Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.     

Identification of a Cd2+- and Zn2+-binding site in cytochrome c using FTIR coupled to an ATR microdialysis setup and NMR spectroscopy

Fourier transform infrared (FTIR) difference spectroscopy allows the study of molecular changes occurring at active sites in proteins with high sensitivity. Reactions are triggered by light, potential, or temperature steps and more recently by the diffusion of buffers containing effectors above membrane proteins deposited as films on ATR crystals. We have adapted a microdialysis system to an ATR, to study metal sites in soluble proteins. In this study, we identified a Cd(2+)- or Zn(2+)-binding site in cytochrome c with dissociation constants of 17 and 42 microM, respectively, which affects the oxidation rate of ferrocytochrome c by hydrogen peroxide. Using the microdialysis ATR-FTIR setup, we determined that a histidine and the carboxylate group of a glutamate are involved in Zn(2+) binding. The implication of His 33 and Glu 104 in the binding site was deduced from the comparison of FTIR data recorded with horse heart and the variant tuna cytochrome c lacking these two amino acids. A two-dimensional NMR analysis of the Zn(2+)-binding site in horse heart cytochrome c confirmed that His 33 and residues close to the C terminus are sensitive to Zn(2+) binding. This study demonstrates that the microdialysis ATR-FTIR setup is promising for the analysis of metal sites in proteins. From H(2)O/(2)H(2)O exchange experiments, we concluded that the impact of Zn(2+) and Cd(2+) binding on the oxidation kinetics of ferrocytochrome c by H(2)O(2) is associated to the perturbation of a hydrogen-bonding network involving His 33 that is sensitive to the redox state of cytochrome c.     

Phosphotransferase-mediated transport of the osmolyte 2-O-alpha-mannosyl-D-glycerate in Escherichia coli occurs by the product of the mngA (hrsA) gene and is regulated by the mngR (farR) gene product acting as repressor

2-O-alpha-mannosyl-D-glycerate (MGs) has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strainMC4100. Uptake is mediated by the P-enolpyruvate-dependent phosphotransferase system with the MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent Km of MG uptake in induced cells was 10 microm, and the Vmax was 0.65 nmol/min/10(9) cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with alpha-mannosidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system.     

Modeling of arabinofuranose and arabinan,II. Nmr and Conformational analysis of arabinobiose and arabinan

The disaccharide arabinobiose (5-O -α-L -arabinofuranosyl-α-L -arabinofuranose) constitutes the basic repeating structures found in such polysaccharides as arabinan or in the side chains of the hairy regions of pectins. The conformational behavior of aqueous arabinobiose has been investigated by high resolution nmr and computerized molecular modeling. The complete conformational analysis of the, disaccharide has been achieved with the MM3 molecular mechanics methods using the flexible residue method. In this study, both the puckering of the arabinofuranose, rings and the orientations about the glycosidic torsion angles ?, ψ, and ω; were considered. Some insights into conformational transitions were obtained through molecular dynamics simulation using the CHARMM force field. In parallel, transient nuclear Overhauser effects at 400.13 MHz and long-range vicinal homonuclear and heteronuclear coupling constants have been measured. The theoretical nmr data were calculated taking into account all accessible conformations and using averaging methods for both slow and fast internal motions models. The data do not support a single conformational model, and only conformational averaging yields the excellent agreement between the observed and simulated parameters. Within the potential energy surfaces computed for the disaccharide, several low energy conformers can be identified. When these conformations are extrapolated to regular polysaccharide structures, they generate chains of arabinan displaying right- and left-handed chirality and a wide range of repeating units per turn of helix. © 1994 John Wiley & Sons, Inc.     

Automated High-Throughput Quantification of Mitotic Spindle Positioning from DIC Movies of Caenorhabditis Embryos

The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes.To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics.Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.     

Comparing aspirations: intercalibration of ecological status concepts across European lakes for littoral diatoms

Eleven European countries participated in an exercise to harmonise diatom-based methods used for status assessment in lakes. Lakes were divided into low, medium and high alkalinity types for this exercise. However, it was not possible to perform a full intercalibration on low alkalinity lakes due to the short gradient and confounding factors. Values of the Trophie Index were computed for all samples in order that national datasets could all be expressed on a common scale. Not all participants had reference sites against which national methods could be standardised and, therefore, a Generalised Linear Modelling approach was used to control the effect of national differences in datasets. This enabled the high/good and good/moderate status boundaries to be expressed on a common scale and for deviations beyond ±0.25 class widths to be identified. Those countries which had relaxed boundaries were required to adjust these to within ±0.25 class widths whilst the intercalibration rules allowed those countries with more stringent boundaries to retain these. Despite biogeographical and typological differences between countries, there was broad agreement on the characteristics of high, good and moderate status diatom assemblages, and the exercise has ensured consistent application of Water Framework Directive assessments around Europe.     

Central Domain of DivIB Caps the C-terminal Regions of the FtsL/DivIC Coiled-coil Rod

DivIB(FtsQ), FtsL, and DivIC(FtsB) are enigmatic membrane proteins that are central to the process of bacterial cell division. DivIB(FtsQ) is dispensable in specific conditions in some species, and appears to be absent in other bacterial species. The presence of FtsL and DivIC(FtsB) appears to be conserved despite very low sequence conservation. The three proteins form a complex at the division site, FtsL and DivIC(FtsB) being associated through their extracellular coiled-coil region. We report here structural investigations by NMR, small-angle neutron and x-ray scattering, and interaction studies by surface plasmon resonance, of the complex of DivIB, FtsL, and DivIC from Streptococcus pneumoniae, using soluble truncated forms of the proteins. We found that one side of the “bean”-shaped central β-domain of DivIB interacts with the C-terminal regions of the dimer of FtsL and DivIC. This finding is corroborated by sequence comparisons across bacterial genomes. Indeed, DivIB is absent from species with shorter FtsL and DivIC proteins that have an extracellular domain consisting only of the coiled-coil segment without C-terminal conserved regions (Campylobacterales). We propose that the main role of the interaction of DivIB with FtsL and DivIC is to help the formation, or to stabilize, the coiled-coil of the latter proteins. The coiled-coil of FtsL and DivIC, itself or with transmembrane regions, could be free to interact with other partners.Cell division is one of the defining features of life. Understanding the division of bacteria is also required to find novel antibiotic strategies. Numerous studies, carried out mostly with the model organisms Escherichia coli and Bacillus subtilis have uncovered several components of the divisome, which can be defined as the ensemble of proteins localized at the division site and participating in the process. Comparison of genomes and deletion studies indicate that the core of the divisome comprises eight conserved, mostly essential proteins: FtsZ, FtsA, FtsK, FtsQ(DivIB), FtsL, FtsB(DivIC), FtsW, and FtsI. Fts nomenclature applies to Gram-negative organisms, whereas Div nomenclature applies to Gram-positive bacteria. These proteins are listed here in the conditional order of their recruitment to the division site of E. coli (1–4).Processes in which they participate have been attributed to several division proteins. FtsZ forms polymers with an annular distribution on the cytoplasmic side of the membrane and governs the recruitment of the other proteins. FtsA may mediate the interaction of FtsZ with the membrane. FtsK participates to the resolution of chromosome dimers, and possibly to the membrane fission. FtsI, and likely FtsW, participate to septal cell wall formation (1–4). In contrast, the roles of FtsQ(DivIB), FtsL, and FtsB(DivIC) have not been firmly linked to any particular process.FtsQ(DivIB), FtsL, and FtsB(DivIC) are positioned in the middle of the conditional order of recruitment in E. coli and B. subtilis. When the temporality of the recruitment was examined, FtsQ(DivIB) was found to belong to the late recruits, together with the proteins involved in cell wall assembly (5). In E. coli, the presence of FtsL and FtsB at the division site is mutually dependent, and their localization depends on that of FtsQ (6, 7). In B. subtilis, the presence of FtsL and DivIC at mid-cell depends on that of DivIB, at the temperature at which DivIB is essential, and reciprocally (8, 9). A complex comprising FtsQ, FtsL, and FtsB was isolated from E. coli by co-immunoprecipitation (10), and reconstituted in vitro with recombinant soluble forms of pneumococcal DivIB, FtsL, and DivIC (11). The interaction of the three proteins was also confirmed by yeast and bacterial triple hybrid (12, 13).The genes ftsL and ftsB(divIC) are essential in E. coli and B. subtilis (6, 14–16) and presumably in Streptococcus pneumoniae (17). The essentiality of ftsQ(divIB) in laboratory conditions varies between species. The gene ftsQ is essential in E. coli (18), but divIB is essential only at high temperatures in B. subtilis (9, 19), or in a chemically defined medium in S. pneumoniae (17). Under these conditions, the essentiality of DivIB appears to be a consequence of the protection from proteolysis that it provides to FtsL (8, 17).FtsQ(DivIB), FtsL, and FtsB(DivIC) are bitopic membrane proteins with an N-terminal cytoplasmic region, a single transmembrane segment, and an extracytoplasmic region. The extracellular part is necessary and sufficient for the localization and function of FtsQ(DivIB), provided that it is anchored to the membrane (e.g. Refs. 20 and 21)), although the transmembrane segment also contributes to the localization (22, 23). The extracellular part is organized in three regions termed α, β, and γ. The crystal structure of a region consisting of the α- and β-domains was solved for FtsQ from E. coli and Yersinia enterocolitica (24). The α-domain, comprising about 70 amino acids proximal to the cytoplasmic membrane, corresponds to the POTRA (for polypeptide transport-associated) domain first identified by sequence analysis and proposed to function as a molecular chaperone (25). The α- and β-domains form the conserved region of the FtsQ(DivIB) protein. The γ-region constitutes a C-terminal tail. It is highly variable in length and sequence and predicted to be unfolded. The γ-region was not observed in the structures from E. coli and Y. entercolitica, thus confirming its flexible nature (24).The α-domain in the recombinant soluble form of the extracellular part of DivIB from Geobacillus stearothermophilus was digested by trypsin and therefore considered to be largely unfolded (26). The γ-region was also removed by trypsin digestion, together with a C-terminal fragment of the β-domain. The structure of the resulting shorter β-domain from G. stearothermophilus was solved by NMR (26) and lacks the two C-terminal β-strands.Localization epitopes have been identified in the transmembrane segment, the α-domain, and a region encompassing the C-terminal part of the β-domain and γ-tail of DivIB from B. subtilis (23). Likewise in E. coli, a region in the α-domain is required for localization of FtsQ, whereas the C-terminal region of the β-domain and the last α-helix are required for recruitment of FtsL and FtsB (24). In S. pneumoniae, the essentiality of DivIB in defined medium was found to reside in the C-terminal region of the β-domain (17).No experimental structure is known for FtsL or FtsB(DivIC). Both are small proteins comprising between 90 and 140 amino acids. The number of residues is sometimes larger, as in Mycobacterium tuberculosis (384 for FtsL and 228 for FtsB), due to N- and/or C-terminal extensions consisting of mostly charged and polar amino acids or proline-rich sequences. The major part of FtsL or FtsB(DivIC) is extracellular and contains a region proximal to the transmembrane segment, predicted to form a coiled-coil of about five heptads. Coiled-coil helices associate longitudinally to mediate protein association. It is possible that the coiled-coil helices are continuations of the transmembrane helices, although a proline (known to break helices) is present in some species between the two segments. Following the coiled-coil region is a 25–35-residue long C-terminal region in both FtsL and DivIC(FtsB). This region was recently shown in FtsB to be required for interaction with FtsQ in E. coli (27).We report here the results of structural studies in solution of a ternary complex consisting of the β- and γ-segments of DivIB, and a constrained dimer of the extracellular parts of FtsL and DivIC from S. pneumoniae. Despite the coiled-coil predictions, the recombinant extracellular domains of FtsL and DivIC did not interact in vitro (11, 28). Forced dimerization was obtained by fusion with artificial coiled-coil peptides k5 and e5 (35 residues long), which are known to form a heterodimer due to their complementarity of charge, with a nanomolar dissociation constant (29). The k5- and e5-coils were fused to the extracellular domain of FtsL and DivIC, to give rise to KL and EC fusion proteins, respectively. The constrained dimer (KL/EC) was shown to interact with the extracellular part of DivIB (DivIB_ext), yielding a soluble complex amenable to structural studies (11).The overall shape of the complex and its constituents was probed using small-angle x-ray scattering (SAXS)² and small-angle neutron scattering (SANS). NMR was used to investigate the interface between the proteins by chemical shift mapping. The interaction was further investigated using surface plasmon resonance with truncated forms of the proteins. The complex of DivIB, FtsL, and DivIC is formed by the interaction of one face of the β-domain of DivIB with the C-terminal regions of FtsL and DivIC, at the tip of an elongated rod formed by the coiled-coil segments. The α-domain of DivIB and the coiled-coil regions of FtsL and DivIC remain free to interact with other proteins of the division apparatus.     

Proton NMR detection of porphyrins and cytochrome C in small unilamellar vesicles: role of the dissociation kinetic constant

The molecular tumbling of small unilamellar vesicles is not fast enough to enable the detection of (1)H NMR signals of molecules associated with phospholipids. We show that relatively fast kinetic exchange of the interacting molecules is able to induce a strong decrease of the residual homonuclear dipolar coupling, allowing the acquisition of sharp signals. At low molecule/lipids molecular ratio, this can be lead to signal broadening due to exchange at intermediate rates on the NMR chemical timescale. However, proton resonances can be easily detected when sufficient lipids are added to prevent the occurrence of any free compounds in solution. This is demonstrated, using lipid signal suppression, in the case of paramagnetic porphyrin derivatives as well as diamagnetic hematoporphyrin. Since several peptides and proteins are expected to be associated with lipids having relatively fast dynamics, this study addresses, as a first example, the interaction of cytochrome c.