Amino-acid sequence of LC-1 light chain of squid mantle muscle myosin

Tryptic and chymotryptic peptides of the LC-1 light chain of squid mantle muscle myosin were isolated and sequenced by conventional methods, so that the whole amino-acid sequence of the light chain was established. The light chain consisted of 159 amino-acid residues, and its N-terminal alpha-amino group is blocked. Comparing the established sequence with those of vertebrate muscle myosin light chains, only 35% sequence homology was recognized, but a conservative structure was observed in the third domain.     

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

Carbon-13 NMR studies of C2H5N13C binding to various hemoproteins

The addition of an excess of C₂H₅N¹³C to myoglobin and human adult and fetal hemoglobins, gives three characteristic NMR spectra with new ¹³C resonances respectively at δ = ?10,56 ppm, δ = ?7,03 and ?7,95 ppm and δ = ?6,28 and ?7,95 ppm (CH₃CO₂Na as external standard). These signals correspond to the C₂H₅N¹³C bound to the Fe(II) of the different heme units, according to CO exchange experiments. Characteristic resonances can be assigned to C₂H₅N¹³C bound to α, β and γ subunits. C₂H₅N¹³C appears as a more sensitive probe than ¹³CO for hemoprotein NMR studies.     

Studies on cardiac myosin light chains: Comparison of the sequences of cardiac and skeletal myosin LC-2

Partial sequence analysis of bovine cardiac myosin LC-2 indicates that it is closely related to LC-2, the “DTNB light chain” of skeletal muscle myosin. The results suggest that myosins from a variety of sources have related light chains of two distinct types, although the sizes and properties of the light chains can vary substantially.     

18 kDa microtubule-associated protein: identification as a new light chain (LC-3) of microtubule-associated protein 1 (MAP-1)

SDS gel electrophoresis of microtubule proteins obtained from bovine brain by polymerization cycles revealed a new protein of 18 kDa. This protein was copolymerized with tubulin and its stoichiometry to tubulin remained constant for at least 5 cycles of assembly. Moreover, this protein remained bound to microtubules stabilized with 10 μM taxol and pelleted through a 4 M glycerol cushion. The same 18 kDa protein was found in a purified preparation of the high molecular mass microtubule-associated protein 1 (MAP-1). The 18 kDa protein copurified with the MAP-1 heavy chains during column chromatography on phosphocellulose, DEAE-cellulose, hydroxyapatite and Bio-Gel A-15m. Incubation of the MAP-1 preparation with a mouse monoclonal antibody to the light chain 1 (LC-1) of MAP-1 and with a second precipitating antibody (a rabbit antibody to mouse IgG) immunoprecipitated from the solution all the known components of MAP-1 (heavy chains, LC-1, LC-2), as well as the 18 kDa protein. Immunoblotting showed, however, that this antibody does not interact directly with the 18 kDa protein. These results indicate that the 18 kDa protein forms a complex with all other components of MAP-1. This polypeptide, therefore, is a new light chain (LC-3) of M AP-1.     

Application of diffusion‐edited and solvent suppression 1H‐NMR to the direct analysis of markers in valerian‐hop liquid herbal products

Introduction – The rising trend to consume herbal products for the treatment and/or prevention of minor ailments together with their chemical and pharmacological complexity means there is an urgent need to develop new approaches to their quality and stability. Objectives – This work looks at the application of one‐dimensional diffusion‐edited ¹H‐NMR spectroscopy (1D DOSY) and ¹H‐NMR with suppression of the ethanol and water signals to the characterisation of quality and stability markers in multi‐component herbal medicines/food supplements. Material and Methods – The experiments were performed with commercial tinctures of Valeriana officinalis L. (valerian), expired and non‐expired, as well as its combination with Hummulus lupulus L. (hops), which is one of the most popular blends of relaxant herbs. These techniques did not require purification or evaporation of components for the qualitative analysis of the mixture, but only the addition of D₂O and TSP. Results – The best diagnostic signals were found at δ 7 ppm (H‐11, valerenic acid), δ 4.2 ppm (H‐1, hydroxyvalerenic acid) and δ 1.5‐1.8 ppm (methyl groups in prenylated moieties, α‐acids/prenylated flavones). Conclusion – This work concludes on the potential value of 1D DOSY ¹H‐NMR to provide additional assurance of quality in complex natural mixtures. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and multinuclear magnetic resonance spectroscopy of the novel ionic Pt(II) mixed-ligands complexes cis- and trans-[Pt(amine)2(pyrimidine)2](NO3)2

Novel ionic mixed-ligands complexes of the types cis- and trans-[Pt(amine)₂(pm)₂](NO₃)₂ (where pm = pyrimidine) were synthesized and studied in the solid state by IR spectroscopy and in aqueous solution by multinuclear (¹⁹⁵Pt, ¹H and ¹³C) magnetic resonance spectroscopy. The results of the solution NMR characterization have shown that the isolated compounds are pure. In ¹⁹⁵Pt NMR, the cis RNH₂ complexes were observed at slightly lower fields (ave. −2441 ppm) than the equivalent trans analogues (ave. −2448 ppm). For Me₂NH, the difference between the two isomers is larger (29 ppm). The complexes are observed at lower fields (difference of 100 ppm) than the corresponding [Pt(amine)₄]²⁺ complexes, which might indicate the presence of π-backdonation in the Pt-pm bond. In ¹H NMR, the coupling constants ³J(¹⁹⁵Pt-¹H_amine) are larger in the cis compounds (38-48 Hz) than in the trans analogues (30-36 Hz). The ³J(¹⁹⁵Pt-¹H_pm) values are also larger for the cis isomers. In ¹³C NMR spectroscopy, the coupling constants ³J(¹⁹⁵Pt-¹³C_amine) are 36 Hz (ave.) for the cis complexes and 26 Hz (ave.) for the trans isomers, while the ²J(¹⁹⁵Pt-¹³C_amine) are 18 Hz (cis) and 14 Hz (trans), respectively. The ³J(¹⁹⁵Pt-¹³C_5(pm)) values are 36 Hz (cis) and 28 Hz (trans). A few ²J(¹⁹⁵Pt-¹³C_pm) couplings were observed (7-10 Hz).     

Chemotaxonomic Profiling Through NMR1

A metabolite screening of cyanobacteria was performed by nuclear magnetic resonance (NMR) analysis of the soluble material obtained through sequential extraction of the biomass with three different extractive ability solvents (hexane, ethyl acetate, and methanol). Twenty-five strains from the Coimbra Collection of Algae (ACOI) belonging to different orders in the botanical code that represent three subsections of the Stainer-Rippka classification were used. The ¹H NMR spectra of hexane extracts showed that only two strains of Nostoc genus accumulated triacylglycerols. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols were the major components of the ethyl acetate extracts in a mono- to digalactosyldiacylglycerols ratio of 4.5 estimated by integration of the signals at δ 3.99 and 3.94 ppm (sn3 glycerol methylene). Oligosaccharides of sucrose and mycosporine-like amino acids, among other polar metabolites, were detected in the methanolic extracts. Strains of Nostocales order contained heterocyst glycolipids, whereas sulphoquinovosyldiacylglycerols were absent in one of the studied strains (Microchaete tenera ACOI 1451). Phosphathidylglycerol was identified as the major phospholipid in the methanolic extracts together with minor amounts of phosphatidylcholine based on ¹H, ³¹P 2D correlation experiments. Chemotaxonomic information could be easily obtained through the analysis of the δ 3.0–0.5 ppm (fatty acid distribution) and δ 1.2–1.1 ppm (terminal methyl groups of the aglycons in heterocyst glycolipids) regions of the ¹H NMR spectra of the ethyl acetate and methanol extracts, respectively.     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

Study of Pt(II)-aromatic amines complexes of the types cis- and trans-Pt(amine)2I2, [Pt(amine)4]I2 and I(amine)Pt(μ-I)2Pt(amine)I

Pt(II) complexes of the types cis- and trans-Pt(amine)₂I₂ with amines containing a phenyl group were synthesized and studied mainly by IR and multinuclear (¹⁹⁵Pt, ¹H and ¹³C) magnetic resonance spectroscopies. The compounds are not very soluble. In ¹⁹⁵Pt NMR spectroscopy, the cis isomers were observed at slightly lower fields than the trans analogues (average Δδ = 11 ppm) in acetone. In ¹H NMR, the NH groups were also found at slightly lower fields in the cis isomers. The coupling constants ²J(¹⁹⁵Pt-¹HN) varied from 53 to 85 Hz and seem slightly smaller in the trans configuration. The ¹³C NMR spectra of most of the complexes were measured. No coupling constants J(¹⁹⁵Pt-¹³C) were detected due to the low solubility of the compounds. The cis isomers containing a phenyl group on the N atom could not be isolated except for Ph-NH₂ which was shown to be a mixture of isomers in acetone. The tetrasubstituted ionic compounds [Pt(amine)₄]I₂ for the less crowded ligands were also studied mainly by NMR spectroscopy in aqueous solution. The ¹⁹⁵Pt chemical shifts vary between −2855 and −2909 ppm. The coupling constants ³J(¹⁹⁵Pt-¹H) are about 40 Hz. The iodo-bridged dinuclear species I(amine)Pt(μ-I)₂Pt(amine)I were also synthesized and characterized. Two isomers are present in acetone solution for most of the compounds. Their δ(Pt) signals were observed at about −4000 ppm and their coupling constants ²J(¹⁹⁵Pt-¹HN) are around 69 Hz.     

Synthesis and multinuclear magnetic resonance spectroscopy of the novel ionic Pt(II) mixed-ligands complexes cis- and trans-[Pt(pyrazine)2(Ypy)2](NO3)2 where Ypy = pyridine derivative

Novel ionic mixed-ligands complexes of the types cis- and trans-[Pt(pz)₂(Ypy)₂](NO₃)₂ (where Ypy is a pyridine derivative and pz = pyrazine) were synthesized and studied mainly in the solid state by IR spectroscopy and in aqueous solution by multinuclear (¹⁹⁵Pt, ¹H and ¹³C) magnetic resonance spectroscopy. The trans isomers with ligands containing a methyl group in ortho position on the pyridine ring could not be synthesized. The results of the solution NMR characterization have shown that the isolated compounds are pure. In ¹⁹⁵Pt NMR, the cis complexes containing a methyl group in ortho positions were observed at lower field (average −2337 ppm) than the other cis compounds (average −2427 ppm), which is explained by the solvent effect. The trans isomers were observed at very slightly lower fields (average −2422 ppm) than the equivalent cis complexes (average −2427 ppm). In ¹H NMR, the coupling constants ³J(¹⁹⁵Pt-¹H_Ypy) and ³J(¹⁹⁵Pt-¹H_pz) are larger in the cis compounds (∼40 Hz) than in the trans complexes (∼31 Hz). A few ⁴J(¹⁹⁵Pt-¹H_pz) were observed (∼16 Hz). In ¹³C NMR spectroscopy, the coupling constants ³J(¹⁹⁵Pt-¹³C_pz) and ³J(¹⁹⁵Pt-¹³C_Ypy) are also larger in the cis configuration (∼30 and ∼38 Hz, respectively) than in the trans isomers (∼20 Hz). One ⁴J(¹⁹⁵Pt-¹³C_pz) could be calculated (17 Hz). The presence of the syn and anti rotamers were observed in all the cis complexes containing a pyridine derivative with a -CH₃ group in ortho position. They were observed in ¹⁹⁵Pt, ¹H and ¹³C NMR spectroscopy. The proportion of the two rotamers is about 55% and 45%.     

Synthesis, 95Mo NMR spectra and x-ray crystal structure of MoO2(C14H11N2O)2(C2H5H)1 and Mo2O5(C14H11N2O)2(C2H5OH)1(H2O)1

The crystal structures and ⁹⁵Mo NMR spectra of two complexes formed between 2-α-hydroxybenzyl- benzimidazole (C₆H₅·CHOH·C₇H₅N₂=HOBB), as its sodium salt, and MoO₂Cl₂ are reported. [MoO₂- (OBB)₂]·EtOH (OBB=deprotonated HOBB) crystallizes in space group P2₁/n, with a=12.8441(7), b=15.917(3), c=13.314(2) Å, β=97.163(8)° and Z =4. The structure was determined from 3096 observed reflections and refined to a final R value of 0.030. The complex is a six coordinate cis-dioxo species, the ⁹⁵Mo spectrum of which shows a single sharp peak at 56 ppm in dimethylformamide (DMF). The second complex, [Mo₂O₅(OBB)₂]·EtOH·H₂O, crystallizes in space group Pbca, with a=22.482(4), b=16.442(3), c=18.407(3) Å and Z=8. The structure was determined from 2936 observed reflections and refined to a final R value of 0.061. The complex is a binuclear doubly bridged species in which one metal atom is six coordinate while the other is five coordinate. Its ⁹⁵Mo NMR spectrum in DMF shows a sharp peak at 124 ppm and a second broader much weaker peak at 51 ppm.     

Multinuclear magnetic resonance spectroscopy and crystal structures of iodo-bridged dinuclear Pt(II) complexes with amines

Complexes of the types cis-Pt(amine)₂I₂ were transformed into the iodo-bridged dimers, which were characterized mainly by multinuclear (¹⁹⁵Pt, ¹H and ¹³C) magnetic resonance spectroscopy. For bulby amines, the dinuclear species were synthesized directly from K₂[PtI₄]. Compounds with several primary aliphatic and cyclic amines and two secondary amines were studied. In ¹⁹⁵Pt NMR, two signals were observed between −3899 and −4080 ppm in acetone. These species were assigned to the cis and trans dinuclear compounds I(amine)Pt(μ-I)₂PtI(amine). We suggest that the most shielded compound is the trans isomer. The difference between the two isomers is 12-13 ppm for the primary amine system and 26-27 ppm for the two secondary amines. There seems to be a slight dependence of the proton affinity in the gas phase of the amine (linear amines) with the δ(Pt) chemical shifts of the dinuclear Pt(II) compounds. The ²J(¹⁹⁵Pt-¹HN) coupling constants are slightly larger for the trans isomers (average 67 Hz, vs. 56 Hz). The ³J(¹⁹⁵Pt-¹H) coupling constants were detected only for the dimethylamine compounds, 46 Hz (trans) and 44 Hz (cis). In ¹³C NMR, the values of ²J(¹⁹⁵Pt-¹³C) and ³J(¹⁹⁵Pt-¹³C) were also found to be very slightly larger for the trans complexes (average 19 and 25 Hz vs. 15 and 18 Hz). The structures were confirmed by X-ray diffraction studies of the n-butylamine and diethylamine compounds. The two crystals were those of the trans dinuclear complexes.     

Synthesis of Haemoglobin Lepore

HAEMOGLOBIN Lepore is a class of haemoglobin variants composed of α-chains and hybrids of δ and β-chains, which can be designated as α₂(δβ)₂. The N-terminal amino-acid sequence of the hybrid chains corresponds to that of δ-chains, while the C-terminal sequence corresponds to that of β-chains. A misplaced synapse of the δ and β gene loci is thought to have occurred during meiosis, followed by a non-homologous cross-over¹. Three types are known which differ in the position of the cross-over: Hb Lepore Hollandia^2,3, Hb Lepore Baltimore⁴ and Hb Lepore Boston (Washington)^5,6.     

Multinuclear magnetic resonance study of Pt(II) compounds with sulfoxide ligands and crystal structures of complexes of the types [Pt(R2SO)X3] and Pt(R2SO)2Cl2

Pt(II) complexes of the types K[Pt(R₂SO)X₃], NR₄[Pt(R₂SO)X₃] and Pt(R₂SO)₂Cl₂ (where X = Cl or Br) were characterized by multinuclear magnetic resonance spectroscopy (¹⁹⁵Pt, ¹H and ¹³C). In ¹⁹⁵Pt NMR, the chloro ionic compounds have shown signals between −2979 and −3106 ppm, while the cis disubstituted complexes were observed at higher fields, between −3450 and −3546 ppm. The signal of the compound trans-Pt(DPrSO)₂Cl₂ was found at higher field (−3666 ppm) than its cis analogue (−3517 ppm), since π-back-donation is considerably less effective in the trans geometry. In ¹H NMR, a single signal was observed for the sulfoxide in [Pt(DMSO)Cl₃]⁻, but for the other more sterically hindered ligands, two series of resonances were observed for the protons in α and β positions. The coupling constant ³J(¹⁹⁵Pt-¹H) are between 15 and 33 Hz. The ¹³C NMR results were interpreted in relation to the concept of inversed polarization of the π sulfoxide bond. The ²J(¹⁹⁵Pt-¹³C) values vary between 35 and 66 Hz, while a few ³J(¹⁹⁵Pt-¹³C) couplings were observed (13-26 Hz). The crystal structures of five monosubstituted ionic compounds N(n-Bu)₄[Pt(TMSO)Cl₃], N(Me)₄[Pt(DPrSO)Cl₃], K[Pt(EMSO)Cl₃], K[Pt(TMSO)Br₃] · H₂O and N(Et)₄[Pt(DPrSO)Br₃] and one disubstituted complex cis-Pt(DBuSO)₂Cl₂ were determined. The trans influence of the different ligands is discussed.     

The growth of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> as influenced by CO<Subscript>2</Subscript>, light,and flue gases

In order to achieve recognition as environmentally friendly production, flue gases should be used as a CO₂ source for growing the microalgae Chlorella sorokiniana when used for hydrogen production. Flue gases from a waste incinerator and from a silicomanganese smelter were used. Before testing the flue gases, the algae were grown in a laboratory at 0.04, 1.3, 5.9, and 11.0 % (v/v) pure CO₂ gas mixed with fresh air. After 5 days of growth, the dry biomass per liter algal culture reached its maximum at 6.1 % CO₂. A second experiment was conducted in the laboratory at 6.2 % CO₂ at photon flux densities (PFD) of 100, 230, and 320 μmol photons m^?2 s^?1. After 4 days of growth, increasing the PFD increased the biomass production by 67 and 108 % at the two highest PFD levels, as compared with the lowest PFD. A bioreactor system containing nine daylight-exposed tubes and nine artificial light-exposed tubes was installed on the roof of the waste incinerator. The effect of undiluted flue gas (10.7 % CO₂, 35.8 ppm NO _x , and 38.6 ppm SO₂), flue gas diluted with fresh air to give 4.2 % CO₂ concentration, and 5.0 % pure CO₂ gas was studied in daylight (21.4?±?9.6 mol photons m^?2 day^?1 PAR, day length 12.0 h) and at 135 μmol photons m^?2 s^?1 artificial light given 24 h day^?1 (11.7?±?0.0 mol photons m^?2 day^?1 PAR). After 4 days’ growth, the biomass production was the same in the two flue gas concentrations and the 5 % pure CO₂ gas control. The biomass production was also the same in daylight and artificial light, which meant that, in artificial light, the light use efficiency was about twice that of daylight. The starch concentration of the algae was unaffected by the light level and CO₂ concentration in the laboratory experiments (2.5–4.0 % of the dry weight). The flue gas concentration had no effect on starch concentration, while the starch concentration increased from about 1.5 % to about 6.0 % when the light source changed from artificial light to daylight. The flue gas from the silicomanganese smelter was characterized by a high CO₂ concentration (about 17 % v/v), low oxygen concentration (about 4 %), about 100 ppm NO _x , and 1 ppm SO₂. The biomass production using flue gas significantly increased as compared with about 5 % pure CO₂ gas, which was similar to the biomass produced at a CO₂ concentration of 10–20 % mixed with N₂. Thus, the enhanced biomass production seemed to be related to the low oxygen concentration rather than to the very high CO₂ concentration.     

Carbon-13 NMR studies of the lysine side chains of calmodulin and its proteolytic fragments

ThepH-titration and dynamic behaviour of the seven lysine side chains in bovine calmodulin were studied by carbon-13 NMR. The amino groups of the calcium saturated protein and its proteolytic fragments TR₁C(1–75) and TR₂C (78–148) were dimethylated with carbon-13 labeled formaldehyde; this modification did not alter the protein's structure or its ability to activate the enzyme cyclic nucleotide phosphodiesterase. Tentative assignments for 5 out of the 7 dimethyl lysine resonances could be obtained by comparing spectra of the fully and partially modified protein, with those of the proteolytic fragments. ThepK_a values measured for calcium saturated calmodulin ranged between 9.5 (Lys 75) and 10.2 (Lys 13); two residues (Lys 94 and Lys 13) showed a biphasic titration curve suggesting their possible involvement in ion-pairs. The dynamic behavior of the lysine side chains was deduced from spin lattice relaxation measurements. All side chains were flexible and this was not influenced by the removal of calcium, or the addition of the calmodulin antagonist trifluoperazine. The latter data suggest that the lysine side chains are not directly involved in calmodulin's target binding sites.     

Toxicity of flue gas components from cement plants in microalgae CO2 mitigation systems

Microalgae-based CO₂ capture from flue gas is an attractive mitigation strategy in the cement industry. However, NO _x and SO _x components might be harmful to microalgae. We performed toxicity assays, under 2 % (v/v) CO₂ and using nitrite, sulfite, or bisulfite salts, on an environmental isolate, identified as Desmodesmus abundans (The University of Texas at Austin (UTEX), no. 2976) and Scenedesmus sp. UTEX1589. Nitrite and sulfite did not inhibit growth at the tested concentrations (0–1,067 ppm (w/v) NO₂ ^? and 0–254 ppm (w/v) SO₃ ^2?); however, bisulfite was toxic above 39 ppm. Non-toxic concentrations of both sulfur-based compounds stimulated growth, but significantly higher growth rates were only observed for HSO₃ ^?. Within a narrower range, NO _x and SO _x served as a sole nutrient source. Overall, biomass production and growth rates of the environmental isolate were greater. A novel strategy to buffer high concentrations of HSO₃ ^? (200 ppm) was developed by adding cement kiln dust (CKD), a byproduct and flue gas component. The results suggest that CKD also provided other beneficial growth components and that sulfur optimization of the culture medium significantly increased carbon assimilation, particularly in D. abundans. In additional simulations of typical flue gas conditions in a modern cement plant (320, 40, and 40 ppm (w/v) of NO₂ ^?, SO₃ ^2?, and HSO₃ ^?, respectively, and 25 % (v/v) CO₂), along with the incorporation of 300 ppm CDK, growth of D. abundans was supported. Although further studies are needed, direct utilization of flue gas might be possible with the environmental isolate, where NO _x , SO _x , and CKD are all beneficial components of the mitigation system.     

Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G1-59) of the Saccharomyces cerevisiae V1VO ATPase

Understanding the structural traits of subunit G is essential, as it is needed for V₁V_O assembly and function. Here solution NMR of the recombinant N- (G_1-59) and C-terminal segment (G_61-114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G_1-59 only. The residues of G_1-59 involved in d binding are Gly7 to Lys34. The structure of G_1-59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G_1-59 are flexible. The surface charge distribution of G_1-59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G_1-59-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E_18-38 of subunit E of yeast V-ATPase and the presently solved structure of G_1-59, both proteins have been docked and binding epitopes have been analyzed.