Growth of the Stress-Bearing and Shape-Maintaining Murein Sacculus of Escherichia coli

To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a covalently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of a multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.     

Tertiary structure of bacterial murein: the scaffold model

Although the chemical structure and physical properties of peptidoglycan have been elucidated for some time, the precise three-dimensional organization of murein has remained elusive. Earlier published computer simulations of the bacterial murein architecture modeled peptidoglycan strands in either a regular (D. Pink, J. Moeller, B. Quinn, M. Jericho, and T. Beveridge, J. Bacteriol. 182: 5925-5930, 2000) or an irregular (A. Koch, J. Theor. Biol. 204: 533-541, 2000) parallel orientation with respect to the plasma membrane. However, after integrating published experimental data on glycan chain length distribution and the degree of peptide side chain cross-linking into this computer simulation, we now report that the proposed planar network of murein appears largely dysfunctional. In contrast, a scaffold model of murein architecture, which assumes that glycan strands extend perpendicularly to the plasma membrane, was found to accommodate published experimental evidence and yield a viable stress-bearing matrix. Moreover, this model is in accordance with the well-established principle of murein assembly in vivo, i.e., sequential attachment of strands to the preexisting structure. For the first time, the phenomenon of division plane alternation in dividing bacteria can be reconciled with a computer model of the molecular architecture of murein.     

Tertiary structure of Staphylococcus aureus cell wall murein

The recently described scaffold model of murein architecture depicts the gram-negative bacterial cell wall as a gel-like matrix composed of cross-linked glycan strands oriented perpendicularly to the plasma membrane while peptide bridges adopt a parallel orientation (B. A. Dmitriev, F. V. Toukach, K. J. Schaper, O. Holst, E. T. Rietschel, and S. Ehlers, J. Bacteriol. 185:3458-3468, 2003). Based on the scaffold model, we now present computer simulation studies on the peptidoglycan arrangement of the gram-positive organism Staphylococcus aureus, which show that the orientation of peptide bridges is critical for the highly cross-linked murein architecture of this microorganism. According to the proposed refined model, staphylococcal murein is composed of glycan and oligopeptide chains, both running in a plane that is perpendicular to the plasma membrane, with oligopeptide chains adopting a zigzag conformation and zippering adjacent glycan strands along their lengths. In contrast to previous models of murein in gram-positive bacteria, this model reflects the high degree of cross-linking that is the hallmark of the staphylococcal cell wall and is compatible with distinguishing features of S. aureus cytokinesis such as the triple consecutive alteration of the division plane orientation and the strictly centripetal mode of septum closure.     

Molecular dynamics simulation of glycoprotein-glycans of immunoglobulin G and Immunoglobulin M

Glycoprotein-glycans have recently been implicated to play a variety of functional roles. The same glycan chain have been found complexed with proteins of diverse functions. In this article two such glycan chains found attached to Fc regions of immunoglobulin G and immunoglobulin M have been studied. An extensive simulated annealing procedure have been adopted to arrive at a low-energy minimum of the two oligosaccharides. Molecular dynamics simulations have been performed to study the flexibility of the glycosidic linkages. It was found that both glycan chains can undergo conformational transitions and adopt folded and extended conformations. The two β(1–2) linkages of complex-type glycan had been found to prefer different conformational regime and the terminal fucose linked to the GlcNAc residue drastically modifies the GlcNAc β(1–4)GlcNAc linkage conformation. In the high-mannose type glycan chain α(1–3) linkages can induce flexibility in addition to the α(1–6) linkages. The results have been compared with recent experimental nmr and fluorescence energy transfer data. © 1998 John Wiley & Sons, Inc. Biopoly 45: 177–190, 1998     

Synthesis and processing of a type I procollagen containing shortened pro-alpha 1(I) chains by fibroblasts from a patient with osteogenesis imperfecta

Skin fibroblasts from a patient with a lethal form of osteogenesis imprefecta were found to synthesize equal amounts of normal pro-alpha 1(I) chains and pro-alpha 1(I) chains which are about 10% shorter because of a deletion of about 100 amino acids in the middle of the alpha chain domain. The pro-alpha 1(I) chains were incorporated into three different kinds of trimers: a normal type I trimer with normal length pro-alpha 1(I) chains; a type Is trimer with one shortened pro-alpha 1(I) chain and two normal length chains; and a type Iss trimer containing two shortened pro-alpha 1(I) chains and one normal length pro-alpha 2(I) chain. As judged by resistance to digestion by chymotrypsin and trypsin, the type Is and Iss trimers denatured at a temperature at least 3 degrees C lower than normal type I procollagen. Procollagen containing the shortened pro-alpha 1(I) chains was slowly secreted by the cells but was degraded by extracellular proteinases within 6 h of chase into the medium. The results indicated that the presence of the shortened pro-alpha 1(I) chains in procollagen trimers produces a delay in rate of helix formation, overmodification of the polypeptides by post-translational enzymes, a decrease in the thermal stability of the trimers, and increased susceptibility of the protein to endogenous proteinases. Additionally, the fibroblasts of this patient synthesized and secreted a type III-like species of procollagen with unusual chromatographic properties.     

Effect of glycosylation on structure and dynamics of MHC class I glycoprotein: a molecular dynamics study

Complex carbohydrates linked to glycoproteins are recently being implicated to play a variety of biological roles. The lack of well-resolved crystallographic coordinates of the carbohydrates makes it difficult to assess the contributions of the glycan chain on protein structure and dynamics. We have modeled two different oligosaccharides NeuNAc2Gal3Man3GlcNAc5Fuc and Man3GlcNAc4 to generate two glycosylation variants of major histocompatibility complex (MHC) class I glycoprotein. Molecular dynamics simulations of the isolated fourteen- and seven-residue oligosaccharides have been done in vacuo and in solution. The dynamics of the two glycoforms of MHC class I protein have been simulated in solution in the free as well as in the peptide-bound form. Good agreement between the calculated solution conformations of the oligosaccharides in isolated and conjugated forms and the average conformations obtained from x-ray or NMR data was observed for most of the glycosidic linkages. These molecular dynamics simulations of the isolated glycan chains and the glycoconjugates reveal the details of the conformational flexibility of the glycan chains; they also provide atomic level details of protein-carbohydrate interactions and the effect of the ligand binding on the carbohydrate structure and dynamics. It was found that though there is some flexibility in some of the glycosidic linkages in the isolated oligosaccharides, in the protein-conjugated form the linkages adopt more restricted conformations. The glycan chains protrude out into the solvent and might hinder the lateral association of the proteins. The presence of the bulky glycan chains does not affect the average backbone fold of the protein but induces local changes in protein structure and dynamics. It has been noted that the extent of the changes depends upon the nature of the attached glycan chain. The glycan chains do not appear to influence the peptide binding property of the protein directly, but may stabilize the protein residues that are involved in ligand binding.     

Structural studies on the bacterial cell wall peptidoglycan pseudomurein. I. Conformational energy calculations on the glycan strands in C1 conformation and comparison with murein

Conformational energy calculations have been used to explore the conformations which may be realized for the sugar moiety of murein and pseudomurein. For the building blocks of the pseudomurein sugar strands, i.e. for the monosaccharides beta-D-N-acetylglucosamine (NAG) and alpha-L-N-acetyltalosaminuronic acid (NAT), both in C1 ring conformation, as well as for their 1,3 and 1,4 linked disaccharides, the favoured conformations were obtained. The helical parameters of sugar strands of both linkage types, which describe the regular structure of the corresponding polysaccharides, poly-(1,3-NAT-NAG) and poly-(1,4-NAT-NAG), were calculated. Both types of polysaccharides poly-(NAG-NAT) considered in this study favoured extended conformations, which in the case of 1,3 linked polymers showed less gain of length per saccharide unit compared to 1,4 linked poly-(NAG-NAT) residues. For a 1,3 linked sugar moiety of pseudomurein every pair of neighbouring peptides attached to glycan chain pointed in favoured conformations approximately to opposite sides of the strands, whereas in a 1,4 linked poly-(NAG-NAT) the peptides protruded approximately to the same side of the glycan moiety. A comparison between pseudomurein and murein revealed that the sugar moieties of both peptidoglycans have similar features in respect to their overall structure, i.e. both favoured more or less extended structures. In contrast to these data the shapes of the resulting polysaccharide moieties were remarkably different. In poly-(1,3-NAG-NAT) the glycan chains possessed a zig-zag-like arrangement, whereas for glycan chains of the murein type relatively flat structures were preferred. These remaining differences in the conformational arrangement between both peptidoglycans depend strongly on the C1 chair conformation of NAT. It is, therefore, attractive to speculate about an hypothetical pseudomurein sugar chain configuration comprising beta-L-N-acetyltalosaminuronic acid in its 1C conformation.     

Length distribution of the peptidoglycan chains in the sacculus of Escherichia coli

The stress-bearing fabric of bacteria is made of peptidoglycan. This crosslinked fabric is formed from disaccharide pentapeptide units that are transported through the cytoplasmic membrane and then polymerized in two directions: (i) to form oligoglycan chains; and (ii) to cross link these chains by tail-to-tail bonds from the muropeptides to the protruding peptides of other chains. The distribution of the glycan chain lengths is reminiscent of the "most probable distribution of polymer chemistry. Of course, the process is more complex than solely the random addition of units to growing chains. The complexity precludes mathematical analysis, but computer modeling of the Monte Carlo type is capable of including a range of possibilities. At each time point a specified number of disaccharides are singly added to the muramic acid residue ends of existing chains chosen at random. The transfer is in exchange for the cleavage of pyrophosphate bactoprenol that transported the disaccharide pentapeptide through the membrane. The progam then selects, again at random, which chain to cleave and between which two disaccharides of the chain the cleavage event is to occur. The cleavage generates an N -acetyl 1,6 anhydro-muramic acid end and a non-reducing N -acetyl glucosamine end. The simulation can be modified so that the program does not cleave off a disaccharide next to either end of the chain. Comparisons are shown with the experimental results of Obermann & H]oltje (1994. Microbiology140, 79-87.) They obtained their data by taking the results with normal growing cells and subtracting the similar data from minicells to estimate the chain length distribution in the cylinder part of the cell. In its most basic form the computer simulation has only one fitted parameter, K, which is the number of disaccharides added to the murein for every internal cleavage event. In this form the fitting to the experimental results is poor. One possible reason for this is that the tension on the chains, and therefore the probability of being cleaved by autolysins varies with orientation of the chain on the cylinder surface. It is well known that the tension in the cylindrical wall is twice as large in the circumferential direction as in the axial one, so one class would consist of those chains aligned longitudinally, subject to lower stress, and would have a higher energy of activation for autolysis than chains aligned circumferentially. A good fit is obtained on the assumption that there are only two classes of chains; one more likely to be cleaved than the other. The key point is that only two processes: adding of disaccharide pentapeptides at random to glycan chains and cleavage between the disaccharides at random, together with the assumption that the wall is less easily hydrolysed in the axial direction is sufficient to account for the experimental distribution.     

Molecular determinants of self-association and rearrangement of a trimeric intermediate during the assembly of a parvovirus capsid

The minute virus of mice (MVM) provides a simple model for the dissection of the molecular determinants of the self-assembly, stability, and dynamics of a biological supramolecular complex. MVM assembly involves the trimerization of capsid subunits in the cytoplasm; trimers are transported to the nucleus, where they suffer a conformational change and are made competent for capsid formation. Our previous study revealed that capsid assembly from trimers is dependent on stronger intertrimer interactions that are equally spaced in an equatorial belt surrounding each trimer. We have now targeted the interfaces between monomers within each trimer to identify the molecular determinants of trimerization and the rearrangement needed for capsid assembly. Twenty-eight amino acid residues per monomer were individually mutated to alanine to remove most of the stronger intersubunit interactions. The effects on trimer and capsid assembly and virus infectivity in cells were analyzed. No side chain was individually required for trimer assembly in the cytoplasm; in contrast, half of them were required to make the trimers competent for nuclear capsid assembly, even though none was close to intertrimer interfaces. These critical side chains are conserved and participate in extensive hydrophobic contacts, buried hydrogen bonds, or salt bridges between subunits. This study on MVM capsid assembly reveals that: (i) trimerization is a robust process, insensitive to removal of individual intersubunit interactions; and (ii) the rearrangement of the trimer intermediate required for capsid assembly is a global process that depends on the establishment of many interactions along the protein-protein interfaces within each trimer.     

Murein biosynthesis in synchronized cells of Proteus mirabilis. Quantitative analysis of O-acetylated murein subunits and of chain terminators incorporated into the sacculus during the cell cycle

Cells of Proteus mirabilis, synchronized by sucrose density gradient centrifugation, were grown in complex medium containing radioactive N-acetylglucosamine. At various times, labelled murein sacculi were isolated and digested with endo-N,O-acetylmuramidase from Chalaropsis. The murein fragments thus obtained were separated into disaccharide peptides as the monomeric subunits and into peptide-cross-linked subunits by gel filtration. The subunits were further differentiated into O-acetylated and non-O-acetylated species, and into subunits containing anhydro-N-acetylmuramic acid which were glycan chain terminators in the native sacculi. Quantification of the subunit species gave the following results. At specific times during the cell cycle, murein subunits were lost from the polymer and a transient decrease in cross-linkage was observed. The overall degree of cross-linkage in mature murein, i.e. the ratio of peptide-cross-linked subunits versus uncross-linked subunits, was 1.15 as determined by regression analysis. Anhydro-N-acetylmuramic-acid-containing murein subunits representing glycan chain terminators were found either peptide-cross-linked or uncross-linked as monomers. Since these two subunit species were recovered in a defined ratio of 1.6, mature murein consisted of at least two different types of glycan chains. On average, each chain contained 15.4 murein subunits. About 60% of the murein subunits in mature murein were O-acetylated and showed a higher degree of cross-linkage than the non-O-acetylated portion. Finally, following the composition of the sacculus during the cell cycle revealed a complex precursor-product relationship between non-O-acetylated and O-acetylated subunits during murein maturation. The data allowed us to deduce several features of the assembly process of murein sacculi.     

A model for the three-dimensional structure of peptidoglycan in staphylococci

Although the monomeric units of peptidoglycan in Staphylococcus aureus and other staphylococci are well known, the complete structure of the peptidoglycan has not been elucidated. The peptidoglycan monomeric unit may be divided into three parts: (1) glycan chain piece, consisting of N-acetylglucosaminyl-N-acetylmuramic acid; (2) connecting peptide extending from L-alanine to the alpha-amino group of L-lysine; (3) peptide chain piece, consisting of D-alanine, the remainder of L-lysine not included in the connecting peptide, and pentaglycine (S. aureus) or mixed glycine and serine residues (other staphylococci) attached to the epsilon amino group of lysine. The deformation of cross wall into hemisphere in the course of cell division, the distensibility of peptidoglycan, and the appearance of circular (? spiral) lines in the cross wall and on the surface of the newly-formed hemisphere are clues to the structure of peptidoglycan. In the proposed model, cross wall is formed as a linear spiral with 20 turns extending in a plane from periphery to center of the cell. During cell division, the cross wall is bisected. The cross wall spiral becomes a spiral forming the peripheral wall of a new hemisphere. The width of the spiral on the cell surface is maintained by rigid glycan chains and by covalent bonds linking turns of the spiral. The length of the spiral is about 30 times the diameter of the cell. Flexible polypeptide sheets consisting of parallel polypeptide chains run along the length of the spiral. Individual polypeptides contain an average of ten peptide chain pieces. The glycan chain is a helix with two disaccharide residues per turn; consequently consecutive connecting peptides project in opposite directions and are perpendicular both to the glycan chain and to the peptide chain. In cross wall, hydrogen bonding between polypeptide chains enables the polypeptide sheet to transmit changes in tension. The deformation of cross wall into peripheral wall requires doubling of the external surface area of the peptidoglycan. A change in the angle of the glycan chain with respect to the peptide chain results in an increase of the distance between peptide chains, causing the doubling of surface area. Implications of the model include explanations for the initiation of cell division and for the existence of osmotically growth-dependent staphylococci.     

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.     

Changes in the composition of Escherichia coli murein as it ages during exponential growth

Escherichia coli murein was specifically labeled with [14C]diaminopimelic acid in the mutant strains W7 (dap lysA) and BUG6. Pulse-labeled heat-denatured E. coli cells were digested with 2 mg of egg-white lysozyme per ml to degrade the murein completely and free any lipoprotein-bound muropeptide trimers, dimers, and monomers. Pulse-chase experiments showed that the relative percentage of trimers and dimers found in the newly synthesized murein increased somewhat with time at the expense of monomers. The increase in cross-links indicated that the radioactive monomers served as acceptors in multisite transpeptidations occurring after the labeling period. The content of nonreducing monomers (C7 and C8) remained unaltered, indicating that the oligosaccharide chain length did not change with time. A gradual conversion of the reducing disaccharide tetrapeptide monomer to its tripeptide analog occurred during chasing. Braun lipoprotein was linked to about 2% of the murein subunits within 30 s of the incorporation of subunits into insoluble murein, and after one-half a generation of chase, lipoprotein-associated muropeptides had approached the maximum (16% of the total murein subunits). The distribution of muropeptides was similar in lipoprotein-linked and lipoprotein-free murein, showing that the enzyme that links Braun lipoprotein to murein does not discriminate between monomers, dimers, and trimers. No evidence for a chasable, soluble polymer of murein was found in our experiments. Hence, our data support the idea that new murein is incorporated directly into the sacculus without first existing as a soluble intermediate.     

Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12

Bacterial peptidoglycan (PG or murein) is a single, large, covalently cross‐linked macromolecule and forms a mesh‐like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre‐existing cross‐links for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross‐bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross‐link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.     

Two proposed general configurations for bacterial cell wall peptidoglycans shown by space-filling molecular models

Bacterial cell wall peptidoglycans are built from unbranched β-(1 → 4)-linked glycan chains composed of alternately repeating units of N-acetylglucosamine and N-acetylmuramic acid residues, with peptide side chains attached to the muramic acid residues. The glycan chains are interconnected by peptide bonds formed between the peptide side chains. Through the use of three-dimensional molecular models, two configurations of the glycan strands and the peptide side chains are described, which by their constancy of form reflect the fundamental constancies of the covalent structures. Each of these two models will accommodate any chemical modification that has been observed in bacteria without change in the configuration of the peptide backbone. Some alterations in the chemical structure, which have been sought in bacteria, but not found, would not be tolerated by the models. In these models, glycan strands are parallel, with their lengths and widths predominantly in the plane of the cell wall. The cross-bridging portions of the peptide side chains are at right angles to the glycan strand, in a separate, parallel plane. A compact model is presented in which the peptide side chain is closely appressed to the glycan strand and is stabilized by three hydrogen bonds per disaccharide–peptide subunit. In a second model, the peptide side chain is raised away from the glycan strand in an entirely extended configuration. The compact and extended forms are interconvertible. The thickness of a sheet of peptidoglycan would be from 10.6 to 11.1 Å for the compact model, and 19.1 Å for the extended model.     

Incorporation of diaminopimelic acid into the old poles of Escherichia coli

The surface stress theory of the ontogeny of the bacterial rod depends critically on whether the old poles continue to incorporate new material into the stress-bearing murein. If insertion of peptidoglycan continues, then seemingly the shape must become gradually rounder due to the surface stress resulting from the internal hydrostatic pressure. We have reanalysed our earlier experimental data by classifying grains with respect to distance from the nearest pole, and not from the cell centre as was done previously, and conclude that old poles do incorporate new diaminopimelic acid residues. This eliminates the model we have proposed for Gram-positive rods, which assumed diffuse growth on the cylindrical sides and that poles once formed would be rigid. The new results are consistent with another model (presented elsewhere) in which insertion of new murein occurs all over the surface, although not equally. This new model leads to elongation and division if the energetics of wall expansion is altered by the cell in a control region at a particular point of the cycle by the cell.     

Isolation and analysis of cell wall components from Streptococcus pneumoniae

The complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is composed of peptidoglycan and a covalently attached wall teichoic acid. The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides. We have developed a method to purify the glycan chains, and we show that they are longer than approximately 25 disaccharide units. From purified peptidoglycan, we released 50 muropeptides that differ in the length of their peptides (tri-, tetra-, or pentapeptides with or without mono- or dipeptide branch), the degree of peptide crosslinking (monomer, dimer, or trimer), and the presence of modifications in the glycan chains (N-deacetylation, O-acetylation, or lack of GlcNAc or GlcNAc-MurNAc) or peptides (glutamic acid instead of glutamine). We also established a method to isolate wall teichoic acid chains and show that the most abundant chains have 6 or 7 repeating units. Finally, we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls. These novel tools will help to characterize mutant strains, cell wall-modifying enzymes, and protein-cell wall interactions.     

Molecular modelling of MHC class I carbohydrates

In this article we present the results of molecular modelling of four complex carbohydrates which have been found in the MHC class I proteins. Though these proteins show diversity in their sequences, the glycosylation sites are highly conserved indicating a possible structural/functional role of the glycan chain. Similar glycan chains have been found linked with other proteins of completely different function, such as IgG, and erythropoeitin. Thus, the molecular modelling of these carbohydrates will not only provide structural/dynamic information of these complex molecules but will also provide conformational information which can be utilised to build the glycoprotein models. The results presented here indicate that although several linkages show less conformational flexibility, terminal linkages can be quite flexible.     

The molecular defect in an autosomal dominant form of osteogenesis imperfecta. Synthesis of type I procollagen containing cysteine in the triple-helical domain of pro-alpha 1(I) chains

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Proteolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two alpha 1(I) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded alpha 1(I) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in alpha 1(I)CB8, a fragment containing amino acid residues 124-402 of the alpha 1(I) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous nature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-alpha(I) chains, a type I trimer with one mutant pro-alpha 1(I) chain and two normal chains, and a type I trimer containing two mutant pro-alpha 1(I) chains and one normal pro-alpha 2(I) chain. The presence of one or two mutant pro-alpha 1(I) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 degree C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-alpha 1(I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-alpha 1(I) chains synthesized by the patient's fibroblasts.     

Analysis of the length distribution of murein glycan strands in ftsZ and ftsI mutants of E. coli

The chain length distribution of murein glycan strands was analyzed in wild-type cells and in cells in which preseptal and/or septal murein synthesis was prevented in ftsZ84 and ftsI36 mutants of E. coli. This revealed a significant change in glycan chain lengths in newly synthesized murein associated with inactivation of the ftsZ gene product but not with inactivation of the ftsI gene product. This is the first reported abnormality in murein biosynthesis associated with mutation of an essential cell division gene.