Biomedical subjects

Shirley A Müller

Publications and source records attributed to Shirley A Müller.

18 recordsLinked to original sources

PubMed2006

Molecular anatomy of a trafficking organelle.

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.

Animals↗

PubMed2006

Impact of disease mutations on the desmin filament assembly process.

It has been documented that mutations in the human desmin gene lead to a severe type of myofibrillar myopathy, termed more specifically desminopathy, which affects cardiac and skeletal as well as smooth muscle. We showed recently that 14 recombinant versions of these disease-causing desmin variants, all involving single amino acid substitutions in the alpha-helical rod domain, interfere with in vitro filament formation at distinct stages of the assembly process. We now provide mechanistic details of how these mutations affect the filament assembly process by employing analytical ultracentrifugation, time-lapse electron microscopy of negatively stained and glycerol-sprayed/low-angle rotary metal-shadowed samples, quantitative scanning transmission electron microscopy, and viscometric studies. In particular, the soluble assembly intermediates of two of the mutated proteins exhibit unusually high s-values, compatible with octamers and other higher-order complexes. Moreover, several of the six filament-forming mutant variants deviated considerably from wild-type desmin with respect to their filament diameters and mass-per-length values. In the heteropolymeric situation with wild-type desmin, four of the mutant variants caused a pronounced "hyper-assembly", when assayed by viscometry. This indicates that the various mutations may cause abortion of filament formation by the mutant protein at distinct stages, and that some of them interfere severely with the assembly of wild-type desmin. Taken together, our findings provide novel insights into the basic intermediate filament assembly mechanisms and offer clues as to how amino acid changes within the desmin rod domain may interfere with the normal structural organization of the muscle cytoskeleton, eventually leading to desminopathy.

Amino Acid Substitution↗

PubMed2006

Immunogenicity and structural characterisation of an in vitro folded meningococcal siderophore receptor (FrpB, FetA).

The iron-limitation-inducible protein FrpB of Neisseria meningitidis is an outer-membrane-localized siderophore receptor. Because of its abundance and its capacity to elicit bactericidal antibodies, it is considered a vaccine candidate. Bactericidal antibodies against FrpB are, however, type-specific. Hence, an FrpB-based vaccine should comprise several FrpB variants to be capable of providing broad protection. To facilitate the development of a meningococcal subunit vaccine, we have established a procedure to obtain large quantities of the protein in a native-like conformation. The protein was expressed without its signal sequence in Escherichia coli, where it accumulated in inclusion bodies. After in vitro folding, the protein was biochemically, biophysically and biologically characterised. Our results show that in vitro folded FrpB assembles into oligomers, presumably dimers, and that it induces high levels of bactericidal antibodies in laboratory animals.

Animals↗

PubMed2006

The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis.

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.

Alzheimer Disease↗

PubMed2006

Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.

ClyA is a pore-forming toxin from virulent Escherichia coli and Salmonella enterica strains. Here, we show that the intrinsic hemolytic activity of ClyA is independent of its redox state, and that the assembly of both reduced and oxidized ClyA to the ring-shaped oligomer is triggered by contact with lipid or detergent. A rate-limiting conformational transition in membrane-bound ClyA monomers precedes their assembly to the functional pore. We obtained a three-dimensional model of the detergent-induced oligomeric complex at 12 A resolution by combining cryo- and negative stain electron microscopy with mass measurements by scanning transmission electron microscopy. The model reveals that 13 ClyA monomers assemble into a cylinder with a hydrophobic cap region, which may be critical for membrane insertion.

Cysteine↗

PubMed2006

Double hexameric ring assembly of the type III protein translocase ATPase HrcN.

The specialized type III secretion (T3S) apparatus of pathogenic and symbiotic Gram-negative bacteria comprises a complex transmembrane organelle and an ATPase homologous to the F1-ATPase beta subunit. The T3S ATPase HrcN of Pseudomonas syringae associates with the inner membrane, and its ATP hydrolytic activity is stimulated by dodecamerization. The structure of dodecameric HrcN (HrcN12) determined to 1.6 nm by cryo-electron microscopy is presented. HrcN12 comprises two hexameric rings that are probably stacked face-to-face by the association of their C-terminal domains. It is 11.5 +/- 1.0 nm in diameter, 12.0 +/- 2.0 nm high and has a 2.0-3.8 nm wide inner channel. This structure is compared to a homology model based on the structure of the F1-beta-ATPase. A model for its incorporation within the T3S apparatus is presented.

Adenosine Triphosphatases↗

PubMed2005

Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C.

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase with essential functions in mitosis, meiosis, and G1 phase of the cell cycle. APC/C recognizes substrates via coactivator proteins such as Cdh1, and bound substrates are ubiquitinated by E2 enzymes that interact with a hetero-dimer of the RING subunit Apc11 and the cullin Apc2. We have obtained three-dimensional (3D) models of human and Xenopus APC/C by angular reconstitution and random conical tilt (RCT) analyses of negatively stained cryo-electron microscopy (cryo-EM) preparations, have determined the masses of these particles by scanning transmission electron microscopy (STEM), and have mapped the locations of Cdh1 and Apc2. These proteins are located on the same side of the asymmetric APC/C, implying that this is where substrates are ubiquitinated. We have further identified a large flexible domain in APC/C that adopts a different orientation upon Cdh1 binding. Cdh1 may thus activate APC/C both by recruiting substrates and by inducing conformational changes.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2005

The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles.

Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.

Antigens, Bacterial↗

PubMed2005

Multiple assembly pathways underlie amyloid-beta fibril polymorphisms.

The amyloid beta-protein transiently forms low and high molecular mass oligomers and protofibrils in vitro, and after longer incubation times assembles into polymorphic mature fibrils. The precursor-to-product relationship of these species remains to be understood. Protofibrils are up to approximately 600 nm in length and have mass-per-lengths of 19(+/-2) kDa/nm measured by scanning transmission electron microscopy. Two predominant mature fibril types, several microns in length and with mass-per-lengths of 18(+/-3) and 27(+/-3) kDa/nm, are identified after longer incubation times. The difference of approximately 9 kDa/nm between the two fibril types indicates a bona fide elementary protofilament subunit of this mass-per-length. Fibrils in the 18(+/-3) kDa/nm group often exhibited distinct coiling with axial cross-over spacings of approximately 25 nm. Although strikingly different in morphology, the mass-per-length (MPL) of these coiled fibrils is equivalent to that measured for protofibrils. They could therefore arise from a conformational change in the protofibril concurrent with coiling and rapid elongation. Alternatively, we cannot rule out an assembly pathway not directly related to protofibrils. In contrast, the 27(+/-3) kDa/nm fibrils correspond to a MPL of approximately 1.5 x the protofibril and thus can neither arise from a simple conformational transition nor from lateral association of 19 kDa/nm protofibril precursors. Twisted ribbons with axial periodicities ranging from approximately 80 nm to 130 nm were prominent in the 27(+/-3) kDa/nm group as well as more tightly coiled fibrils. Individual fibril ribbons had elongation rates of 20(+/-12) nm/min when imaged by time-lapse atomic force microscopy. Protofibrils exhibited growth rates approximately 15 x slower at 1.3(+/-0.5) nm/min. The data support a model where concurrent multiple assembly pathways give rise to the various polymorphic fibril types.

Amyloid↗

PubMed2005

Structural insights into the secretin PulD and its trypsin-resistant core.

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.

Bacterial Outer Membrane Proteins↗

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

Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II.

In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.

Aminopeptidases↗

PubMed2005

Characterization of the in vitro co-assembly process of the intermediate filament proteins vimentin and desmin: mixed polymers at all stages of assembly.

We have investigated the co-assembly properties of the intermediate filament (IF) proteins vimentin and desmin. First, the soluble complexes formed by both proteins separately in 5 mM Tris-HCl, pH 8.4, were characterized by analytical ultracentrifugation. In both cases, s-values of around 5 S were obtained corresponding to the formation of tetramers. However, at pH 7.5 and in the presence of 1 mM EDTA, both proteins behaved quite differently; whereas vimentin sedimented at 7.2 S, desmin assembled into much larger complexes of about 13 S. A mixture of equimolar amounts of vimentin and desmin in 8 M urea yielded, after reconstitution into 5 mM Tris-HCl, pH 7.5, and 1 mM EDTA, complexes exhibiting a sharp peak at 10.9 S. This intermediate s-value indicated that co-assembly into a distinct new set of complexes had occurred. As judged by electron microscopy and viscometry, these mixtures assembled into IFs with characteristics similar to those of pure vimentin and desmin. Furthermore, when vimentin and desmin tetramers were mixed in 5 mM Tris-HCl, pH 8.4, and subsequently subjected to IF assembly conditions, again "hybrid" filaments were obtained. Most interestingly, after 10 min of assembly, mass-per-length (MPL) measurements by scanning transmission electron microscopy yielded IFs with an MPL-peak value of 36 +/- 5 kDa/nm, hence closer to that of vimentin IFs (33 +/- 4 kDa/nm) than to that of desmin IFs (48 +/- 8 kDa/nm). Finally, when unit length-filaments (ULF) of vimentin and desmin were mixed and assembled further, the diameters of individual mature IFs formed exhibited a significantly higher degree of width inhomogeneity along their length than vimentin and desmin IFs as might be expected for a modular mode of assembly. Last but not least, atomic force microscopy provided further direct evidence that desmin IFs are able to fuse end-to-end with vimentin IFs. In summary, we have shown that vimentin and desmin are able to co-assemble at the dimer, tetramer, ULF and even the mature IF level.

Desmin↗

PubMed2004

Exploring amyloid formation by a de novo design.

Protein deposition as amyloid fibrils underlies many debilitating human disorders. The complexity and size of disease-related polypeptides, however, often hinders a detailed rational approach to study effects that contribute to the process of amyloid formation. We report here a simplified peptide sequence successfully designed de novo to fold into a coiled-coil conformation under ambient conditions but to transform into amyloid fibrils at elevated temperatures. We have determined the crystal structure of the coiled-coil form and propose a detailed molecular model for the peptide in its fibrillar state. The relative stabilities of the two structural forms and the kinetics of their interconversion were found to be highly sensitive to small sequence changes. The results reveal the importance of specific packing interactions on the kinetics of amyloid formation and show the potential of this exceptionally favorable system for probing details of the molecular origins of amyloid disease.

Amino Acid Sequence↗

PubMed2004

Structure and electrophysiological properties of the YscC secretin from the type III secretion system of Yersinia enterocolitica.

YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.

Bacterial Outer Membrane Proteins↗

PubMed2002

Exploring the 3D molecular architecture of Escherichia coli type 1 pili.

An integrated approach combining information gained by Fourier transformation, linear Markham superposition (real space) and mass-per-length measurement by scanning transmission electron microscopy was used to analyze the helical structure of the rod-like type 1 pili expressed by uropathogenic Escherichia coli strain W3110. The 3D reconstruction calculated from the experimental data showed the pili to be 6.9nm wide, right-handed helical tubes with a 19.31(+/-0.34)nm long helical repeat comprising 27 FimA monomers associated head-to-tail in eight turns of the genetic one-start helix. Adjacent turns of the genetic helix are connected via three binding sites making the pilus rod rather stiff. In situ immuno-electron microscopy experiments showed the minor subunit (FimH) mediating pilus adhesion to bladder epithelial cells to be the distal protein of the pilus tip, which had a spring-like appearance at higher magnification. The subunits FimG and FimF connect FimH to the FimA rod, the sequential orientation being FimA-FimF-FimG-FimH. The electron density map calculated at 18A resolution from an atomic model of the pilus rod (built using the pilin domain FimH together with the G1 strand of FimC as a template for FimA and applying the optimal helical parameters determined to the head-to-tail interaction model for pilus assembly) was practically identical with that of the actual 3D reconstruction.

Adhesins, Escherichia coli↗

PubMed2002

Imaging and manipulation of biological structures with the AFM.

Many biologists have dreamt of physically touching and manipulating the biomolecules they were investigating. With the invention of the atomic force microscope (AFM), this dream has come true. Here, recent applications of the AFM to image and to manipulate biological systems at the nanometer scale are reviewed. Macromolecular biological assemblies as well as individual biomolecules can be subjected to controlled nanomanipulation. Examples of AFM application in imaging and nanomanipulation include the extraction of chromosomal DNA for genetic analysis, the disruption of antibody--antigen bonds, the dissection of biological membranes, the nanodissection of protein complexes, and the controlled modulation of protein conformations. Also reviewed is the novel combination of single molecule imaging and force spectroscopy which allows biomolecules to be imaged, and inter- and intramolecular forces to be measured. Future application of these nanotechniques will reveal new information on the structure, function and assembly of biomolecules.

Antigen-Antibody Complex↗

PubMed2002

The reaction centre from green sulphur bacteria: progress towards structural elucidation.

The reaction centre (RC) of green sulphur bacteria is a FeS-type RC, as are the RCs of Photosystems I (PS I) of oxygenic photosynthetic organisms and of heliobacteria. The core domains of both green sulphur bacterial and heliobacterial RCs are considered to be homodimeric, in contrast to those of purple bacteria, PS I and Photosystem II (PS II). This paper briefly describes the techniques of electron microscopy and image processing suited to investigate the structure of these proteins. Recent advances in the study of the structure of the green sulphur bacterial RC, primarily achieved by the application of scanning transmission electron microscopy, are reviewed.

Journal Article↗