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A Engel

Publications and source records attributed to A Engel.

At least 19 recordsLinked to original sources
PubMed1997

The three-dimensional structure of aquaporin-1.

The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6A resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning alpha-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.

Animals↗
PubMed1997

The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution.

The surfaces of two- and three-dimensional phi29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm x 16.5 nm. High resolution images of connectors from the two-dimensional crystal surface show two connectors per unit cell confirming the p42(1)2 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right-handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco-conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of approximately 150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three-dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing.

Bacillus Phages↗
PubMed1997

The Campylobacter jejuni porin trimers pack into different lattice types when reconstituted in the presence of lipid.

Purified major outer membrane protein of Campylobacter jejuni exhibited different classes of molecules by SDS/PAGE and immunoblotting. A high-molecular-mass product (120-140 kDa) was observed under mild conditions of solubilization, a folded monomeric form of 35 kDa was seen when treated at high SDS concentrations and finally, a single band around 45 kDa occurred when the sample was heated to 96 degrees C [Bolla, J. M., Loret, E., Zalewski. M. & Pages, J. M. (1995) J. Bacteriol. 177, 4266-4271]. The high-molecular-mass product was reconstituted into two-dimensional crystals in the presence of phospholipids and Mg2+. The C. jejuni porin required different conditions for successful reconstitution into two-dimensional crystals than the Escherichia coli porin OmpF. Electron microscopy and digital image processing of negatively stained specimens revealed a rectangular lattice with a unit cells size of a = 8.9 nm, b = 14.9 nm, an oblique lattice with a = 8.9 nm, b = 30.1 nm, gamma = 98 degrees, and a trigonal lattice with a = b = 9.6 nm. Projection maps were calculated to a resolution of 2 nm, and exhibited a trimeric protein with three stain-filled indentations.

Bacterial Outer Membrane Proteins↗
PubMed1997

[Effectiveness of selective hepatic artery embolization in a child after blunt hepatic trauma].

A 9-year-old boy was admitted after a bicycle fall. Abdominal CT-scan revealed severe liver injury (stage IV according to the liver injury scale of the American Association for Surgery Trauma), including ruptured intraparenchymal hematoma with active bleeding. The patient was hemodynamically stable and was treated conservatively for the first 2 days. On the 3rd day selective hepatic artery angiography was performed because of abdominal distension and the need for 7 pints of packed red blood cells. Active right hepatic artery bleeding was identified and treated successfully by embolization. We think that early angiography and selective embolization should always be considered for acute or continuous bleeding after liver injury.

Child↗
PubMed1997

Polymorphic fibrillar assembly of human amylin.

Human amylin forms fibrillar amyloid between pancreatic islet cells in patients with non-insulin-dependent (type 2) diabetes mellitus. Fibrillar assemblies also form in vitro in aqueous solutions of synthetic human amylin. We now report on the structural polymorphism of these fibrils. The thinnest fibril, referred to as the protofibril, has an apparent width of 5 nm but is only rarely observed by itself. These protofibrils spontaneously assemble into higher order fibrillar structures with distinct morphologies. Prominent among these is an 8-nm fibril with a distinct 25-nm axial crossover repeat which is formed by left-handed coiling of two 5-nm protofibrils. Coiling of more than two 5-nm protofibrils results in cable-like structures of variable width depending on the number of protofibrils involved. Lateral (side-by-side) assembly of 5-nm protofibrils is also observed and produces ribbons which may contain two, three, four, or more protofibrils and occasionally large single-layered sheets. The mass-per-length (MPL) of the 5-nm protofibril is 10 kDa/nm. This has been established in two ways: first, the 8-nm fibril, which is formed by coiling two 5-nm protofibrils around each other, has an MPL of 20 kDa/nm. Second, higher order fibrils differ by increments of 10 kDa/nm. Hence, about 2.6 human amylin molecules (3904 Da) are packed in 1 nm of protofibril length. Similarities exist between amylin fibrils and those formed from other amyloid proteins, suggesting that the in vitro assembly of synthetic protein may serve as a useful model system in advancing our understanding of amyloid formation in disease.

Amyloid↗
PubMed1997

Adsorption of biological molecules to a solid support for scanning probe microscopy.

Scanning probe microscopes are now established tools to study the surface structure of biological macromolecules under physiological conditions. Sample preparation methods for this microscopy all have the objective to attach the specimen firmly to a support. Here we analyse the commonly used method of adsorbing biological specimens to freshly cleaved mica. This is facilitated by adjusting the electrolyte concentration and the pH of the buffer solution. Native macromolecular systems absorbed to mica in this way can be reproducibly imaged at submolecular resolution.

Adsorption↗
PubMed1997

Structural changes in native membrane proteins monitored at subnanometer resolution with the atomic force microscope: a review.

Three membrane proteins, OmpF porin from Escherichia coli, bacteriorhodopsin from Halobacterium salinarium, and the hexagonally packed intermediate (HPI) layer from Deinoccocus radiodurans, were investigated with the atomic force microscope in buffer solution. A resolution of up to 0.8 nm allowed structural differences of individual proteins to be detected. OmpF porin exhibits different static conformations on the outer surface, which possibly represent the two conductive states of the ion channels. Reversible structural changes in the cytoplasmic surface of purple membrane have been induced by changing the force applied to the scanning stylus: doughnut-shaped bacteriorhodopsin trimers transformed into a structure with three pronounced protrusions when the force was reduced from 300 to 100 pN. Furthermore, individual pores of the inner surface of the HPI layer were observed to switch from an "open" to a "closed" state. Together, the structural changes in proteins monitored under physiological conditions suggest that direct observation of function-related conformational changes of biomolecules with the atomic force microscope is feasible.

Bacterial Outer Membrane Proteins↗
PubMed1997

Structural Analysis of Photosystem II: Comparative Study of Cyanobacterial and Higher Plant Photosystem II Complexes

Oxygen evolving photosystem II (PSII-OEC) complexes and PSII core complexes were isolated from spinach and the thermophilic cyanobacterium Synechococcus sp. OD24 and characterized by gel electrophoresis, immunoblotting, and absorbance spectroscopy. The mass of the core complexes was determined by scanning transmission electron microscopy (STEM) and found to be 281 ± 65 kDa for spinach and 313 ± 52 kDa for Synechococcus sp. OD24. The mass of the spinach PSII-OEC complex was 327 ± 64 kDa. Digital images of negatively stained PSII-OEC and PSII core complexes were recorded by STEM and analyzed by single particle averaging. All monomeric complexes showed similar morphologies and were of comparable length (14 nm) and width (10 nm). The averages revealed a pseudo-twofold symmetry axis, which is a prominent structural element of the monomeric form. Difference maps between the averaged projections of the oxygen evolving complexes and the core complexes from both species indicated where the 33-kDa extrinsic manganese stabilizing protein is bound. A symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex, is proposed.

Journal Article↗
PubMed1997

EDITORIAL

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Journal Article↗
PubMed1997

Clinical and sonographic evaluation of the risk of rupture in the Achilles tendon.

Chronic pain in the region of the Achilles tendon is a common problem and often a sign of progressive degeneration of the tendon which may lead to its rupture. We studied the clinical course and sonograms in 36 patients with achillodynia to find a prognostic parameter enabling us to estimate the risk of rupture. The patients were evaluated clinically for swelling and tenderness and by high-resolution real-time sonography. The sonograms were graded according to the tendon thickness as normal (< 6 mm), minimal (6-8 mm), moderate (8-10 mm) to high-grade (> 10 mm) in the sagittal diameter of the transverse section, and alterations of echotexture were described as diffuse, circumscribed, or inhomogenous. At the time of the primary investigation, we found thickening and alterations of the echotexture in 33 of 72 tendons. In 48 tendons we found pain and local or diffuse swelling in the Achilles tendon region (sensitivity 0.58, specificity 0.79). After a follow-up of 48 +/- 8 months, 7 tendons had ruptured spontaneously. Analysis of the sonograms of the patients taken prior to the rupture showed a high-grade thickening in 4 cases, moderate thickening in 2 cases, and a diameter between 6 and 8 nm in one patient. In no case did we find a rupture of a tendon primarily classified as normal. Patients without sonographic changes exhibited a significantly better clinical outcome following conservative treatment. Sonography was found to be a valuable tool for determination of the tendon's thickness and echotexture. In 28% of our patients with thickening, circumscribed lesions of the echotexture, and chronic pain, a spontaneous rupture occurred.

Achilles Tendon↗
PubMed1997

Imaging of avascular necrosis of bone.

The etiology of avascular necrosis (AVN) is multifactorial. Independent of its etiology and localization it shows typical pathologies and radiological images. In the early stages localized subchondral edema is characteristic. In 50 % of all cases accompanying joint effusion may be found. Due to necrosis of the cells of bone marrow and bone fibrovascular, reactions with hyperemia can be delineated. These reactions allow us to visualize necrosis indirectly. The best imaging methods are MRI and, to a lesser extent, bone scintigraphy. In later stages calcification as well as new bone formation and microfractures are typically demonstrated and visualized best with plain X-rays and CT. Why reparations in many cases, particularly in the hip, are incomplete and may stop in any stage is unknown. Over years clinically complete silent AVNs are not an uncommon finding. Prognosis depends on the localization and size of the AVN. The number of repair mechanisms is best outlined with contrast-enhanced MRI and return of fatty marrow.

Bone and Bones↗
PubMed1997

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals.

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.

Aquaporin 1↗
PubMed1997

The height of biomolecules measured with the atomic force microscope depends on electrostatic interactions.

In biological applications of atomic force microscopy, the different surface properties of the biological sample and its support become apparent. Observed height differences between the biomolecule and its supporting surface are thus not only of structural origin, but also depend on the different sample-tip and support-tip interactions. This can result in negative or positive contributions to the measured height, effects that are described by the DLVO (Derjaguin, Landau, Verwey, Overbeek) theory. Experimental verification shows that the electrostatic interactions between tip and sample can strongly influence the result obtained. To overcome this problem, pH and electrolyte concentration of the buffer solution have to be adjusted to screen out electrostatic forces. Under these conditions, the tip comes into direct contact with the surface of support and biological system, even when low forces required to prevent sample deformation are applied. In this case, the measured height can be related to the thickness of the native biological structure. The observed height dependence of the macromolecules on electrolyte concentration makes it possible to estimate surface charge densities.

Aquaporin 1↗
PubMed1997

High resolution imaging of native biological sample surfaces using scanning probe microscopy.

The possibility of acquiring high resolution topographs using scanning probe microscopes under physiological conditions allows the observation of biomolecules at work. Progress has recently been made in imaging protein-DNA complexes, individual oligomers and protein arrays. Scanning probe microscopes are now tools that complement X-ray crystallography and electron microscopy.

Bacterial Outer Membrane Proteins↗
PubMed1997

Electron and atomic force microscopy of membrane proteins.

Electron crystallography is becoming a powerful tool for the resolution of membrane protein structures. The past year has seen the production of a bacteriorhodopsin model at 3.5 A and the structure of aquaporin 1 approaching atomic resolution. Determination of surface topographies of 2D crystals using the atomic force microscope is similarly advancing to a level that reveals submolecular details. As the latter is operated in solution, membrane proteins can be observed at work.

Aquaporin 1↗
PubMed1997

The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome.

HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslIV and the ATPase HsIU. We have used electron microscopy and image analysis to examine the structural organization of HslV and HslU homo-oligomers and the active HslVU enzyme. Electron micrographs of HslV reveal ring-shaped particles, and averaging of top views reveal six-fold rotational symmetry, in contrast to other beta-type proteasome subunits, which form rings with seven-fold symmetry. Side views of HslV show two rings stacked together, thus, HslV behaves as dodecamer. The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide binding, but not hydrolysis, is required for oligomerization. Subunit crosslinking, STEM mass estimation, and analysis of HslU top views indicate that HslU exists both as hexameric and heptameric rings. With AMP-PNP present, maximal proteolytic activity is observed with a molar ratio of HslU to HslV subunits of 1:1, and negative staining electron microscopy shows that HslV and HsIU form cylindrical four-ring structures in which the HsIV dodecamer is flanked at each end by a HslU ring.

ATP-Dependent Proteases↗