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PubMed1999

Interaction of process partitions in phylogenetic analysis: an example from the swallowtail butterfly genus Papilio.

In this study, we explored how the concept of the process partition may be applied to phylogenetic analysis. Sequence data were gathered from 23 species and subspecies of the swallowtail butterfly genus Papilio, as well as from two outgroup species from the genera Eurytides and Pachliopta. Sequence data consisted of 1,010 bp of the nuclear protein-coding gene elongation factor-1 alpha (EF-1 alpha) as well as the entire sequences (a total of 2,211 bp) of the mitochondrial protein-coding genes cytochrome oxidase I and cytochrome oxidase II (COI and COII). In order to examine the interaction between the nuclear and mitochondrial partitions in a combined analysis, we used a method of visualizing branch support as a function of partition weight ratios. We demonstrated how this method may be used to diagnose error at different levels of a tree in a combined maximum-parsimony analysis. Further, we assessed patterns of evolution within and between subsets of the data by implementing a multipartition maximum-likelihood model to estimate evolutionary parameters for various putative process partitions. COI third positions have an estimated average substitution rate more than 15 times that of EF-1 alpha, while COII third positions have an estimated average substitution rate more than 22 times that of EF-1 alpha. Ultimately, we found that although the mitochondrial and nuclear data were not significantly incongruent, homoplasy in the fast-evolving mitochondrial data confounded the resolution of basal relationships in the combined unweighted parsimony analysis despite the fact that there was relatively strong support for the relationships in the nuclear data. We conclude that there may be shortcomings to the methods of "total evidence" and "conditional combination" because they may fail to detect or accommodate the type of confounding bias we found in our data.

Animals↗
PubMed2002

Alternate conformations observed in catalytic serine of Bacillus subtilis lipase determined at 1.3 A resolution.

Bacillus subtilis extracellular lipase (BsL) has an exceptionally low molecular weight (19.4 kDa) for a member of the lipase family. A crystallographic study was performed on BsL in order to design and produce mutant BsL that will be more suitable for industrial uses based on analysis of the three-dimensional structure. Recently, the crystal structure of BsL has been determined at 1.5 A resolution [van Pouderoyen et al. (2001). J. Mol. Biol. 309, 215-226]. In the present study, a new crystal form of BsL which provides diffraction data to higher resolution was obtained and its structure was determined at 1.3 A using the MAD method. It was found that the active-site residue Ser77 has alternate side-chain conformations. The O(gamma) atom of the first conformer forms a hydrogen bond to the N(epsilon) atom of His155, a member of the catalytic triad. In contrast, the second conformer is constructed with a hydrogen bond to the side-chain atom of the adjacent His76. These two conformers presumably correspond to the active and inactive states, respectively. Similar alternate conformations in the catalytic serine residue have been observed in Fusarium solani cutinase determined at 1.0 A resolution and Penicillium purpurogenum acetylxylan esterase at 0.9 A resolution. In addition, a glycerol molecule, which was used as a cryoprotectant, is found to be located in the active site. On the basis of these results, a model for substrate binding in the reaction-intermediate state of BsL is proposed.

Amino Acids↗
PubMed2006

DADOS-Prospective: an open source application for Web-based prospective data collection.

BACKGROUND: Randomized, prospective trials involving multi-institutional collaboration have become a central part of clinical and translational research. However, data management and coordination of multi-center studies is a complex process that involves developing systems for data collection and quality control, tracking data queries and resolutions, as well as developing communication procedures. We describe DADOS-Prospective, an open-source Web-based application for collecting and managing prospective data on human subjects for clinical and translational trials. DADOS-Prospective not only permits users to create new clinical research forms (CRF) and supports electronic signatures, but also offers the advantage of containing, in a single environment, raw research data in downloadable spreadsheet format, source documentation and regulatory files stored in PDF format, and audit trails. RESULTS: Feedback from formal and field usability tests was used to guide the design and development of DADOS-Prospective. To date, DADOS-Prospective has been implemented in five prospective clinical studies at our institution. Four of these studies are still in the CRF creation phase and one study has been entirely launched. CONCLUSION: DADOS-Prospective has significant advantages over existing Web-based data collecting programs. At our institution, it has been demonstrated to be an efficient tool for prospective clinical studies.

Journal Article↗
PubMed1987

Decreasing hydrostatic pressure does not uniformly decrease high-pressure pulmonary edema.

Pulmonary artery wedge pressure (PAWP) of 30 mm Hg with left atrial balloon inflation for 1 1/2 hours produced pulmonary edema in eight dogs. PAWP was then decreased to 10 mm Hg for two hours, and shunt, lung water (extravascular thermal volume, or ETV, by thermal dye), and perfusion distribution (radiomicrosphere technique) were measured and compared with four other dogs (group 1) whose PAWP was maintained at 10 mm Hg. The eight dogs with PAWP of 30 mm Hg for 1 1/2 hours were retrospectively subdivided into two groups of four based on ETV (group 2, double baseline ETV; group 3, triple baseline ETV). Baseline ETV and shunt were similar for all groups and remained unchanged for group 1. At 1 1/2 hours, 2 hours (1/2 hour after decreasing PAWP), 2 1/2 hours, and 3 1/2 hours, respectively, ETV were: 13.9 +/- 1.9, 12.8 +/- 2.0, 9.3 +/- 1.5, and 8.5 +/- 1.0 ml/kg in group 2; and 21.9 +/- 2.1, 22.7 +/- 2.2, 22.5 +/- 2.0, and 22.2 +/- 2.0 ml/kg in group 3. A more variable rate of edema formation was detected in eight additional dogs, but failure to resolve higher levels of edema after decreasing PAWP was also demonstrated in this group. Edema was greatest in lower lobes and decreased lobar perfusion. Shunt was higher in group 3 than in group 2 at 1 1/2 hours and decreased in group 2 but not in group 3 at 3 1/2 hours. Changes in colloid osmotic pressure may account for the differences in edema formation and resolution, but our data suggest that, independent of the rate of edema formation, a decrease in vascular exchange surface area at higher levels of edema may inhibit edema resolution when PAWP is decreased.

Animals↗
PubMed2004

3 dimensional modelling of early human brain development using optical projection tomography.

BACKGROUND: As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped. RESULTS: Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (approximately 300 microm by approximately 150 microm) to the rootlets of cranial nerve XII (approximately 20 microm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is approximately 30 microm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated. CONCLUSION: In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of approximately 20 microm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.

Brain↗
PubMed1994

Laue and monochromatic diffraction studies on catalysis in phosphorylase b crystals.

The conversion of substrate, heptenitol, to product, beta-1-C-methyl, alpha-D-glucose-1-phosphate (heptulose-2-P), in crystals of glycogen phosphorylase b has been studied by Laue and monochromatic diffraction methods. The phosphorolysis reaction in the crystal was started following liberation of phosphate from a caged phosphate compound, 3,5-dinitrophenyl phosphate (DNPP). The photolysis of DNPP, stimulated by flashes from a xenon flash lamp, was monitored in the crystal with a diode array spectrophotometer. In the Laue diffraction experiments, data to 2.8 A resolution were collected and the first time shot was obtained at 3 min from the start of reaction, and data collection comprised three 800-ms exposures. Careful data processing of Laue photographs for the large enzyme resulted in electron density maps of almost comparable quality to those produced by monochromatic methods. The difference maps obtained from the Laue measurements showed that very little catalysis had occurred 3 min and 1 h after release of phosphate, and a distinct peak consistent with the position expected for phosphate, in the attacking position was observed. Data collection times with monochromatic crystallographic methods on a home source took 16 h for data to 2.3 A resolution. Sufficient phosphate was released from the caged phosphate in the crystal from 5 flashes with a xenon flashlamp within 1 min for the reaction to go to completion within the time scale of the monochromatic data collection procedures. The heptulose-2-P product complex has been refined and the model agrees with that obtained previously with the major difference that the interchange of an aspartic acid (Asp 283) by an arginine (Arg 569) was not observed at the catalytic site. This change is part of the activation process of glycogen phosphorylase and may not have taken place in the current experiments because the caged compound binds weakly at the inhibitor site, restricting conformational change, and because activators of the enzymic reaction were not present in the crystal. In experiments with monochromatic radiation in which low phosphate concentrations were generated either by fewer photons or by diffusion of known phosphate concentrations, mixtures of substrate and product were observed. It was not possible through crystallographic refinement at 2.3 A resolution to establish the fractional occupancies of the enzyme-substrate and enzyme-product complexes, but the results did indicate that the reaction was proceeding slowly, consistent with approximate calculations for the likely rate of the reaction in the crystal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Progressive EPR imaging with adaptive projection acquisition.

Continuous wave electron paramagnetic resonance imaging (EPRI) of living biological systems requires rapid acquisition and visualization of free radical images. In the commonly used multiple-stage back-projection image reconstruction algorithm, the EPR image cannot be reconstructed until a complete set of projections is collected. If the data acquisition is incomplete, the previously acquired incomplete data set is no longer useful. In this work, a 3-dimensional progressive EPRI technique was implemented based on inverse Radon transform in which a 3-dimensional EPR image is acquired and reconstructed gradually from low resolution to high resolution. An adaptive data acquisition strategy is proposed to determine the significance of projections and acquire them in an order from the most significant to the least significant. The image acquisition can be terminated at any time if further collection of projections does not improve the image resolution distinctly, providing flexibility to trade image quality with imaging time. The progressive imaging technique was validated using computer simulations as well as imaging experiments. The adaptive acquisition uses 50-70% less projections as compared to the regular acquisition. In conclusion, adaptive data acquisition with progressive image reconstruction should be very useful for the accelerated acquisition and visualization of free radical distribution.

Algorithms↗
PubMed1993

Crystallization and preliminary X-ray analysis of vipoxin, a complex between a toxic phospholipase A2 and its natural polypeptide inhibitor.

The toxin vipoxin, which is a complex between a basic toxic phospholipase A2 and an acidic non-toxic protein inhibitor, is found in the venom of the Bulgarian viper (Vipera ammodytes ammodytes), the most toxic snake in Europe. The two polypeptide chains each consist of 122 residues and are highly homologous (62%). The vipoxin complex is the first reported example of a high degree of structural homology between an enzyme and its natural inhibitor. The present crystals diffract in the X-ray beam to 1.8 A resolution. The space group is P2(1)2(1)2(1). The cell dimensions are a = 45.80 A, b = 55.36 A and c = 107.69 A. Native data to a resolution of 2.8 A have been recorded.

Animals↗
PubMed2003

Clinical utility of oral valacyclovir compared with oral acyclovir for the prevention of herpes simplex virus mucositis following autologous bone marrow transplantation or stem cell rescue therapy.

Oral acyclovir has been demonstrated to prevent reactivation of herpes simplex virus (HSV) infections when administered prophylactically to autologous bone marrow transplant (BMT) recipients or patients undergoing stem cell rescue therapy. Oral valacyclovir, which is converted in the body to acyclovir, has greater oral bioavailability than oral acyclovir and compared with oral acyclovir yields similar acyclovir plasma concentrations with less frequent (twice-daily) dosing. This study compared the efficacy of oral valacyclovir with that of oral acyclovir at preventing HSV mucositis in BMT recipients. A total of 60 HSV-1-positive patients scheduled for BMT or stem cell rescue therapy were treated prophylactically with valacyclovir 500 mg twice daily until resolution of neutropenia. Data from these patients were compared with those of a historical control group of 60 patients who had received acyclovir 600 mg every 6 h until resolution of neutropenia or acyclovir 125 mg/m(2) intravenously every 6 h. The results show that none of the patients developed oral or oropharyngeal HSV infection while receiving either treatment. Of the 60 patients receiving valacyclovir, 38 (63%) completed treatment without the need for intravenous acyclovir compared with 12 of 60 (20%) patients in the acyclovir group. Additionally, the total number of doses of drug administered to the valacyclovir group was significantly less than the number received by patients in the acyclovir group. No serious adverse events occurred in either group of patients. This study demonstrates that oral valacyclovir and acyclovir are comparably effective and safe in preventing reactivation of HSV infections in autologous BMT and stem cell recipients. The less frequent dosing schedule with valacyclovir compared with acyclovir offers a potential advantage for patients undergoing BMT who frequently suffer with severe mucositis and have difficulty taking oral medications.

Acyclovir↗
PubMed2003

Crystallization and preliminary X-ray analysis of the E. coli hypothetical protein TdcF.

Crystals of the hypothetical protein TdcF (subunit MW = 14 007) from Escherichia coli were grown by vapour diffusion. The protein crystallizes in space group P2(1)2(1)2, with unit-cell parameters a = 72.67, b = 86.22, c = 62.62 A. Native data to a resolution of 2.35 A were collected from a single crystal at 100 K on a rotating-anode X-ray generator. Preliminary analysis of these data indicated that the asymmetric unit corresponded to a trimer, which was supported by a convincing molecular-replacement solution using the YjgF trimer as the probe structure.

Amino Acid Sequence↗
PubMed2003

Crystallization and preliminary X-ray studies on the putative dTDP sugar epimerase NovW from the novobiocin biosynthetic cluster of Streptomyces spheroides.

Crystals of recombinant NovW (subunit MW = 22 289 Da), a putative dTDP sugar epimerase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallizes in space group P4(3)2(1)2, with unit-cell parameters a = b = 59.20, c = 109.23 A. Native data to a resolution of 2.0 A were collected from a single crystal at 100 K on a rotating-anode X-ray generator. Preliminary analysis of these data indicated that the asymmetric unit corresponded to a monomer, whilst dynamic light scattering (DLS) suggested that NovW was a dimer in solution. NovW is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin, which targets the bacterial enzyme DNA gyrase, and represents the first enzyme to be crystallized from this biosynthetic pathway.

Carbohydrate Epimerases↗
PubMed2002

Beamforming with a reduced sampling rate.

The beamforming process requires a high delay resolution to avoid the deteriorating effects of the delay quantization lobes on the image dynamic range and signal to noise ratio. Wideband transducers require delay resolutions in the order of 1/16 the signal period. If oversampling is used to achieve this timing resolution, a huge data volume has to be acquired and processed in real time. This is usually avoided by sampling just above the Nyquist rate and interpolating to achieve the required delay resolution. However this increases the hardware complexity. Baseband sampling has been alternatively proposed with sampling rates as low as the transducer frequency or even lower. This approach uses two A/D converters and processing chains for every channel, thus doubling the hardware requirements. Quadrature sampling can be used instead with a single A/D converter, but the sampling rate must be a multiple of four times the transducer frequency, decreasing the application flexibility. Furthermore, it produces relatively high errors in the detected envelope if wideband transducers are used. This work presents a new approach, the selective sampling technique (SST), which keeps the lowest sampling rate required by the imaging process or the signal bandwidth (whatever is larger) and, at the same time, provides a high delay resolution to keep the highest image dynamic range. The SST is based on a second order sampling process which, differently from the mentioned approaches, does not pose any constraints in the time interval between samples and produce lower errors in the detected envelope. The hardware requirements are low (a single A/D converter and processing chain for every transducer element), working at the lowest data rate compatible with the Nyquist criterion, thus reducing the data bandwidth. Furthermore, the sampling points can be also freely chosen, so that the SST simplify the usually required scan conversion process to a simple linear interpolation easily carried out by software in real-time.

Journal Article↗
PubMed2007

Time-resolved 3D quantitative flow MRI of the major intracranial vessels: initial experience and comparative evaluation at 1.5T and 3.0T in combination with parallel imaging.

Exact knowledge of blood flow characteristics in the major cerebral vessels is of great relevance for diagnosing cerebrovascular abnormalities. This involves the assessment of hemodynamically critical areas as well as the derivation of biomechanical parameters such as wall shear stress and pressure gradients. A time-resolved, 3D phase-contrast (PC) MRI method using parallel imaging was implemented to measure blood flow in three dimensions at multiple instances over the cardiac cycle. The 4D velocity data obtained from 14 healthy volunteers were used to investigate dynamic blood flow with the use of multiplanar reformatting, 3D streamlines, and 4D particle tracing. In addition, the effects of magnetic field strength, parallel imaging, and temporal resolution on the data were investigated in a comparative evaluation at 1.5T and 3T using three different parallel imaging reduction factors and three different temporal resolutions in eight of the 14 subjects. Studies were consistently performed faster at 3T than at 1.5T because of better parallel imaging performance. A high temporal resolution (65 ms) was required to follow dynamic processes in the intracranial vessels. The 4D flow measurements provided a high degree of vascular conspicuity. Time-resolved streamline analysis provided features that have not been reported previously for the intracranial vasculature.

Adult↗
PubMed2005

Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes.

Human Cu-Zn superoxide dismutase (SOD1) protects cells from the effects of oxidative stress. Mutations in SOD1 are linked to the familial form of amyotrophic lateral sclerosis. Several hypotheses for their toxicity involve the mis-metallation of the enzyme. We present atomic-resolution crystal structures and biophysical data for human SOD1 in three metallation states: Zn-Zn, Cu-Zn and as-isolated. These data represent the first atomic-resolution structures for human SOD1, the first structure of a reduced SOD1, and the first structure of a fully Zn-substituted SOD1 enzyme. Recombinantly expressed as-isolated SOD1 contains a mixture of Zn and Cu at the Cu-binding site. The Zn-Zn structure appears to be at least as stable as the correctly (Cu-Zn) metallated enzyme. These data raise the possibility that in a cellular environment with low availability of free copper, Zn-Zn may be the preferred metallation state of SOD1 prior to its interaction with the copper chaperone.

Animals↗
PubMedDate not supplied

A method for measurement of cross sectional area, segment length, and branching angle of airway tree structures in situ.

Accurate quantitative measurements of airway and vascular dimensions are essential for evaluating function in both the normal and in the diseased lung. This report describes a new integrated method for three-dimensional (3D) extraction and analysis of pulmonary tree structures using data from High Resolution Computed Tomography (HRCT). Serially scanned two-dimensional (2D) slices of the lower left lobe of isolated dog lungs were stacked to create a volume of data. Airway and vascular trees were extracted using a 3D seeded region-growing algorithm based on differences in CT number between wall and lumen. In the region-growing step, voxels in the lumen are tagged with a distance descriptor to identify points along the tree structure equidistant from the seed point. To obtain quantitative data, we reduced each tree to its central axis. From the central axis, branch length was measured as the distance between two successive branch points, branch angle was measured as the angle produced by two daughter branches, and cross-sectional area was measured from a plane perpendicular to the central axis point. Data derived from these methods can be used to localize and quantify structural differences both during different physiologic conditions and in pathologic lungs.

Algorithms↗
PubMed2003

Away from the edge: SAD phasing from the sulfur anomalous signal measured in-house with chromium radiation.

Anomalous scattering with soft X-ray radiation opens new possibilities in phasing for macromolecular crystallography. Anomalous scattering from S atoms collected on an in-house chromium radiation source (lambda = 2.29 A) was used to phase the X-ray diffraction data of thaumatin (22 kDa) and trypsin (24 kDa) crystals. The contribution to the anomalous term, Deltaf" = 1.14 e(-), from sulfur for Cr Kalpha radiation is doubled compared with that for Cu Kalpha radiation, Deltaf" = 0.56 e(-). The direct-methods programs RANTAN or SHELXD successfully found sulfur positions using data sets with resolution limited to 3.5 A. The statistical phasing program SHARP was used to produce the electron-density maps using the sulfur anomalous signal alone at low resolution ( approximately 3.5 A). An interpretable electron-density map for each structure was obtained solely from the phases derived from single-wavelength anomalous dispersion (SAD) data obtained using Cr Kalpha radiation. Much fewer data (that is, lower redundancy) are required for this sulfur SAD phasing procedure compared with the highly redundant data reported in the sulfur SAD phasing procedure with Cu Kalpha radiation. Cr Kalpha radiation can also improve the strength of anomalous scattering of many other intrinsic elements in macromolecules, such as calcium, zinc and phosphorus, because of the increased Deltaf". Furthermore, the anomalous scattering of selenium is increased substantially from 1.14 e(-) with Cu Kalpha radiation to 2.28 e(-) with Cr Kalpha radiation. In order to measure the small Bijvoet differences accurately, several devices were developed for the experiment, including an Osmic Confocal MaxFlux optic optimized for Cr Kalpha radiation, a helium path and a beam stop. In the cases studied here, radiation damage to the samples and reduction of anomalous signal were observed in some long exposure time data sets. Therefore, an adequate data-collection strategy to maximize the completeness in a short scan range was used in subsequent data collections. The results show that the anomalous signal of S atoms can be collected quickly. Since the absorption of solvent and the loop may no longer be negligible with Cr Kalpha radiation, the orientation of the crystal and exposure time were taken into account in order to minimize the effects of radiation damage and absorption. This experimental study shows that using Cr Kalpha radiation from an in-house rotating-anode X-ray generator can provide sufficient phasing power from sulfur anomalous signals to routinely phase protein diffraction data.

Chromium↗
PubMed1996

MAD Phasing Strategies Explored with a Brominated Oligonucleotide Crystal at 1.65A Resolution.

The crystal structure of a brominated oligonucleotide d(CGCG(Br)CG), chemical formula C(114)N(48)O(68)P(10)Br(2), has been analysed by multiwavelength anomalous dispersion (MAD) methods. The oligonucleotide crystallizes in space group P2(1)2(1)2(1) with a = 17.97, b = 30.98, c = 44.85 A, alpha = beta = gamma 90 degrees . Data to a resolution of 1.65 A were collected at four wavelengths about the K-absorption edge of the bromine atom (lambda(1) = 0.9323 A, a reference wavelength at the long-wavelength side of the edge; lambda(2) = 0.9192 A, at the absorption-edge inflection point; lambda(3) = 0.9185 A, at the ;white line' absorption maximum; lambda(4) = 0.8983 A, a reference wavelength at the short-wavelength side) using synchrotron radiation at Station PX9.5, SRS, Daresbury. Multiwavelength data could be collected on a single-crystal as the sample was radiation stable. Anomalous and dispersive Patterson maps were readily interpretable to give the bromine anomalous scatterer positions. Phase calculations to 1.65 A, resolution, using all four wavelengths, gave a figure of merit of 0.825 for 2454 reflections. The electron-density map was readily interpretable showing excellent connectivity for the sugar/phosphate backbone and each base was easily characterized. The two nucleotide strands paired up as expected in an antiparallel Watson-Crick-type manner. The structure was refined to 1.65 A using all the data (R-factor = 17.0% based on 3151 reflections, with a data-to-parameter ratio of 2.6). In addition to the four-wavelength analysis, a variety of other phasing strategies, and the associated quality of the resulting electron-density maps, were compared. These included use of either of the reference wavelength data sets in the two possible three-wavelength phasing combinations to assess their relative effectiveness. Moreover, the time dependence upon measuring the Bijvoet differences and its effect upon phasing was also investigated. Finally, the use of only two wavelengths, including Friedel pairs, is demonstrated (the theoretical minimum case); this is of particular interest when considering overall beam time needs and is clearly a feasible experimental strategy, as shown here.

Journal Article↗
PubMed1992

Refinement of the structure of Escherichia coli-derived rat intestinal fatty acid binding protein with bound oleate to 1.75-A resolution. Correlation with the structures of the apoprotein and the protein with bound palmitate.

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel beta strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation with C1-C12 more ordered than C13-C18. Two of the eight ordered waters in I-FABP:oleate are part of a hydrogen bond network that includes the carboxylate of oleate, the guanidinium group of Arg106, the nitrogen of the indole group of Trp82, and the side chain of Gln115. Most of the methylenes of bound oleate reside in a crevice formed by hydrophobic and aromatic side chains. Tyr70 and Tyr117 envelop the acyl chain from C3 to C8 forming contacts with both the convex and concave faces of its van der Waals surface. The hydroxyls of each phenolic side chain hydrogen bond to ordered water molecules. Two ordered waters make van der Waals contact with the concave face of the bound fatty acid. The omega-terminal methyl of oleate is oriented so that it points toward the center of the benzene of Phe55 allowing it to form van der Waals interactions with its component methylenes. Comparison of the structure of I-FABP:oleate with a recently refined 1.19-A model of apoI-FABP and an earlier 2.0-A model of I-FABP:palmitate revealed a remarkable degree of similarity in the positions of their main chain and side chain atoms and in the conformations of the bound oleate and palmitate. The principal differences were confined to a few discrete regions of the protein. The helical domain, the type I turn between beta strands C and D, and the ring of Phe55 together form a solvent-accessible portal to the interior of the protein. They are repositioned in I-FABP:oleate (and I-FABP:palmitate) so that the binding cavity is even more accessible to solvent and its volume is increased. The side chain of Phe55 which shows discrete disorder in the apoprotein functions as an omega-terminal "sensing device": moving progressively outward toward the surface as the chain length of the bound fatty acid increases by 2 methylenes. Tyr70 and Tyr117 which also show discrete disorder in the apoprotein structure due to rotation around their C alpha-C beta bonds, are stabilized in a single, well ordered position in the holoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗