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The sulphur‐dependent archaebacterium Thermoproteus tenaxhas a cylindrical cell shape variable in length, but constant in diameter. Its whole surface is covered by a regular protein layer (S‐layer). The lattice has p6 symmetry and a lattice constant of 32.8 nm. The three‐dimensional reconstruction from a tilt series of isolated and negatively stained S‐layer shows a complex mass distribution of the protein: a prominent, pillar‐shaped protrusion is located at the 6‐fold crystallographic axis with radiating arms connecting neighbouring hexamers in the vicinity of the 3‐fold axis. The base vectors of the S‐layer lattice have a preferred orientation with respect to the longitudinal axis of the cell. The layer can be seen as a helical structure consisting of a right‐handed, two‐stranded helix, with the individual chains running parallel. Supposing that new S‐layer protein is inserted at lattice faults (wedge disclinations) near the poles, growing of the layer would then proceed by moving a disclination at the end of the helix. The constant shape of the cell, as well as the particular structure of the layer, strongly suggest that this S‐layer has a shape‐maintaining function.

Porin from Escherichia coli outer membranes has been analysed by high angle diffuse X‐ray diffraction, and by attenuated total reflection infrared spectroscopy. These methods demonstrate independently that the majority of the polypeptide backbone is arranged in anti‐parallel beta‐pleated sheet structure. The average length of the beta‐strands, which are oriented nearly normal to the membrane plane, is estimated to be 10‐12 residues, independent of the method used. Although the details of strand arrangement (beta‐barrels or stacked sheets) are not as yet known, porin represents the first transmembrane protein for which beta‐structure has been established unequivocally.

The stalks (hyphae) of a prosthecate bacterium, directly sampled from the water surface of a hot pond, show extended regular patterns on their envelope in the electron microscope. Image processing revealed a structure of the crystalline complexes which is very similar to the gross morphology of the Escherichia coliporins OmpC and OmpF. The natural two-dimensional crystal of the outer membrane protein has p3 symmetry and a lattice constant of 7.95 nm. The three-dimensional structure of the stalk porin has been determined to an almost isotropic resolution of 1.7 nm. The reconstruction revealed a complex network of channels within the membrane matrix with a triplet of pores merging into a common outlet, similar to the structure of the E. coliporin OmpF in reconstituted membranes. In addition, a blindly ending pore exists which appears to be connected to the continuous pores via small channels. The significance of the regularly arrayed porin cylinders with respect to the shape and function of the stalks is discussed.

We have studied the mechanism of inhibition of the recombinant Rhodococcus proteasome by four different chemical classes of active site-directed small molecule inhibitors. Clasto-lactacystin beta-lactone is a time-dependent inhibitor of the Rhodococcus proteasome's ability to hydrolyze Suc-Leu-Leu-Val-Tyr-AMC, a substrate for this proteasome's single type of active site, and proceeds with a kinact/[I] of 1,700 M-1 s-1. Using peptide mapping of tryptic digests, LC/MS, and amino acid sequence analysis, we have established that the Ogamma of the hydroxyl group on the N-terminal threonine of the beta-subunit is the sole modification made by the beta-lactone. Active site titrations of the Rhodococcus proteasome with reversible peptide aldehydes show the expected stoichiometry of one inhibitor molecule per beta-subunit. Prior modification with beta-lactone completely abrogates the binding of peptidyl boronic acid inhibitors, suggesting that these inhibitors also inactivate the enzyme by reacting with the Ogamma moiety on Thr1. High performance liquid chromatography analysis of peptidyl vinyl sulfone-modified intact Rhodococcus proteasome beta-subunit and its tryptic peptides suggests that the peptidyl vinyl sulfone modifies a residue in the N-terminal 20 amino acids. This modification is also blocked by prior treatment with beta-lactone.

A gene has been cloned from the archaebacterium, Thermoplasma acidophilum, which, on the basis of the deduced amino acid sequence, encodes a homologue of the eukaryotic large subunit ribosomal proteins, L15/YL10. This gene, rpl15, was identified as an open reading frame (ORF) located 2.3 kb upstream of the gene encoding the α-subunit of the T. acidophilumproteasome. The putative translation product of rpl15(RPL15) contains 197 amino acid residues, with a Mrof 22,928 and a basic pIof 11.59. The RPL15 amino acid sequence shows significant similarity (>35% identity) to the L15/YL10 proteins of various eukaryotes.

On electron micrographs, negatively stained multicatalytic proteinase molecules are viewed end-on (ring shaped) or side-on (rectangular shaped). For aurothioglucose, ammonium molybdate- and phosphotungstate-stained molecules, the dimensions measured are consistent. In contrast, uranyl acetate-staining reveals ring-shaped particles which vary in diameter between 12 and 16 nm. This is due to a partial collapse and substantial flattening of the structure. Digital image analysis of side-on views of the particles reveals a tripartite, reel-shaped structure. Within the ring-like, end-on projections of ammonium molybdate-stained molecules six local centres of mass can be discerned; their position appears to depart, however, from a true six-fold symmetry.

The paracrystalline outer membraneous layer (HPI layer) of Micrococcus radiodurans has been investigated by negative- and positive-staining electron microscopy and subsequent digital image processing. The subunit structure of the major HPI layer protein complex and the lipid-protein distribution in the plane of the membrane have been determined. The HPI layer was found to be highly asymmetric in a transmembrane direction, with the major protein complex only partly penetrating into a lipid-containing backing layer intimately associated with it.

Acetogenium kivui is anaerobically growing thermophilic bacterium with a gram-positive type of cell wall structure. The outer surface is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer polypeptide was cloned in Escherichia coli on two overlapping fragments by using the plasmid pUC18 as the vector. It was expressed under control of a cloned Acetogenium promoter or the lacZ gene. We determined the complete sequence of the structural gene. The mature polypeptide comprises 736 amino acids and is preceded by a typical procaryotic signal sequence of 26 amino acids. It i weakly acidic, weakly hydrophilic, and contains a relatively high proportion of hydroxyamino acids, including two clusters of serine and threonine residues. An N-terminal region of about 200 residues is homologous to the N-terminal part of the middle wall protein, one of the two S-layer proteins of Bacillus brevis, and there is also an internal homology within the N-terminal region of the A. kivui polypeptide.

Cell-free culture supernatants of a lipopolysaccharide (LPS) O-polysaccharide-deficient, single-insertion transposon mutant of the tetragonal surface protein array (S layer)-containing fish pathogen Aeromonas salmonicida were examined by electron microscopy. Negative staining showed that the S layer was released as sheets of tetragonal material, indicating that although surface retention of assembled S layer requires the presence of wild-type LPS oligosaccharides, initial assembly of S-layer subunits into sheets does not require the presence of O-polysaccharide chains. The three-dimensional structure of the S layer was reconstructed from tilted micrographs of the released sheets. Horizontal sections through this reconstruction showed that the released sheets were composed of two identical S layers that were perfectly in register. The reconstructed layer had a lattice constant of 12.5 nm. At a resolution of 1.6 nm, the layer consisted of a major tetragon at one fourfold axis of symmetry and a minor tetragon at the second fourfold axis of symmetry. The core, composed of four of the major domains, contained a large depression and was located toward the inside of the layer. The minor tetragon provided connectivity within the layer and was located toward the outer surface of the layer. Projections through the double layer gave a type I (closed) pattern (M. Stewart, T. J. Beveridge, and T. J. Trust, J. Bacteriol. 166:120-127, 1986), yet projections through the single layer indicated that the type II (open) pattern was present. This open pattern was indistinguishable from that seen in S layer released from the surfaces of wild-type cells.

The spherical cells of the thermophilic, sulfur-dependent archaebacterium Desulfurococcus mobilis are completely covered with a relatively poorly ordered, tetragonally arrayed surface protein. The structure of this surface protein was examined by using three-dimensional electron microscopy. The protein lattice forms an open meshwork composed of cross-shaped morphological units, which are released when glycerol is added. These subunits make contact at the distal ends of their four arms. The p4 symmetry requires that each of these morphological subunits represents a tetramer. The strong interaction of the monomers within the crosses and the relatively weak interaction of the intersecting arms of the crosses within the lattice structure suggest that the tetramers are assembled before their incorporation into the lattice.

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.

Fragments of the Azotobacter vinelandii tetragonal surface (S) layer, free of outer membrane material, were obtained by treating whole cells with 100 microM EDTA. The three-dimensional structure of the S layer was reconstructed from tilted-view electron micrographs of the S-layer fragments, after computer-assisted image processing by correlation averaging. At a resolution of 1.7 nm, the S layer exhibited funnel-shaped subunits situated at one fourfold-symmetry axis and interconnected at the other fourfold-symmetry axis to form prominent cruciform linking structures. These data, in conjunction with a relief reconstruction of the surface of freeze-etched whole cells, indicated that the apex of the funnel-shaped subunit was associated with the outer membrane, while the funnel "opening" faced the environment; the cruciform linking structures were formed at the outermost surface of the S layer. Electron microscopy and image enhancement were used to compare the structure of the outer membrane-associated S layer with that of fragments of the S layer dislodged from the outer membrane. This analysis revealed an increase in the lattice constant of the S layer from 12.5 to 13.6 nm and an alteration in the position of the cruciform linking structures in the z direction. These conformational changes resulted in a reduction in the thickness of the S layer (minimum estimate, 5 nm) and an apparent increase in the size of the gaps between the subunits. In terms of the porosity of the S layer, this gave the appearance of a transition from a closed to a more open structure.

The three-dimensional structure of the regular surface layer of Sporosarcina ureae has been determined to a resolution of 1.7 nm by electron microscopy and image reconstruction. The S-layer has p4 symmetry, a lattice constant of 12.9 nm, and a minimum thickness of 6.6 nm. The reconstruction reveals a distinct domain structure: a massive core, arms connecting adjacent unit cells, and spurs which make contact at the subsidiary fourfold symmetry axes. In the z-direction the domains appear to be arranged in three planes, creating two entirely different surface reliefs. The S-layer has a complex pattern of pores and gaps that are 2 to 3 nm wide. In addition, the secondary-structure composition has been determined by infrared spectroscopy: about 35% of the polypeptide appears to have a beta-structure conformation.

The HPI protein of Deinococcus radiodurans belongs to the class of surface layer proteins which form crystalline two-dimensional arrays on bacterial cell envelopes. We have cloned and expressed the gene for this protein of Mr about 100,000 by using plasmid pUC8 in Escherichia coli. As judged by immunoreaction with monospecific antibodies, apparent Mr, and limited proteolysis, a single clone contained the gene encoding the complete polypeptide on an 8.9-kilobase (kb) insert. The insert was reduced to a 5.7-kb HindIII fragment, cloned in pUC18 in both orientations, and subjected to unilateral processive deletion with exonuclease III. The library of deletion derivatives was mapped and, in conjunction with protein immunoblotting of expressed polypeptides, was used to locate the positions of the structural gene and the Deinococcus promoter region that was responsible for expression of the HPI polypeptide. The HPI gene was confined to a stretch 2.95 kb in length.

Two-dimensional (2-D) crystallization experiments at the water−air interface with recombinant apoferritin (horse liver) are described using a recently developed protein spreading technique, which is based on density and surface tension differences of the subphase and the injected protein solution. The preparation protocol was modified to investigate the influence of additives such as glutaraldehyde and CdSO4 on the crystallization process. The crystalline arrays were examined by electron crystallographic techniques. It turned out that addition of the cross-linker glutaraldehyde to the subphase buffer was crucial for maintaining the integrity of a fragile 2-D crystal of the square lattice type, which was not observed previously during transfer onto electron microscopy grids.