Expression, purification, crystallization and preliminary X-ray diffraction analysis of conjugated bile salt hydrolase from Bifidobacterium longum

Conjugated bile salt hydrolase (BSH) catalyses the hydrolysis of the amide bond that conjugates bile acids to glycine and to taurine. The BSH enzyme from Bifidobacterium longum was overexpressed in Escherichia coli BL21(DE3), purified and crystallized. Crystallization conditions were screened using the hanging-drop vapour-diffusion method. Crystal growth, with two distinct morphologies, was optimal in experiments carried out at 303 K. The crystals belong to the hexagonal system, space group P622 with unit-cell parameters a = b = 124.86, c = 219.03 Angstrom, and the trigonal space group P321, with unit-cell parameters a = b = 125.24, c = 117.03 Angstrom. The crystals diffracted X-rays to 2.5 Angstrom spacing. Structure determination using the multiple isomorphous replacement method is in progress

Expression, purification, crystallization and preliminary X-ray diffraction analysis of conjugated bile salt hydrolase from Bifidobacterium longum

Conjugated bile salt hydrolase (BSH) catalyses the hydrolysis of the amide bond that conjugates bile acids to glycine and to taurine. The BSH enzyme from Bifidobacterium longum was overexpressed in Escherichia coli BL21(DE3), purified and crystallized. Crystallization conditions were screened using the hanging-drop vapour-diffusion method. Crystal growth, with two distinct morphologies, was optimal in experiments carried out at 303 K. The crystals belong to the hexagonal system, space group P622 with unit-cell parameters a = b = 124.86, c = 219.03 Angstrom, and the trigonal space group P321, with unit-cell parameters a = b = 125.24, c = 117.03 Angstrom. The crystals diffracted X-rays to 2.5 Angstrom spacing. Structure determination using the multiple isomorphous replacement method is in progress

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

The crystallization of three catalytically inactive mutants of penicillin Vacylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide threedimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme

Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k(cat)/K-m values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K-s. Values of k(cat/)K(m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k(cat)/K-m for the CySB71 mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 X resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S-1 subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity

The structures of Micrococcus lysodeikticus catalase, its ferryl intermediate (compound II) and NADPH complex

The crystal structure of the bacterial catalase from Micrococcus lysodeikticus has been refined using the gene-derived sequence both at 0.88 Angstrom resolution using data recorded at 110 K and at 1.5 Angstrom resolution with room-temperature data. The atomic resolution structure has been refined with individual anisotropic atomic thermal parameters. This has revealed the geometry of the haem and surrounding protein, including many of the H atoms, with unprecedented accuracy and has characterized functionally important hydrogen-bond interactions in the active site. The positions of the H atoms are consistent with the enzymatic mechanism previously suggested for beef liver catalase. The structure reveals that a 25 Angstrom long channel leading to the haem is filled by partially occupied water molecules, suggesting an inherent facile access to the active site. In addition, the structures of the ferryl intermediate of the catalase, the so-called compound II, at 1.96 Angstrom resolution and the catalase complex with NADPH at 1.83 Angstrom resolution have been determined. Comparison of compound II and the resting state of the enzyme shows that the binding of the O atom to the iron (bond length 1.87 Angstrom) is associated with increased haem bending and is accompanied by a distal movement of the iron and the side chain of the proximal tyrosine. Finally, the structure of the NADPH complex shows that the cofactor is bound to the molecule in an equivalent position to that found in beef liver catalase, but that only the adenine part of NADPH is visible in the present structure

A GAIN in understanding autoproteolytic G protein‐coupled receptors and polycystic kidney disease proteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102153/1/embj201251.pd

Brain-Wide Analysis of the Supraspinal Connectome Reveals Anatomical Correlates to Functional Recovery After Spinal Injury

The supraspinal connectome is essential for normal behavior and homeostasis and consists of numerous sensory, motor, and autonomic projections from brain to spinal cord. Study of supraspinal control and its restoration after damage has focused mostly on a handful of major populations that carry motor commands, with only limited consideration of dozens more that provide autonomic or crucial motor modulation. Here, we assemble an experimental workflow to rapidly profile the entire supraspinal mesoconnectome in adult mice and disseminate the output in a web-based resource. Optimized viral labeling, 3D imaging, and registration to a mouse digital neuroanatomical atlas assigned tens of thousands of supraspinal neurons to 69 identified regions. We demonstrate the ability of this approach to clarify essential points of topographic mapping between spinal levels, measure population-specific sensitivity to spinal injury, and test the relationships between region-specific neuronal sparing and variability in functional recovery. This work will spur progress by broadening understanding of essential but understudied supraspinal populations

Evidence for CO depletion in the inner regions of gas-rich protoplanetary disks

We investigate the physical properties and spatial distribution of Carbon Monoxide (CO) gas in the disks around the Herbig Ae/Be stars HD 97048 and HD 100546. Using high-spectral-resolution 4.588-4.715 $\mu$m spectra containing fundamental CO emission taken with CRIRES on the VLT, we probe the circumstellar gas and model the kinematics of the emission lines. By using spectro-astrometry on the spatially resolved targets, we constrain the physical size of the emitting regions in the disks. We resolve, spectrally and spatially, the emission of the $^{13}$CO v(1-0) vibrational band and the $^{12}$CO $v=1-0, v=2-1, v=3-2$ and $v=4-3$ vibrational bands in both targets, as well as the $^{12}$CO $v=5-4$ band in HD 100546. Modeling of the CO emission with a homogeneous disk in Keplerian motion, yields a best fit with an inner and outer radius of the CO emitting region of 11 and $\geq$ 100 AU for HD 97048. HD 100546 is not fit well with our model, but we derive a lower limit on the inner radius of 8 AU. The fact that gaseous [OI] emission was previously detected in both targets at significantly smaller radii suggests that CO may be effectively destroyed at small radii in the surface layers of these disksComment: v2: Letter format has been changed to Paper format; Change in the focus of the paper towards CO depletion; Major changes in text; Change of title. Submitted to A&A, 14/10/2008. Accepted by A&A, 17/04/200

Fire-induced structural failure: the World Trade Center, New York

Fire investigation has generally concentrated on determination of the cause and origin of a fire. Methodologies developed for this purpose have thus focused on the dynamics of fire growth and investigation of its effect on different objects within the structure affected by the fire. It is unusual to see a fire investigation emphasising structural damage as a way to obtain information for fire reconstruction. The series of dramatic fire events that occurred on 11 September 2001 within the World Trade Center, New York complex have emphasised the need to introduce structural analysis as a companion to evaluation of a fire timeline. Only a combined analysis is capable of providing a complete reconstruction of the event and therefore a solid determination of causality. This paper presents a methodology to establish, by means of modern structural and fire analysis tools, the sequence of events leading to a structural failure. This analysis will be compared with classic cause and origin techniques, emphasising the importance of a comprehensive study. Specific structural features and fire conditions that lead to unique forms of failure will be discussed, establishing the complexity of linking fire, structure characteristics and failure mode. The collapse of buildings 1 and 2 of the World Trade Center will be used to illustrate different forms of failure and the fires that cause them