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PubMed1999

Detection of vascular haemodynamics through a high-speed velocity profiler.

OBJECTIVE: This paper aims at demonstrating that ultrasound Doppler multigate spectral analysis performed with advanced equipment may provide detailed and significant haemodynamic information. METHODS: A novel multigate system was recently introduced and shown capable of performing real-time spectral analysis of Doppler data from 64 resolution cells located at different depths from the transducer. The system extends the typical capabilities of conventional Pulsed Wave (PW) equipment by displaying the full spectral content of Doppler signals over an ultrasound scan line rather than in a single resolution cell. In cases where it is appropriate to display the available information in a simpler form, parameters such as the maximum frequency can be extracted from each spectrum, by using conventional or advanced image processing methods. RESULTS: In-vitro experiments show that the multigate system can perform velocity measurements with good accuracy and precision. Examples of in vivo profiles detected from carotid, femoral and radial arteries are presented. In particular, the first results obtained from the aorta are shown. CONCLUSIONS: Blood flow behavior can be accurately investigated using a real-time multigate system which extends Doppler spectral analysis to a whole scan line.

Aorta↗
PubMed2000

Crystal structure of the molybdenum cofactor biosynthesis protein MobA from Escherichia coli at near-atomic resolution.

BACKGROUND: All mononuclear molybdoenzymes bind molybdenum in a complex with an organic cofactor termed molybdopterin (MPT). In many bacteria, including Escherichia coli, molybdopterin can be further modified by attachment of a GMP group to the terminal phosphate of molybdopterin to form molybdopterin guanine dinucleotide (MGD). This modification reaction is required for the functioning of many bacterial molybdoenzymes, including the nitrate reductases, dimethylsulfoxide (DMSO) and trimethylamine-N-oxide (TMAO) reductases, and formate dehydrogenases. The GMP attachment step is catalyzed by the cellular enzyme MobA. RESULTS: The crystal structure of the 21.6 kDa E. coli MobA has been determined by MAD phasing with selenomethionine-substituted protein and subsequently refined at 1. 35 A resolution against native data. The structure consists of a central, predominantly parallel beta sheet sandwiched between two layers of alpha helices and resembles the dinucleotide binding Rossmann fold. One face of the molecule bears a wide depression that is lined by a number of strictly conserved residues, and this feature suggests that this is where substrate binding and catalysis take place. CONCLUSIONS: Through comparisons with a number of structural homologs, we have assigned plausible functions to several of the residues that line the substrate binding pocket. The enzymatic mechanism probably proceeds via a nucleophilic attack by MPT on the GMP donor, most likely GTP, to produce MGD and pyrophosphate. By analogy with related enzymes, this process is likely to require magnesium ions.

Amino Acid Sequence↗
PubMed1996

The structure of simian virus 40 refined at 3.1 A resolution.

BACKGROUND: The structure of simian virus 40 (SV40), previously determined at 3.8 degree resolution, shows how its pentameric VP1 assembly units are tied together by extended C-terminal arms. In order to define more precisely the possible assembly mechanisms, we have refined the structure at 3.1 degree resolution. RESULTS: New data from a high-intensity synchrotron source have been used for phase extension by electron-density averaging and refinement, exploiting only the strict 5-fold non-crystallographic symmetry for the real-space averaging steps. The accurate model enables us to study important structural features of the virus particle in detail. The remarkably invariant core of the VP1 pentamer bears the docking sites for the C-terminal arms from other pentamers. These contacts are the principal way in which pentameric assembly units are linked together in the capsid. Only at the interface between five-coordinated and six-coordinated pentamers do the pentamer cores appear to interact strongly. There are two cation-binding sites per VP1 monomer, seen in a soaking experiment with gadolinium nitrate. These sites are quite close to each other at the interfaces between pentamers. CONCLUSION: We propose that the contact between five-coordinated and six-coordinated pentamers may help to generate a six-pentamer nucleus, with which further pentamers can assemble to generate the complete particle. Calcium ions probably stabilize the structure of the assembled particle, rather than direct its assembly.

Amino Acid Sequence↗
PubMed1998

Zinc enzymes.

The number of zinc enzymes for which detailed structural and mechanistic data, including high resolution crystal structures, are available is increasing rapidly. The new findings continue to support the conclusion that the majority of zinc enzymes catalyze hydrolysis or closely related transfer reactions. In a protein environment, tetrahedral or 5-coordinate Zn2+ is ideally suited to activate a coordinated water (frequently a Zn2+--OH) as a nucleophile attacking the carbonyl carbon of a peptide bond, the carbon of carbon dioxide or the phosphorus of a phosphate ester. Protein-bound Zn2+ can function catalytically by forming mixed complexes with the substrate, either by expanding its coordination sphere or by exchanging a ligand. Formation of protein-Zn2+-substrate bonds can position the substrate or polarize its electron distribution to facilitate further steps in the reaction.

Alkaline Phosphatase↗
PubMedDate not supplied

Binocular depth perception following early experience with interocular torsional disparity.

The relationship between the behavioral and physiological consequences of rearing with optically induced cyclotropia was assessed. Beginning at the age of 4 weeks, kittens wore goggles that rotated the visual field in opposite directions in each eye for several hours each day over a period of several weeks. The amounts of interocular rotation were 0 deg (control), 16 deg, and 32 deg. Subsequently, they were tested to determine their monocular and binocular depth thresholds and, in some cases, visual acuity. In several kittens recordings were also made from the visual cortex. Binocular performance of all kittens in the 0-deg condition and three out of six kittens in the 16-deg condition was comparable to, although slightly lower than, that of normally reared kittens. In contrast, none of the 32-deg kittens showed any evidence of the binocular superiority that would suggest the presence of stereopsis. Extracellular unit recordings from the visual cortex confirmed our earlier results with goggle-reared kittens. In 16-deg kittens, the distribution of the cells' preferred interocular disparities (IOD) in receptive-field orientation showed a compensating shift so that the mean matched the experienced rotational disparity. In the 32-deg kittens, binocularity was greatly disrupted and there was no compensatory shift in the IOD distribution. Two 32-deg kittens were afforded 3 years of subsequent normal visual experience. Both the behavioral and the physiological findings were unaffected by normal visual exposure in adulthood. Control measurements of acuity indicated that any deficits in depth perception were not due to reduced spatial-resolution abilities. The data indicate that the kitten visual system is able to maintain functional binocularity sufficient to subserve a moderate level of stereoacuity with interocular rotations of up to at least 16 deg.

Animals↗
PubMed2006

Cross-correlation algorithm for calculation of peptide molecular weight from tandem mass spectra.

We have developed an approach to identify the molecular weight of a peptide ion directly from its corresponding tandem mass spectrum using a cross-correlation function. We have shown that the monoisotopic molecular weight can be calculated for approximately 90% of tandem mass spectra identified from tryptic digests of complex protein mixtures. The accuracy of the calculated monoisotopic masses was dependent on the resolution and mass accuracy of the spectra analyzed, but was typically <0.25 amu for linear ion trap mass spectra. The ability to calculate accurate monoisotopic molecular weights for low-resolution ion trap data should significantly improve both the speed and performance of database searches for which typical mass accuracies of approximately 3 amu are employed. In addition, this strategy can also be used to identify the precursor ion for tandem mass spectra acquired using large ion selection windows in data-independent collision-activated dissociation and has the potential to identify multiplexed tandem mass spectra.

Algorithms↗
PubMed1989

Histone-histone interaction mediates chromatin unfolding at physiological ionic strength.

High-resolution thermal denaturation data on chicken erythrocyte chromatin are reported over 4 orders of magnitude in NaCl concentration which includes the physiological region. A novel technique using critical-point polyacrylamide sols instead of ordinary solvents effectively stabilizes chromatin against precipitation at high salt concentrations. These sols are optically transparent from 260 to 320 nm and are thermally stable over the temperature ranges studied. At Na+ ion concentrations below 10 mM, the polyacrylamide slightly destabilizes chromatin at the nucleosome level, possibly through interactions of histones H1 and H5 with the carboxylic acid residues. At the same low salts, polyacrylamide stabilizes pure DNA against denaturation, presumably by mechanically stabilizing it against helix-distorting thermal fluctuations. In both cases, however, the polyacrylamide sols are entirely noninvasive at higher salts. Prominent low-temperature thermal transitions are observed in chromatin at and above 100 mM NaCl which evidently are associated with conformational changes in DNA. Our results are in accord with the idea that histone-histone interactions at physiological ionic strengths (approximately 100 mM Na+) may be comparable to histone-DNA interactions and hence may be sufficient to promote the destabilization of the DNA helix in chromatin under these conditions. The biological implications of this are discussed, and a possible model for the local decondensation of chromatin under physiological conditions is proposed.

Animals↗
PubMed1989

Gramicidin cation channel: an experimental determination of the right-handed helix sense and verification of beta-type hydrogen bonding.

Due to the difficulty of obtaining protein/lipid cocrystals for diffraction studies, structural research on intrinsic membrane proteins and polypeptides has been largely restricted to indirect experimental techniques. Hence, many fundamental questions associated with peptide/lipid systems remain unanswered. In particular, the handedness of the gramicidin A transmembrane ion channel incorporated into lipid bilayers has been an open question for nearly two decades. In this study, solid-state 15N NMR spectroscopy is employed to probe directly the secondary structure of the polypeptide backbone. Recent determinations of the 15N chemical shift anisotropy tensor with respect to the molecular frame enable the quantitative evaluation of the 15N chemical shift resonances obtained from oriented dimyristoylphosphatidylcholine (DMPC) bilayer samples containing specific site 15N labeled gramicidin. This direct structural approach verifies the beta-sheet hydrogen-bonding pattern proposed by Urry [Urry, D. W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676] and determines that in our DMPC bilayer preparations the gramicidin channel is right-handed. Additional structural information is provided by the 15N chemical shift data in the form of orientational constraints on the C alpha-C alpha axis orientation of individual peptides relative to the helix axis. The significance of these solid-state NMR results lies in the direct determination of the helix sense and the verification of the beta-type hydrogen bonding, in the development of the solid-state NMR methods for obtaining such information, and in emphasizing the importance of having direct structural data at atomic resolution.

Chemical Phenomena↗
PubMed1990

Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin.

The crystal and molecular structure of trypsin at a transiently stable intermediate step during catalysis has been determined by X-ray diffraction methods. Bovine trypsin cleaved the substrate p-nitrophenyl p-guanidinobenzoate during crystallization under conditions in which the acyl-enzyme intermediate, (guanidinobenzoyl)trypsin, was stable. Orthorhombic crystals formed in space group P2(1)2(1)2(1), with a = 63.74, b = 63.54, and c = 68.93 A. This is a crystal form of bovine trypsin for which a molecular structure has not been reported. Diffraction data were measured with a FAST (Enraf Nonius) diffractometer. The structure was refined to a crystallographic residual of R = 0.16 for data in the resolution range 7.0-2.0 A. The refined model of (guanidinobenzoyl)trypsin provides insight into the structural basis for its slow rate of deacylation, which in solution at 25 degrees C and pH 7.4 exhibits a t1/2 of 12 h. In addition to the rotation of the Ser-195 hydroxyl away from His-157, C beta of Ser-195 moves 0.7 A toward Asp-189 at the bottom of the active site, with respect to the native structure. This allows formation of energetically favorable H bonds and an ion pair between the carboxylate of Asp-189 and the guanidino group of the substrate. This movement is dictated by the rigidity of the aromatic ring in guanidinobenzoate--model-building indicates that this should not occur when arginine, with its more flexible aliphatic backbone, forms the ester bond with Ser-195. As a consequence, highly ordered water molecules in the active site are no longer close enough to the scissile ester bond to serve as potential nucleophiles for hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Structural mechanism of enoyl-CoA hydratase: three atoms from a single water are added in either an E1cb stepwise or concerted fashion.

We have determined the crystal structure of the enzyme enoyl-CoA hydratase (ECH) from rat liver with the bound substrate 4-(N,N-dimethylamino)cinnamoyl-CoA using X-ray diffraction data to a resolution of 2.3 A. In addition to the thiolester substrate, the catalytic water, which is added in the hydration reaction, has been modeled into well-defined electron density in each of the six active sites of the physiological hexamer within the crystallographic asymmetric unit. The catalytic water bridges Glu(144) and Glu(164) of the enzyme and has a lone pair of electrons poised to react with C(3) of the enzyme-bound alpha,beta-unsaturated thiolester. The water molecule, which bridges two glutamate residues, is reminiscent of the enolase active site. However, unlike enolase, which has a lysine available to donate a proton, there are no other sources of protons available from other active site residues in ECH. Furthermore, an analysis of the hydrogen-bonding network of the active site suggests that both Glu(144) and Glu(164) are ionized and carry a negative charge with no reasonable place to have a protonated carboxylate. This lack of hydrogen-bonding acceptors that could accommodate a source of a proton, other than from the water molecule, leads to a hypothesis that the three atoms from a single water molecule are added across the double bond to form the hydrated product. The structural results are discussed in connection with details of the mechanism, which have been elucidated from kinetics, site-directed mutagenesis, and spectroscopy of enzyme-substrate species, in presenting an atomic-resolution mechanism of the reaction. Contrary to the previous interpretation, the structure of the E-S complex together with previously determined kinetic isotope effects is consistent with either a concerted mechanism or an E1cb stepwise mechanism.

Animals↗
PubMed1996

Co-crystallization and characterization of the photosynthetic reaction center-cytochrome c2 complex from Rhodobacter sphaeroides.

The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c2 (cyt c2), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c2 was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 angstroms. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 angstroms. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 angstroms and c = 254.8 angstroms. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c2 by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c2 was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c2, and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c2 amino acid residues. The RC-cyt c2 structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c2 reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 micros in the co-crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC-cyt c2 complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c2 on the RC. The cyt c2 position with the lowest electrostatic energy is very similar to that of the cyt c2 in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M184 and L155) on the periplasmic surface of the RC. Cyt c2 binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of th RC-cyt c2 complex.

Binding Sites↗
PubMed1999

The crystal structure of a 2-fluorocellotriosyl complex of the Streptomyces lividans endoglucanase CelB2 at 1.2 A resolution.

Glycoside hydrolases have been classified into over 66 families on the basis of amino acid sequence. Recently a number of these families have been grouped into "clans" which share a common fold and catalytic mechanism [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. Glycoside hydrolase Clan GH-C groups family 11 xylanases and family 12 cellulases, which share the same jellyroll topology, with two predominantly antiparallel beta-sheets forming a long substrate-binding cleft, and act with net retention of anomeric configuration. Here we present the three-dimensional structure of a family 12 endoglucanase, Streptomyces lividans CelB2, in complex with a 2-deoxy-2-fluorocellotrioside. Atomic resolution (1.2 A) data allow clear identification of two distinct species in the crystal. One is the glycosyl-enzyme intermediate, with the mechanism-based inhibitor covalently linked to the nucleophile Glu 120, and the other a complex with the reaction product, 2-deoxy-2-fluoro-beta-D-cellotriose. The active site architecture of the complex provides insight into the double-displacement mechanism of retaining glycoside hydrolases and also sheds light on the basis of the differences in specificity between family 12 cellulases and family 11 xylanases.

Cellulase↗
PubMedDate not supplied

StructSorter: a method for continuously updating a comprehensive protein structure alignment database.

Advances in protein crystallography and homology modeling techniques are producing vast amounts of high resolution protein structure data at ever increasing rates. As such, the ability to quickly and easily extract structural similarities is a key tool in discovering important functional relationships. We report on an approach for creating and maintaining a database of pairwise structure alignments for a comprehensive database comprising the PDB and homology models for the human and select pathogen genomes. Our approach consists of a novel, multistage method for determining pairwise structural similarity coupled with an efficient clustering protocol that approximates a full NxN assessment in a fraction of the time. Since biologists are commonly interested in recently released structures, and the homology models built from them, an automatically updating database of structural alignments has great value. Our approach yields a querying system that allows scientists to retrieve databank-wide protein structure similarities as easily as retrieving protein sequence similarities via BLAST or PSI-BLAST. Basic, noncommercial access to the database can be requested at https://tip.eidogen-sertanty.com/.

Databases, Protein↗
PubMed2005

High time resolution and size-segregated analysis of aerosol bound polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are an ubiquitous class of compounds in the environment, mostly generated by anthropogenic processes. High time resolution measurements are necessary to gain further knowledge on the fate and diurnal pattern of these often carcinogenic and mutagenic compounds in the atmosphere. It is expected to find a strong correlation of the PAH levels with the strength and proximity to sources, as well as with meteorological parameters. To determine the fate of particle-bound PAHs, they were sampled in this study at an urban background site in Zurich, Switzerland, during summer 2002 and winter 2003. Particle-bound PAHs were collected with a rotating drum impactor and subsequently analyzed with two-step laser mass spectrometry. Using this combination of sampling and measurement, size-segregated (10-1.1, 1.1-0.3, and 0.3-0.1 microm) and high time resolution (20 min) data were obtained. The pronounced diurnal cycle (with day/night ratios of 0.1) was only altered during intensive atmospheric mixing periods (resulting in day/night ratios of up to 8) by cleaner air from upper atmospheric layers which was mixed into the boundary layer. During summer, signal intensities due to particle-bound PAHs were about a factor of 2-10 lower than during winter.

Aerosols↗
PubMedDate not supplied

Acetylation of phenolic derivatives of 7H-dibenzo[c,g]carbazole: identification and quantitation of major metabolites by rat liver microsomes.

Acetylation stabilized the phenolic metabolites of 7H-dibenzo[c,g]carbazole (DBC) and made it possible to accumulate greater amounts of metabolites for comprehensive chemical structural elucidation and quantification without the use of radiolabeled DBC. High-resolution mass spectral data and 1H NMR and fluorescence spectra were used to confirm the existence of 5-OH-DBC, 3-OH-DBC, 1-OH-DBC, and the oxidative dimer, 6,6'-bis-(5-OH-DBC), in the acetylated metabolite mixture formed in vitro by 3-methylcholanthrene-induced rat liver microsomes. Using the synthesized acetoxy-DBC derivatives as standards, the HPLC external standard method was employed for quantitation of the major DBC metabolites after acetylation. The quantities of 5-OH-DBC, 3-OH-DBC, 1-OH-DBC, and DBC in the metabolite mixture determined using the external standard method were found to agree with those calculated using the radiometric method. Acetylation is a promising nonradiometric qualitative and quantitative technique for further metabolism studies of DBC and analogues which produce unstable monohydroxylated metabolites.

Acetylation↗
PubMed2001

A dusty torus around the luminous young star LkH alpha101.

A star forms when a cloud of dust and gas collapses. It is generally believed that this collapse first produces a flattened rotating disk, through which matter is fed onto the embryonic star at the centre of the disk. When the temperature and density at the centre of the star pass a critical threshold, thermonuclear fusion begins. The remaining disk, which can still contain up to 0.3 times the mass of the star, is then sculpted and eventually dissipated by the radiation and wind from the newborn star. But this picture of the structure and evolution of the disk remains speculative because of the lack of morphological data of sufficient resolution and uncertainties regarding the underlying physical processes. Here we present images of a young star, LkH alpha101, in which the structure of the inner accretion disk is resolved. We find that the disk is almost face-on, with a central gap (or cavity) and a hot inner edge. The cavity is bigger than previous theoretical predictions, and we infer that the position of the inner edge is probably determined by sublimation of dust grains by direct stellar radiation, rather than by disk-reprocessing or viscous-heating processes as usually assumed.

Journal Article↗
PubMed1999

Automated protein model building combined with iterative structure refinement.

In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.

Adaptor Protein Complex alpha Subunits↗
PubMed2005

Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme.

The biosynthetic pathway of the clinically important antibiotic fosfomycin uses enzymes that catalyse reactions without precedent in biology. Among these is hydroxypropylphosphonic acid epoxidase, which represents a new subfamily of non-haem mononuclear iron enzymes. Here we present six X-ray structures of this enzyme: the apoenzyme at 2.0 A resolution; a native Fe(II)-bound form at 2.4 A resolution; a tris(hydroxymethyl)aminomethane-Co(II)-enzyme complex structure at 1.8 A resolution; a substrate-Co(II)-enzyme complex structure at 2.5 A resolution; and two substrate-Fe(II)-enzyme complexes at 2.1 and 2.3 A resolution. These structural data lead us to suggest how this enzyme is able to recognize and respond to its substrate with a conformational change that protects the radical-based intermediates formed during catalysis. Comparisons with other family members suggest why substrate binding is able to prime iron for dioxygen binding in the absence of alpha-ketoglutarate (a co-substrate required by many mononuclear iron enzymes), and how the unique epoxidation reaction of hydroxypropylphosphonic acid epoxidase may occur.

Apoenzymes↗