Biomedical subjects
Publications and source records attributed to W Baumeister.
The two genes encoding the constituent subunits of the Thermoplasma acidophilum proteasome were expressed in Escherichia coli yielding fully assembled molecules as shown by electron microscopy. The recombinant proteasomes were purified to homogeneity and were shown to have proteolytic activity indistinguishable from proteasomes isolated from T. acidophilum.
We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90 degrees C. The enzyme activity is enhanced severalfold by Mg2+ and Ca2+ at 25-500 mM. This increase in activity results primarily from a change in Km. The serine-proteinase inhibitors diisopropylfluorophosphate and 3,4-dichloroisocoumarin irreversibly inhibit the enzyme, obviously by modification of both the alpha and beta subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz-Leu-Leu-CH2Cl and Cbz-Ala-Ala-Phe-CH2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a core of 3 alpha subunits. The preparation of reconstituted hollow capsids consisting of 60 beta subunits and their crystallization in a hexagonal (space group P6(3)22) and in a monoclinic (space group C2) modification have been described. The rotational and translational parameters of the protein molecules in both crystal forms were studied by electron microscopy of freeze-etch replicas and by Patterson correlation techniques. Decoration with silver and image processing provided images with the positions of the 3-fold and 5-fold molecular axes being labelled by metal clusters. This allowed the unequivocal determination of the orientation and translational position of the protein molecules with respect to the crystallographic axes in the hexagonal modification. From inspection of the decoration images it was immediately obvious that the hexagonal crystal forms of alpha 3 beta 60 and of beta 60 are isomorphous. In the monoclinic crystals, a local icosahedral 2-fold coincides with the crystallographic 2-fold axis. The exact solution of the particle orientation was determined by interpretation of Patterson self-rotation functions for the icosahedral symmetry axes. Rotational and translational parameters for the monoclinic modification are given. A rational procedure for the efficient application of freeze-etching techniques in order to elucidate the packing in crystals of large proteins is described.
The proteasome or multicatalytic proteinase is a high molecular mass multisubunit complex ubiquitous in eukaryotes but also found in the archaebacterial proteasome is made of two different subunits only, and yet the complexes are almost identical in size and shape. Cloning and sequencing the gene encoding the small (beta) subunit of the T. acidophilum complex completes the primary structure of the archaebacterial proteasome. The similarity of the derived amino acid sequences of 233 (alpha) and 211 (beta) residues, respectively, indicates that they arose from a common ancestral gene. All the sequences of proteasomal subunits from eukaryotes available to date can be related to either the alpha-subunit or beta-subunit of the T. acidophilum "Urproteasome", and they can be distinguished by means of a highly conserved N-terminal extension, which is characteristic for alpha-type subunits. On the basis of circumstantial evidence we suggest that the alpha-subunits have regulatory and targeting functions, while the beta-subunits carry the active sites.
The proteasome or multicatalytic proteinase from the archaebacterium Thermoplasma acidophilum is a 700 kDa multisubunit protein complex. Unlike proteasomes from eukaryotic cells which are composed of 10-20 different subunits, the Thermoplasma proteasome is made of only two types of subunit, alpha and beta, which have molecular weights of 25.8 and 22.3 kDa, respectively. In this communication we present a three-dimensional stoichiometric model of the archaebacterial proteasome deduced from electron microscopic investigations. The techniques which we have used include image analysis of negatively stained single particles, image analysis of metal decorated small three-dimensional crystals after freeze-etching and STEM mass measurements of freeze-dried particles. The archaebacterial and eukaryotic proteasomes are almost identical in size and shape; the subunits are arranged in four rings which are stacked together such that they collectively form a barrel-shaped complex. According to a previous immunoelectron microscopic investigation, the alpha-subunits form the two outer rings of the stack, while the two rings composed of beta-subunits, which are supposed to carry the active sites, are sandwiched between them. Each of the alpha- and beta-rings contains seven subunits; hence the stoichiometry of the whole proteasome is alpha 14 beta 14 and the symmetry is 7-fold. Image simulation experiments indicate that the alpha- and beta-subunits are not in register along the cylinder axis; rather it appears that the beta-rings are rotated with respect to the alpha-rings by approximately 25 degrees. In contrast to some previous reports we have not been able to find stoichiometric amounts of RNA associated with highly purified proteolytically active proteasome preparations.
We have discovered a new oligomeric protein component associated with the outer membrane of the ancestral eubacterium Thermotoga maritima. In electron micrographs, the protein, Omp alpha, appears as a rod-shaped spacer that spans the periplasm, connecting the outer membrane to the inner cell body. Purification, biochemical characterization and sequencing of Omp alpha suggest that it is a homodimer composed of two subunits of 380 amino acids with a calculated M(r) of 43,000 and a pI of 4.54. The sequence of the omp alpha gene indicates a tripartite organization of the protein with a globular NH2-terminal domain of 64 residues followed by a putative coiled-coil segment of 300 residues and a COOH-terminal, membrane-spanning segment. The predicted length of the coiled-coil segment (45 nm) correlates closely with the spacing between the inner and outer membranes. Despite sequence similarity to a large number of coiled-coil proteins and high scores in a coiled-coil prediction algorithm, the sequence of the central rod-shaped domain of Omp alpha does not have the typical 3.5 periodicity of coiled-coil proteins but rather has a periodicity of 3.58 residues. Such a periodicity was also found in the central domain of staphylococcal M protein and beta-giardin and might be indicative of a subclass of fibrous proteins with packing interactions that are distinct from the ones seen in other two-stranded coiled-coils.
The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.
Regularly arrayed surface (glyco) proteins--often referred to as S layers--are a common feature of the cell envelopes of almost all archaebacteria. We have selected some examples (Halobacterium, Sulfolobus, Thermoproteus, Pyrobaculum, Staphylothermus), and we describe the structure of their surface layers as revealed primarily by electron crystallography. In spite of a considerable diversity in shapes and dimensions, some common structural features emerge from the comparison. The glycoprotein arrays are composed of oligomeric units which are anchored in the plasma membrane; extended spacer or linker domains maintain the bulk of the more or less porous surface layers at a constant distance above the membrane surface, thus creating a quasi-periplasmic compartment. Functions ascribed to surface layers, such as compartmentalization, shape maintenance and determination, and adhesion are discussed.
The techniques as well as the principles of the 2D crystallization of membrane and water-soluble proteins for electron crystallography are reviewed. First, the biophysics of the interactions between proteins, lipids and detergents is surveyed. Second, crystallization of membrane proteins in situ and by reconstitution methods is discussed, and the various factors involved are addressed. Third, we elaborate on the 2D crystallization of water-soluble proteins, both in solution and at interfaces, such as lipid monolayers, mica, carbon film or mercury surfaces. Finally, techniques and instrumentations that are required for 2D crystallization are described.
Transmission electron microscopy of proteins has provided molecular- and in a few cases near-atomic-resolution structural information. In this review, we critically evaluate the potential and the limitations in obtaining molecular resolution, particularly with negatively stained specimens, and put these into perspective with cryomicroscopy of unstained frozen-hydrated and sugar-embedded preparations.
The solubilized surface protein of the Gram-negative bacterium Comamonas acidovorans was reconstituted on lipid vesicles by means of controlled dialysis. To this end, a multichamber dialysis apparatus was built which allows one to control the temperature and the dialysis rate, to apply various temperatures or buffer systems and sample conditions in a single experiment, and to monitor the turbidity of the sample by means of light scattering. The reconstitution conditions were optimized such that the surface protein formed two-dimensional crystals suitable for electron crystallography. The recrystallized surface protein arrays gave a resolution of approximately 1.3 nm in projection after correlation averaging of negatively stained preparations. The surface protein assembled into partially self-contained two-dimensional crystals which possess a strong shape-determining effect and formed cylinders and various cone-shaped vesicles. The development of the various vesicle forms is described in a model.
The regular surface protein structure (S-layer) of Caulobacter crescentus was analyzed by electron microscopy and three-dimensional image reconstruction to a resolution of 2 nm. Projections showed that the S-layer is an array of ring structures, each composed of six subunits that are arranged on a lattice with p6 symmetry. Three-dimensional reconstructions showed that the ring subunits were approximately rod-shaped structures and were perpendicular to the plane of the array, with a linker arm emanating from approximately the middle of the rod, accounting for the connections between the rings. The calculated subunit mass was ca. 100 kDa, very close to the size of RsaA (the protein known to be at least the predominant species in the S-layer) predicted from the DNA sequence of the rsaA gene. The core region of the rings creates an open pore 2.5 to 3.5 nm in diameter. The size of the gaps between the neighboring unit cells is in the same range, suggesting a uniform porosity predicted to exclude molecules larger than ca. 17 kDa. Attempts to remove membrane material from S-layer preparations with detergents revealed that the structure spontaneously rearranged into a mirror-image double layer. Negative-stain and thin-section electron microscopy examination of colonies of C. crescentus strains with a mutation in a surface molecule involved in the attachment of the S-layer showed that shed RsaA protein organized into large sheets. The sheets in turn organized into stacks that tended to accumulate near the upper surface of the colony. Image reconstruction indicated that these sheets were also precise mirror-image double layers, and thickness measurements obtained from thin sections were consistent with this finding. The sheets were absent when these mutant strains were grown without calcium, supporting other data that calcium is involved in attachment of the S-layer to a surface molecule and perhaps in subunit-subunit interactions. We propose that when the membrane is removed from S-layer fragments by detergents or the attachment-related surface molecule is absent, the attachment sites of the S-layer align precisely to form a double layer via a calcium interaction.
The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.
The subunit topography of the Thermoplasma acidophilum proteasome was determined by immunoelectron microscopy using monospecific antibodies directed against the two constituent subunits (alpha,beta). Anti-alpha-subunit IgG was found to bind to the outer disks of the cylinder- or barrel-shaped molecule, while the binding sites of the anti-beta-subunit IgG were mapped on the two inner rings. Probably the homologues of the two subunits in the compositionally more complex but isomorphous eukaryotic proteasomes occupy equivalent positions.
On the surface of protein molecules the formation of metal clusters during vacuum condensation is controlled by topochemical features of the substrate and by specific properties of the decorating material. The resulting metal distribution (decoration pattern) can be mapped by electron microscopy in conjunction with image processing. We have applied this technique to freeze-etched crystals of the lumazine synthase-riboflavin synthase complex and its derivative obtained by binding of the heteropolytungstate (NaP5W30O110)(NH4)14.31 H2O. The decoration pattern of the free protein and its heteropolytungstate derivative showed marked differences. The correlation of these data with the X-ray structure of the protein showed an increased affinity of both gold and silver to the location of heteropolytungstate. Decoration sites can, but do not need to, be close to the protein surface. Actually, two of the observed decoration sites are located on a layer of ice as thick as 20 A, which apparently transmits underlying topochemical features. Preferential affinity of a site to a given metal must be seen as a property that depends on specific interaction with the decorating material but also on the differential affinities in adjacent areas.
The three-dimensional structure of proteasomes from the archaebacterium Thermoplasma acidophilum has been determined to a resolution of approximately 2 nm from electron micrographs of negatively stained preparations using the method of 'random conical tilting'. The particles turn out to be essentially cylinder-shaped barrels, 15 nm long and 11 nm wide, enclosing a tripartite inner compartiment. An account is given of some of the present limitations which prevent to attain a higher resolution and possible ways to overcome these limitations are indicated.
The gene encoding the alpha-subunit of the proteasome from the archaebacterium Thermoplasma acidophilum was cloned and sequenced. The gene encodes for a polypeptide with 233 amino acid residues and a calculated molecular weight of 25870. Sequence similarity of the alpha-subunit with the Saccharomyces cerevisiae wild-type suppressor gene scll+ encoded polypeptide, which is probably identical with the subunit YC7-alpha of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression. The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.
We have discovered a large cylindrical protein complex which is an abundant component of the cytoplasm of extremely thermophilic archaebacteria. Structural analysis by image processing of electron micrographs suggests that the complex is composed of two stacked rings of eight subunits each; the rings enclose a central channel. The complex purified from the hyperthermophile Pyrodictium occultum is composed of equal quantities of two polypeptides of Mr 56,000 and 59,000. It exhibits an extremely thermostable ATPase activity with a temperature optimum of 100 degrees C. The basal level of the ATPase complex in the cell is high, and it becomes highly enriched as a result of heat shock (shift from 102 degrees C to 108 degrees C) or balanced growth at temperatures near the physiological upper limit. Immunoblotting results indicate that a related protein is present in most thermophilic archaebacteria and in Escherichia coli. This protein complex may play an important role in the adaptation of thermophilic archaebacteria to life at high temperature.