An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Regulation by molluscan myosins

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.     

A novel small protein associated with a conjugated trienoic chromophore from membranes of scallop adductor muscle: phosphorylation by protein kinase A

Membranes enriched in sarcolemma from the cross-striated adductor muscle of the deep sea scallop have been found to contain a previously undescribed small protein of 6-8 kDa that can be released by treatment with organic solvent mixtures. This proteolipid co-purified with a non-amino acid chromophore containing a conjugated trienoic moiety. Although common in plants and algae, such a stable conjugated trienoic group is unusual for an animal cell. The N-terminal amino acid sequence of the protein was XEFQHGLFGXF/ADNIGLQ, which most strongly resembles sequences in the triacyl glycerol lipase precursor and the product of the human breast cancer susceptibility gene BRCA 1, but does not show similarity to previously described proteolipids. The protein was found to be one of the major substrates in its parent membrane for the catalytic subunit of protein kinase A, which may imply a regulatory function for this molecule.     

A possible role for the dimer ribbon state of scallop sarcoplasmic reticulum. Dimmer ribbons are associated with stabilization of the Ca(2+)-free Ca-ATPase

The Ca-ATPase activity of membranous scallop sarcoplasmic reticulum was found to be unstable when the Ca(2+)-binding sites on the Ca-ATPase were unoccupied. The decay in activity could be slowed or halted by inclusion in the preincubation medium of Na+, K+, nucleotides, ethylene glycol, or high concentrations of choline chloride. Stabilization of the Ca(2+)-free Ca-ATPase by Na+ and K+ showed a markedly different concentration dependence to that seen with activation of the Ca(2+)-activated ATPase activity by the two ions. Examination in the electron microscope of scallop membranes negatively stained in the presence of EGTA under conditions where the enzyme had been stabilized against lack of Ca2+ always showed vesicles containing dimer ribbon structures, whereas unstabilized membranes did not show dimer ribbons. There was an association between the effectiveness of a medium in stabilizing the enzyme in the presence of EGTA and the extent and quality of the dimer arrays seen in the microscope. Comparison of the range of Ca2+ concentration over which the Ca(2+)-binding sites on the scallop Ca-ATPase titrated with the range over which the dimer ribbon structural state was lost indicated that the Ca(2+)-binding sites on the Ca-ATPase must be empty for dimer ribbon formation to occur. Previous studies (Franzini-Armstrong, C., Ferguson, D. G., Castellani, L., and Kenney, L. J. (1987) Ann. N. Y. Acad. Sci. 483, 44-56) have found that the Ca-ATPase molecules in scallop adductor muscle freeze-fractured after fixation under relaxing conditions are arranged in dimer ribbons. Thus, the association of stabilization of the Ca(2+)-free Ca-ATPase with the presence of dimer ribbons implies that one function of the dimer state may be to stabilize the scallop enzyme in situ, when the Ca2+ concentration in the sarcoplasm is low and the muscle is relaxed.     

Variation of scallop sarcoplasmic reticulum Ca2+-ATPase activity with temperature

Methods for preparing native scallop sarcoplasmic reticulum vesicles, largely purified membranous scallop sarcoplasmic reticulum Ca2+-ATPase, and nonionic detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase are described. The effect of a range of polyoxyethylene-based detergents on the solubilized Ca2+-ATPase was tested. Decaethylene glycol dodecyl ether (C12E10) supported the highest levels of activity, although C12E8 and C12E9 were more routinely used. Arrhenius plots of Ca2+-ATPase activity, where the assays were carried out with the same pH at all temperatures (7.4), showed a region of nonlinearity at 10 degrees C. A very similar plot was obtained when no compensation was made for pH variation with temperature. Both the break in the Arrhenius plot and the activation energies for the scallop sarcoplasmic reticulum above and below the break were very similar to those found for lobster sarcoplasmic reticulum (Madeira, V. M. C., Antunes-Madeira, M. C., and Carvalho, A. R. (1974) Biochem. Biophys. Res. Commun. 65, 997-1003). The Arrhenius plot of the scallop Ca2+-ATPase in C12E8 no longer showed the nonlinearity at 10-12 degrees C seen with the native sarcoplasmic reticulum, but instead a break now appeared at 20-21 degrees C. This is close to the Arrhenius break temperature of rabbit Ca2+-ATPase in C12E8 and of a perturbation in C12E8 (Dean, W. L. (1982) Biophys. J. 37, 56-57).     

Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head.

The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre-power stroke or near-rigor state structures; moreover, in contrast to these structures, the SH1 helix is seen to be unwound. Here we compare the overall organization of the myosin head in these three states and show how the conformation of three flexible "joints" produces rearrangements of the four major subdomains in the myosin head with different bound nucleotides. We believe that this novel structure represents one of the prehydrolysis ("ATP") states of the contractile cycle in which the myosin heads stay detached from actin.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.