does not overlap with any substrate sites. An allosteric inhibition mechanism of StaDDI by this compound was proposed. The mechanism provides the basis for developing new antibiotics targeting D-alanine:D-alanine ligase. Because this compound only interacts with residues from the first D-alanine site, inhibitors with this binding mode potentially could overcome vancomycin resistance. antibiotic | crystallography | inhibition mechanism | kinetics | cell wall synthesis

The structures of two mutants of the site-specific recombinase, [gamma][delta] resolvase, that form activated tetramers have been determined. One, at 3.5-A resolution, forms a synaptic intermediate of resolvase that is covalently linked to two cleaved DNAs, whereas the other is of an unliganded structure determined at 2.1-A resolution. Comparisons of the four known tetrameric resolvase structures show that the subunits interact through the formation of a common core of four helices. The N-terminal halves of these helices superimpose well on each other, whereas the orientations of their C termini are more variable. The catalytic domains of resolvase in the unliganded structure are arranged asymmetrically, demonstrating that their positions can move substantially while preserving the four-helix core that forms the tetramer. These results suggest that the precleavage synaptic tetramer of [gamma][delta] resolvase, whose structure is not known, may be formed by a similar fourhelix core, but differ in the relative orientations of its catalytic and DNA-binding domains. site-specific recombination | serine recombinase | hyperactive mutant | cleaved complex | crystallography

The crystal habits of magnetosomes in two coccoid morphotypes of magnetotactic bacteria are studied by using the application of electron holography and high-resolution transmission electron microscopy. It is confirmed that the crystallographic positioning of successive crystals in the magnetsome chain appear to be under strict biological control.

Cyclopentadienyl (Cp)-alkali metal complexes 1-M and 2-M (M=Li, Na, K) were synthesized, and their structures and dynamic behaviors were investigated by means of X-ray crystallography, dynamic (super 13)C NMR, and DFT calculations. The X-ray crystallography results indicated that 1-Li, 1-Na, and 2-Na form monomeric contact ion pairs (CIP) with three THF molecules coordinated to the metal atom.

An examination of synthetic crystalline material by single crystal X-ray diffractometry reveals a unit-cell dimension that was previously ascribed to tincalconite. However, the best fit to the reflection data obtained at 291(2) K is obtained with composition [Na.sub.6][[[B.sub.4][O.sub.5][(OH).sub.4])].sub.3] * 8[H.sub.2]O. This formula is different from the [Na.sub.2][[B.sub.4][O.sub.5][(OH).sub.4])] * 3[H.sub.2]O previously assigned to tincalconite from an earlier crystallographic study. Additionally, our model fits best with non-crystallographic measurements and agrees with the analysis from a more recent low temperature crystallographic analysis. Tincalconite is best formulated as [Na.sub.6][[[B.sub.4][O.sub.5][(OH).sub.4])].sub.3] * 8[H.sub.2]O. It is trigonal, R32, a = 11.1402 (11), c = 21.207 (3) [Angstrom], Z = 3, R = 0.020 for 512 [F.sub.o] > 4[sigma] ([F.sub.o]) and 0.021 for all 529 data at 291(2) K.

Crystallographic studies of the hydrogenases from desulfovibrio gigas and desulfovibrio vulgaris Miyazaki have revealed heterodinuclear nickel-iron active centers in both enzymes. (super 17)O ENDOR is employed to demonstrate the presence of an oxygenic bridge between nickel and iron in the Dg Hase Ni-A dimetallic center and to further indicate that this bridge is present in Ni-B.

A multicomponent paraffin assembly and its structure are studied by using electron crystallography. The result reveals that a model accounting for the actual chain length distribution was more accurate than the best Gaussian distribution of methylene occupancies.

Changes in Fe coordination on reduction of the Fe-only hydrogenase active site from Desulfovibrio desulfuricans are observed.

Research describing studies of complexes of acetylide-alkoxide in solution and in solid state used in the process of manufacturing HIV reverse transcriptase inhibitor efavirenz is presented. The study is carried out bu use of NMR spectroscopy and X-ray crystallography.

Results support the singular boat-boat more compact conformation seen in the benzannelated eight-membered heterocyclic nefopam methochloride crystals. Data indicate that larger halide anions bromide and iodide increase the void distance of the adjacent ammonium cations to facilitate the twist-chair-chair more extended conformation.

Crystallographic analysis of native and mutant forms of cellulase Cel48F and substrate-inhibitor complex crystals reveal seven sub-sites located at two different positions in the enzyme tunnel. Data also indicate that in the processive endo-cellulase, the two sites exhibit different patterns of aromatic residue stacking whereas nonprocessive enzyme show one type of sugar.

Research discusses the direct relationships between metal and flavins in flavoenzymes in mediating intraprotein electron transfer. Results demonstrate that redox orbital of the 1,3-dimethylalloxazine (DMA) heterocyclic pi systems, formed by the complexation of DMA with copper, ruthenium, and tungsten, are related to the isoalloxazines of flavins as revealed by crystallographic studies.

Crystallographic and spectroscopic studies were undertaken on dynamic molecular recognition events in chiral porphyrins.

The preparation and study of unstable tris(bidentate) hexacoordinated phosphorus compounds is discussed. The use of NMR and X-ray crystallography is analyzed.

Results show that the chaperonin GroEL in combination with cochaperonin GroES mediates protein folding in Escherichia coli. The crystal structures of the bound peptide, apical domains of GroEL, and GroEL complexes are presented.

Tetrakis(pyridine)ruthenium trans complexes of phenylcyanamide ligands are discussed based on findings of electronic absorption spectroscopy, crystallography, and cyclic voltammmetry. A series of complexes were made, and one was characterized by crystallography for developing synthetic and interpretive methods needed to make conductive cross-linked polymer systems.

Results demonstrate that interaction between DNA and the very short patch repair protein leads to an intercalation of three aromatic amino acid residues to DNA thereby dislocating base pair staking. The data point out to the structural basis for TG mismatch recognition in bacteria.

Research was conducted to examine the dielectric and piezoelectric properties of BaTiO3 single crystals polarized along the (001) crystallographic axes as a function of temperature and dc bias. The low-temperature phases of BaTiO3 single crystals were poled along (001) to induce an engineered domain state. The derived piezoelectric properties were compared with values predicted by axis transformation for single-domain BaTiO3 crystals using the Devonshire theory.

Research was conducted to examine the importance of a loop structure near the active site of HIV-1 integrase by protein engineering and crystallographic analyses. Amino acid substitution resulted in mutants exhibiting impaired catalysis with the catalytic activity directly related to the degree of conformational flexibility.

Research was conducted to examine active site structural aspects of Humicola insolens cellobiohydrolase II Ce16A by crystallographic analysis. Results show that the enzyme has six binding sites, with aspartic acid 226 and 405 as catalytic sites, and oligosaccharide binding introducing conformational changes in the two active site loops.

A study was conducted to analyze the restacked nature of MoS2 and WS2 using electron crystallography. Experimental results indicated that MoS2 and WS2 support preferred orientation resulting the ab plane to be perpendicular to the electron beam. Findings also showed that the exfoliation/flocculation process would promote turbostratic materials or materials that are susceptible to stacking faults.