Oxygen-containing Side Chains Research Articles

Study on the influence of different side chain structures on the pyrolysis behavior of polyolefin plastic wastes

As an important component of organic solid wastes, disposal of plastic wastes (PWs) has always been a thorny issue. Thermochemical conversion is an efficient method. In-depth research on the influence of structural differences on their pyrolysis behavior is beneficial to promote the development of thermal treatment and conversion technology of PWs. The differences in the chemical structure of different polyolefin plastics at the molecule level are mainly reflected in their side chains. Herein, a series of typical polyolefin plastics with similar backbone structure and different side chain structures, including high-density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyvinyl alcohol (PVA) and ethylene-vinyl acetate copolymer (EVA), were selected for comprehensive pyrolysis characterization in this work. Advanced experimental analysis methods such as in-situ DRIFT and 2D-PCIS were adopted to investigate the evolution of two weak oxygen-containing side chains during pyrolysis of PVA and EVA, respectively. Besides, typical product distribution of six samples were systematically studied by combining SVUV-PIMS and GC/MS. Spectral analysis showed that OH side chain in macromolecular structure of PVA had the weakest thermal stability, and its oxidation reaction occurred earlier than dissociation and dehydration, while the acetoxy side chain in EVA polymers could not only be directly dissociated via deacetoxylation, but also be transformed into OH side chain via deacetylation that occurred simultaneously with deacetoxylation. Further combined with SVUV-PIMS and GC/MS, the distribution characteristics of pyrolysis products of polyolefin plastics and the rules of generation and release of typical products with temperature were obtained in detail. It was found that the release of typical oxygen-containing products was earlier or slightly earlier than the release of hydrocarbons in pyrolysis of PVA or EVA. However, the release of all hydrocarbons was almost simultaneous during the respective pyrolysis process of HDPE, PP and PS, suggesting that the evolution of oxygen-containing side chains with weak thermal stability occurred earlier under the drive of temperature. Based on the characterization results, the main evolution pathways of different side chains were proposed to explain the influence mechanism of different side chain structures on the pyrolysis behavior of polyolefin plastics. The cleavage of CC bonds mainly occurred in HDPE, PP and PS, while the evolution of side chains occurred first in PVC, PVA and EVA, and exerted different influence on the degradation of carbon skeleton.

The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability

Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.

Homology Modeling Identifies C-Terminal Residues that Contribute to the Ca2+ Sensitivity of a BKCa Channel

Activation of BKCa channels by direct Ca2+ binding and membrane depolarization occur via independent and additive molecular processes. The “calcium bowl” domain is critically involved in Ca2+-dependent gating, and we have hypothesized that a sequence within this domain may resemble an EF hand motif. Using a homology modeling strategy, it was observed that a single Ca2+ ion may be coordinated by the oxygen-containing side chains of residues within the calcium bowl (i.e., 912ELVNDTNVQFLD923). To examine these predictions directly, alanine-substituted BKCa channel mutants were expressed in HEK 293 cells and the voltage and Ca2+ dependence of macroscopic currents were examined in inside-out membrane patches. Over the range of 1–10μM free Ca2+, single point mutations (i.e., E912A and D923A) produced rightward shifts in the steady-state conductance-voltage relations, whereas the mutants N918A or Q920A had no effect on Ca2+-dependent gating. The double mutant E912A/D923A displayed a synergistic shift in Ca2+-sensitive gating, as well as altered kinetics of current activation/deactivation. In the presence of 1, 10, and 80mM cytosolic Mg2+, this double mutation significantly reduced the Ca2+-induced free energy change associated with channel activation. Finally, mutations that altered sensitivity of the holo-channel to Ca2+ also reduced direct 45Ca binding to the calcium bowl domain expressed as a bacterial fusion protein. These findings, along with other recent data, are considered in the context of the calcium bowl’s high affinity Ca2+ sensor and the known properties of EF hands.

Phenotypic Screening of Mutations in Pmr1, the Yeast Secretory Pathway Ca2+/Mn2+-ATPase, Reveals Residues Critical for Ion Selectivity and Transport

Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca(2+) and Mn(2+) ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca(2+) pumps and screened for loss of Ca(2+) and Mn(2+) transport on the basis of hypersensitivity to 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn(2+) toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn(2+) toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including (45)Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable (45)Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn(774) and Asp(778) in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca(2+) and Mn(2+) ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps.

Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.

Functional consequences of alterations to polar amino acids located in the transmembrane domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.

Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908 (ligands 1 to 6, respectively) appear to form the high affinity Ca2(+)-binding sites of the Ca2(+)-ATPase. The plasticity of the Ca2(+)-binding sites was tested by separate replacement of each of the ligands with a structurally similar oxygen-containing residue using site-specific mutagenesis. Mutant cDNAs were transfected into COS-1 cells, and ATP-dependent Ca2+ transport or partial reactions were studied in microsomes containing the expressed Ca2(+)-ATPases. In most cases where amino acid substitutions were carried out, the expressed enzymes lacked Ca2+ transport function and Ca2(+)-dependent phosphorylation by ATP. Furthermore, the mutant enzymes were phosphorylated by inorganic phosphate, even in the presence of Ca2+, which inhibits phosphorylation of the wild-type enzyme possessing intact Ca2(+)-binding sites. On mutant, however, containing an isosteric replacement of Glu by Gln at ligand 6, exhibited wild-type levels of Ca2+ transport activity and Ca2+ affinity. Two mutants exhibited properties consistent with a reduction in Ca2+ affinity. In the mutant in which Thr was replaced by Ser at ligand 4, Ca2+ transport activity was 70% of wild-type, while half-maximal activation by Ca2+ occurred at 0.8 microM as compared to 0.3 microM for the wild-type enzyme. In the mutant Glu309----Asp at ligand 1, Ca2+ transport activity was lost, but Ca2(+)-activated phosphorylation by ATP was retained. The concentration of Ca2+ required to activate phosphorylation was increased about 10-fold, however, compared to wild type. These results support our hypothesis that ligands 1 to 6, believed to reside within the transmembrane domain, interact with Ca2+ ions during the transport process. The roles of 12 other oxygen-containing residues and of His278 located in the transmembrane domain were also examined by mutation. Although the oxygen-containing side chains of these residues are potential Ca2+ ligands, their replacement with nonpolar amino acids did not abolish Ca2+ transport function, leading to the conclusion that they are not essential ligands for high affinity Ca2+ binding by the Ca2+ pump.

Restrained least squares refinement of native (calcium) and cadmium-substituted carp parvalbumin using X-ray crystallographic data at 1.6-Å resolution

Carp parvalbumin coordinates calcium through one carbonyl oxygen atom and the oxygen-containing side chains of 5 amino acid residues, or 4 residues and a water molecule, in a helix-loop-helix structural motif. Other calcium-binding proteins, including calmodulin and troponin C, also possess this unique calcium-binding design, which is designated EF-hand or calmodulin fold. Parvalbumin has two such sites, labeled CD and EF. Each of the calcium-binding sites of refined structures of proteins belonging to this group has a 7-oxygen coordination sphere except those of the structure of parvalbumin as it was reported in 1975. This structure had been refined at 1.9 A using difference Fourier techniques on film data. The CD site appeared to be 6-coordinate and the EF site 8-coordinate. Results of NMR experiments using 113Cd-substituted parvalbumin, however, indicate that the sites are similar to one another with coordination number greater than 6. To resolve the inconsistency between crystallographic and NMR results, 1.6 A area detector data was collected for native and cadmium-substituted parvalbumin; the structures have been refined to R factors of 18.7% and 16.4%, respectively, with acceptable geometry and low errors in atomic coordinates. Differences between the parvalbumin structure described in 1975 and the present structure are addressed, including the discovery of 7-coordination for both the CD and EF sites.