PubMed Health⌕ Search

Biomedical subjects

H Stahlberg

Publications and source records attributed to H Stahlberg.

At least 19 recordsLinked to original sources
PubMed2004

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy.

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone'. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner.

Bacillus subtilis↗
PubMed2002

Progress in the analysis of membrane protein structure and function.

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron 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 sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.

Crystallization↗
PubMed2001

ATP synthase: constrained stoichiometry of the transmembrane rotor.

Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.

ATP Synthetase Complexes↗
PubMed2001

Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins.

Electron crystallography and atomic force microscopy allow the study of two-dimensional membrane protein crystals. While electron crystallography provides atomic scale three-dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub-nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function-related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes.

Cell Membrane↗
PubMed2001

Bacterial Na(+)-ATP synthase has an undecameric rotor.

Synthesis of adenosine triphosphate (ATP) by the F(1)F(0) ATP synthase involves a membrane-embedded rotary engine, the F(0) domain, which drives the extra-membranous catalytic F(1) domain. The F(0) domain consists of subunits a(1)b(2) and a cylindrical rotor assembled from 9-14 alpha-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits.

Bacteria, Anaerobic↗
PubMed2001

Early resuscitation with hypertonic saline/dextran in uncontrolled intra-abdominal bleeding in swine combined with a soft tissue gunshot wound.

The effects of hypertonic saline/dextran (HSD) on hemodynamics and on rebleeding were studied during an uncontrolled intra-abdominal hemorrhage combined with a high-energy gunshot wound (GSW) in the hind limb of anesthetized swine. The GSW had instant effects on the central hemodynamics, which were aggravated when the internal hemorrhage was induced. Compared with baseline, cardiac output decreased to about 42%, mean arterial pressure decreased to 52 +/- 4%, and mean flow rates in the splanchnic region, in the upper aorta, and in the kidney decreased to 51 to 15%. The injection of HSD at 10 minutes was followed by a prompt increase in blood flow rates, but rebleeding occurred in five of eight animals, although only two died. In conclusion, GSW induced instant changes in hemodynamics at distance from the injury. When HSD treatment was given in a bolus injection, rebleeding occurred in five of eight animals, although the second hemorrhage became fatal in only one animal.

Animals↗
PubMed2000

Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence.

The lens major intrinsic protein (MIP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight "tongue-and-groove" fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 degrees to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens.

Aluminum Silicates↗
PubMed2000

The aquaporin sidedness revisited.

Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of micro-organisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted.

Aquaporin 1↗
PubMed2000

Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy.

Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gram-negative bacteria. Limited proteolysis of secretin PulD from the Klebsiella oxytoca pullulanase secretion pathway showed that it consists of an N-terminal domain and a protease-resistant C-terminal domain that remains multimeric after proteolysis. The stable C-terminal domain starts just before the region in PulD that is highly conserved in the secretin superfamily and apparently lacks the region at the C-terminal end to which the secretin-specific pilot protein PulS binds. Electron microscopy showed that the stable fragment produced by proteolysis is composed of two stacked rings that encircle a central channel and that it lacks the peripheral radial spokes that are seen in the native complex. Moreover, the electron microscopic images suggest that the N-terminal domain folds back into the large cavity of the channel that is formed by the C-terminal domain of the native complex, thereby occluding the channel, consistent with previous electrophysiological studies showing that the channel is normally closed.

Amino Acids↗
PubMed2000

The 6.9-A structure of GlpF: a basis for homology modeling of the glycerol channel from Escherichia coli.

The three-dimensional structure of GlpF, the glycerol facilitator of Escherichia coli, was determined by cryo-electron microscopy. The 6.9-A density map calculated from images of two-dimensional crystals shows the GlpF helices to be similar to those of AQP1, the erythrocyte water channel. While the helix arrangement of GlpF does not reflect the larger pore diameter as seen in the projection map, additional peripheral densities observed in GlpF are compatible with the 31 additional residues in loops C and E, which accordingly do not interfere with the inner channel construction. Therefore, the atomic structure of AQP1 was used as a basis for homology modeling of the GlpF channel, which is predicted to be free of bends, wider, and more vertically oriented than the AQP1 channel. Furthermore, the residues facing the GlpF channel exhibit an amphiphilic nature, being hydrophobic on one side and hydrophilic on the other side. This property may partially explain the contradiction of glycerol diffusion but limited water permeation capacity.

Aquaporin 1↗
PubMed2000

The 3.7 A projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer.

GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of approximately 80 A side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 A resolution. Cryoelectron microscopy provided a 3.7 A projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.

Aquaporins↗
PubMed1999

High resolution AFM topographs of the Escherichia coli water channel aquaporin Z.

Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning alpha-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change.

Amino Acid Sequence↗
PubMed1999

Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography.

Molecular water channels (aquaporins) allow living cells to adapt to osmotic variations by rapid and specific diffusion of water molecules. Aquaporins are present in animals, plants, algae, fungi and bacteria. Here we present an electron microscopic analysis of the most ancient water channel described so far: the aquaporin Z (AqpZ) of Escherichia coli. A recombinant AqpZ with a poly(histidine) tag at the N terminus has been constructed, overexpressed and purified to homogeneity. Solubilized with octylglucoside, the purified AqpZ remains associated as a homotetramer, and assembles into highly ordered two-dimensional tetragonal crystals with unit cell dimensions a = b = 95 A, gamma = 90 degrees when reconstituted by dialysis in the presence of lipids. Three-dimensional reconstruction of negatively stained lattices revealed the p42(1)2 packing arrangement that is also observed with the human erythrocyte water channel (AQP1). The 8 A projection map of the AqpZ tetramer in frozen hydrated samples is similar to that of AQP1, consistent with the high sequence homology between these proteins.

Aquaporin 1↗
PubMed1999

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy.

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex.

Bacterial Proteins↗
PubMed1999

Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits.

Lon (or La) is a soluble, homooligomeric ATP-dependent protease. Mass determination and cryoelectron microscopy of pure mitochondrial Lon from Saccharomyces cerevisiae identify Lon as a flexible ring-shaped heptamer. In the presence of ATP or 5'-adenylylimidodiphosphate, most of the rings are symmetric and resemble other ATP-driven machines that mediate folding and degradation of proteins. In the absence of nucleotides, most of the rings are distorted, with two adjacent subunits forming leg-like protrusions. These results suggest that asymmetric conformational changes serve to power processive unfolding and translocation of substrates to the active site of the Lon protease.

ATP-Dependent Proteases↗
PubMed1998

Are the light-harvesting I complexes from Rhodospirillum rubrum arranged around the reaction centre in a square geometry?

The basic photosynthetic unit containing the reaction centre and the light-harvesting I complex (RC-LHI) of the purple non-sulphur bacterium Rhodospirillum rubrum was purified and reconstituted into two-dimensional (2D) membrane crystals. Transmission electron microscopy using conventional techniques and cryoelectron microscopy of the purified single particles and of 2D crystals yielded a projection of the RC-LHI complex at a resolution of at least 1.6 nm. In this projection the LHI ring appears to have a square symmetry and packs in a square crystal lattice. The square geometry of the LHI ring was observed also in images of single isolated particles of the RC-LHI complex. However, although the LHI units are packed identically within the crystal lattice, a new rotational analysis developed here showed that the reaction centres take up one of four possible orientations within the ring. This fourfold disorder supports our interpretation of a square ring symmetry and suggests that a hitherto undetected component may be present within the photosynthetic unit.

Bacterial Chromatophores↗
PubMed1998

Friction anisotropy and asymmetry of a compliant monolayer induced by a small molecular tilt

Lateral force microscopy in the wearless regime was used to study the friction behavior of a lipid monolayer on mica. In the monolayer, condensed domains with long-range orientational order of the lipid molecules were present. The domains revealed unexpectedly strong friction anisotropies and non-negligible friction asymmetries. The angular dependency of these effects correlated well with the tilt direction of the alkyl chains of the monolayer, as determined by electron diffraction and Brewster angle microscopy. The molecular tilt causing these frictional effects was less than 15 degrees, demonstrating that even small molecular tilts can make a major contribution to friction.

Journal Article↗