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Biomedical subjects

Andreas Engel

Publications and source records attributed to Andreas Engel.

At least 37 records · Page 2Linked to original sources
PubMed2005

The 4.5 A structure of human AQP2.

Located in the principal cells of the collecting duct, aquaporin-2 (AQP2) is responsible for the regulated water reabsorption in the kidney and is indispensable for the maintenance of body water balance. Disregulation or malfunctioning of AQP2 can lead to severe diseases such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. Here we present the crystallization of recombinantly expressed human AQP2 into two-dimensional protein-lipid arrays and their structural characterization by atomic force microscopy and electron crystallography. These crystals are double-layered sheets that have a diameter of up to 30 microm, diffract to 3 A(-1) and are stacked by contacts between their cytosolic surfaces. The structure determined to 4.5 A resolution in the plane of the membrane reveals the typical aquaporin fold but also a particular structure between the stacked layers that is likely to be related to the cytosolic N and C termini.

Aquaporin 2↗
PubMed2005

Functional and structural characterization of the minimal Sec translocase of the hyperthermophile Thermotoga maritima.

The genome of the hyperthermophilic bacterium Thermotoga maritima contains the genes that encode core subunits of the protein translocase, a complex consisting of the molecular motor SecA and the protein conducting pore SecYE. In addition, we identified an erroneous sequence in the genome encoding for a putative secG gene. The genes of the T. maritima translocase subunits were overexpressed in Escherichia coli and purified to homogeneity. T. maritima SecA showed a basal thermostable ATPase activity that was stimulated up to 4-fold by phospholipids with an optimum at 74 degrees C. Membrane vesicles and proteoliposomes containing SecYE or SecYEG supported 2- to 4-fold stimulation of the precursor dependent SecA ATPase activity. Imaging of small two-dimensional crystals of the SecYE complex using electron microscopy showed square-shaped particles with a side-length of about 6 nm. These results demonstrate that in T. maritima a highly thermostable translocase complex is operational.

Adenosine Triphosphatases↗
PubMed2005

Reconstitution of two recombinant LSm protein complexes reveals aspects of their architecture, assembly, and function.

Sm and Sm-like (LSm) proteins form complexes engaging in various RNA-processing events. Composition and architecture of the complexes determine their intracellular distribution, RNA targets, and function. We have reconstituted the human LSm1-7 and LSm2-8 complexes from their constituent components in vitro. Based on the assembly pathway of the canonical Sm core domain, we used heterodimeric and heterotrimeric sub-complexes to assemble LSm1-7 and LSm2-8. Isolated sub-complexes form ring-like higher order structures. LSm1-7 is assembled and stable in the absence of RNA. LSm1-7 forms ring-like structures very similar to LSm2-8 at the EM level. Our in vitro reconstitution results illustrate likely features of the LSm complex assembly pathway. We prove the complexes to be functional both in an RNA bandshift and an in vivo cellular transport assay.

Cell Nucleus↗
PubMed2005

The VirE1VirE2 complex of Agrobacterium tumefaciens interacts with single-stranded DNA and forms channels.

The VirE2 protein is crucial for the transfer of single-stranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2-ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2-ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector-chaperone complex is multifunctional.

Agrobacterium tumefaciens↗
PubMed2004

Thermal ratchet effects in ferrofluids.

Rotational Brownian motion of colloidal magnetic particles in ferrofluids under the influence of an oscillating external magnetic field is investigated. It is shown that for a suitable time dependence of the magnetic field, a noise-induced rotation of the ferromagnetic particles due to rectification of thermal fluctuations takes place. Via viscous coupling, the associated angular momentum is transferred from the magnetic nanoparticles to the carrier liquid and can then be measured as macroscopic torque on the fluid sample. A thorough theoretical analysis of the effect in terms of symmetry considerations, analytical approximations, and numerical solutions is given which is in accordance with recent experimental findings.

Journal Article↗
PubMed2004

Functional characterization of rhodopsin monomers and dimers in detergents.

Rhodopsin (Rho) is a G protein-coupled receptor that initiates phototransduction in rod photoreceptors. High expression levels of Rho in the disc membranes of rod outer segments and the propensity of Rho to form higher oligomeric structures are evident from atomic force microscopy, transmission electron microscopy, and chemical cross-linking experiments. To explore the structural and functional properties of Rho in n-dodecyl-beta-maltoside, frequently used to purify heterologously expressed Rho and its mutants, we used gel filtration techniques, blue native gel electrophoresis, and functional assays. Here, we show that in micelles containing n-dodecyl-beta-maltoside at concentrations greater than 3 mM, Rho is present as a single monomer per detergent micelle. In contrast, in 12 mM 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), micelles contain mostly dimeric Rho. The cognate G protein transducin (Gt) appears to have a preference for binding to the Rho dimer, and the complexes fall apart in the presence of guanosine 5'-3-O-(thio)triphosphate. Cross-linked Rho dimers release the chromophore at a slower rate than monomers and are much more resistant to heat denaturation. Both Rho(*) monomers and dimers are capable of activating Gt, and both of them are phosphorylated by Rho kinase. Rho expressed in HEK293 cells is also readily cross-linked by a bifunctional reagent. These studies provide an explanation of how detergent influences the oligomer-dimermonomer equilibrium of Rho and describe the functional characterization of Rho monomers and dimers in detergent.

Animals↗
PubMed2004

Rhodopsin signaling and organization in heterozygote rhodopsin knockout mice.

Rhodopsin (Rho) resides within internal membrane structures called disc membranes that are found in the rod outer segments (ROS) of photoreceptors in the retina. Rho expression is essential for formation of ROS, which are absent in knockout Rho-/- mice. ROS of mice heterozygous for the Rho gene deletion (Rho+/-) may have a lower Rho density than wild type (WT) membranes, or the ROS structure may be reduced in size due to lower Rho expression. Here, we present evidence that the smaller volume of ROS from heterozygous mice is most likely responsible for observed electrophysiological response differences. In Rho+/- mice as compared with age-matched WT mice, the length of ROS was shorter by 30-40%, and the average diameter of ROS was reduced by approximately 20%, as demonstrated by transmission and scanning electron microscopy. Together, the reduction of the volume of ROS was approximately 60% in Rho+/- mice. Rho content in the eyes was reduced by approximately 43% and 11-cis-retinal content in the eye was reduced by approximately 38%, as determined by UV-visible spectroscopy and retinoid analysis, respectively. Transmission electron microscopy of negatively stained disc membranes from Rho+/- mice indicated a typical morphology apart from the reduced size of disc diameter. Power spectra calculated from disc membrane regions on such electron micrographs displayed a diffuse ring at approximately 4.5 nm(-1), indicating paracrystallinity of Rho. Atomic force microscopy of WT and Rho+/- disc membranes revealed, in both cases, Rho organized in paracrystalline and raftlike structures. From these data, we conclude that the differences in physiological responses measured in WT and Rho+/- mice are due to structural changes of the whole ROS and not due to a lower density of Rho.

Animals↗
PubMed2004

Double Rosensweig instability in a ferrofluid sandwich structure.

We consider a horizontal ferrofluid layer sandwiched between two layers of immiscible nonmagnetic fluids. In a sufficiently strong vertical magnetic field the flat interfaces between magnetic and nonmagnetic fluids become unstable to the formation of peaks. We theoretically investigate the interplay between these two instabilities for different combinations of the parameters of the fluids and analyze the evolving interfacial patterns. We also estimate the critical magnetic field strength at which thin layers disintegrate into an ordered array of individual drops.

Journal Article↗
PubMed2004

Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition.

The adhesion force and specificity in the first experimental evidence for cell-cell recognition in the animal kingdom were assigned to marine sponge cell surface proteoglycans. However, the question whether the specificity resided in a protein or carbohydrate moiety could not yet be resolved. Here, the strength and species specificity of cell-cell recognition could be assigned to a direct carbohydrate-carbohydrate interaction. Atomic force microscopy measurements revealed equally strong adhesion forces between glycan molecules (190-310 piconewtons) as between proteins in antibody-antigen interactions (244 piconewtons). Quantitative measurements of adhesion forces between glycans from identical species versus glycans from different species confirmed the species specificity of the interaction. Glycan-coated beads aggregated according to their species of origin, i.e., the same way as live sponge cells did. Live cells also demonstrated species selective binding to glycans coated on surfaces. These findings confirm for the first time the existence of relatively strong and species-specific recognition between surface glycans, a process that may have significant implications in cellular recognition.

Animals↗
PubMed2004

The G protein-coupled receptor rhodopsin in the native membrane.

The higher-order structure of G protein-coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi-empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane.

Animals↗
PubMed2004

A concept for G protein activation by G protein-coupled receptor dimers: the transducin/rhodopsin interface.

G protein-coupled receptors (GPCRs) are ubiquitous and essential in modulating virtually all physiological processes. These receptors share a similar structural design consisting of the seven-transmembrane alpha-helical segments. The active conformations of the receptors are stabilized by an agonist and couple to structurally highly conserved heterotrimeric G proteins. One of the most important unanswered questions is how GPCRs couple to their cognate G proteins. Phototransduction represents an excellent model system for understanding G protein signaling, owing to the high expression of rhodopsin in rod photoreceptors and the multidisciplinary experimental approaches used to study this GPCR. Here, we describe how a G protein (transducin) docks on to an oligomeric GPCR (rhodopsin), revealing structural details of this critical interface in the signal transduction process. This conceptual model takes into account recent structural information on the receptor and G protein, as well as oligomeric states of GPCRs.

Animals↗
PubMed2004

Molecular architecture of photosynthetic membranes in Rhodobacter sphaeroides: the role of PufX.

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX- strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX- cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1beta. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.

Bacterial Proteins↗
PubMed2004

Electron and atomic force microscopy of the trimeric ammonium transporter AmtB.

Escherichia coli AmtB is an archetypal member of the ammonium transporter (Amt) family, a family of proteins that are conserved in all domains of life. Reconstitution of AmtB in the presence of lipids produced large, ordered two-dimensional crystals. From these, a 12 A resolution projection map was determined by cryoelectron microscopy, and high-resolution topographs were acquired using atomic force microscopy. Both techniques showed the trimeric structure of AmtB in which each monomer seems to have a pseudo-two-fold symmetry. This arrangement is likely to represent the in vivo structure. This work provides the first views of the structure of any member of the Amt family.

Cation Transport Proteins↗
PubMed2004

Structure and assembly of the pseudopilin PulG.

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.

Bacterial Proteins↗
PubMed2003

Structural analysis of the reaction center light-harvesting complex I photosynthetic core complex of Rhodospirillum rubrum using atomic force microscopy.

The bacterium Rhodospirillum rubrum contains a simple photosynthetic system, in which the reaction center (RC) receives energy from the light-harvesting (LH1) complex. We have used high-resolution atomic force microscopy (AFM) to image two-dimensional crystals of the RC-LH1 complex of R. rubrum. The AFM topographs show that the RC-LH1 complex is approximately 94 A in height, the RC-H subunit protrudes from the cytoplasmic face of the membrane by 40 A, and it sits 21 A above the highest point of the surrounding LH1 ring. In contrast, the RC on the periplasmic side is at a lower level than LH1, which protrudes from the membrane by 12 A. The RC-LH1 complex can adopt an irregular shape in regions of uneven packing forces in the crystal; this reflects a likely flexibility in the natural membrane, which might be functionally important by allowing the export of quinol formed as a result of RC photochemistry. Nanodissection of the RC by the AFM tip removes the RC-H subunit and reveals the underlying RC-L and -M subunits. LH1 complexes completely lacking the RC were also found, providing ideal conditions for imaging both rings of LH1 polypeptides for the first time by AFM. In addition, we demonstrate the ellipticity of the LH1 ring at the cytoplasmic and periplasmic sides of the membrane, in both the presence and absence of the RC. These AFM measurements have been reconciled with previous electron microscopy and NMR data to produce a model of the RC-LH1 complex.

Bacterial Proteins↗
PubMed2003

Ferrofluids as thermal ratchets.

Colloidal suspensions of ferromagnetic nanoparticles, so-called ferrofluids, are shown to be suitable systems to demonstrate and investigate thermal ratchet behavior: By rectifying thermal fluctuations, angular momentum is transferred to a resting ferrofluid from an oscillating magnetic field without net rotating component. Via viscous coupling the noise driven rotation of the microscopic ferromagnetic grains is transmitted to the carrier liquid to yield a macroscopic torque. For a simple setup we analyze the rotation of the ferrofluid theoretically and show that the results are compatible with the outcome of a simple demonstration experiment.

Journal Article↗
PubMed2003

Robert Feulgen Lecture. Microscopic assessment of membrane protein structure and function.

Membrane proteins represent an important class of proteins that are encoded by about 40% of all genes, but compared to soluble proteins structural information is sparse. Most of the atomic coordinates currently available are from bacterial membrane proteins and have been obtained by X-ray crystallography. Recent results demonstrate the imaging power of the atomic force microscope and the accuracy of electron crystallography. These methods allow membrane proteins to be studied while embedded in the bilayer, and thus in a functional state. The low signal-to-noise ratio of cryoelectron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review discusses examples of microscopic membrane protein structure determination and illuminates recent progress.

Animals↗