Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.     

Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch

The S_MK box (SAM-III) translational riboswitches were identified in S-adenosyl-l-methionine (SAM) synthetase metK genes in members of Lactobacillales. This riboswitch switches between two alternative conformations in response to intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound S_MK box riboswitch. In this study, we combined selective 2′-hydroxyl acylation analyzed by primer extension chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo S_MK box RNA molecules exist in an alternatively base-paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which, upon SAM exposure, is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such S_MK variants display a “constitutively OFF” behavior both in vitro and in vivo. Time-resolved and temperature-dependent selective 2′-hydroxyl acylation analyzed by primer extension analyses revealed adaptation of the S_MK box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound S_MK box RNA follows a two-step folding/unfolding process.     

A sensitive mass spectrum assay to characterize engineered methionine adenosyltransferases with S-alkyl methionine analogues as substrates

Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-l-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAMs) into the SAM analogues with engineered MATs. Here we describe a general sensitive activity assay for engineered MATs by converting the reaction products into S-alkylthioadenosines, followed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) quantification. With this assay, 40 human MAT mutants were evaluated against 7 SAAMs as potential substrates. The structure–activity relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower K_m value in contrast to native MATs), the gained activity toward the bulky SAAMs stems from their ability to maintain the desired linear S_N2 transition state (reflected by higher k_cat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAMs.     

Characterisation of Desulfovibrio vulgaris haem b synthase,a radical SAM family member

An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe–4S]^2 +/1 + centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

S-Adenosylmethionine metabolism in HL-60 cells: effect of cell cycle and differentiation

The effect of the cell cycle and differentiation on S-adenosylmethionine (SAM) metabolism in HL-60 cells has been investigated. Synthesis and pool sizes of SAM and S-adenosylhomocysteine (SAH) were cell-cycle-independent (SAM, 315, μM; SAH, 4.6 μM). The SAM-synthase (ATP: l-methionine S-adenosyltransferase) of HL-60 cells has a K_m for methionine of 12.8±2.0 μM and thus appears to be of the intermediate K_m type found in other malignant tissues. The enzyme does not show cell-cycle regulation. Treatment of cells with DMSO resulted in a rapid and marked decrease of SAM and SAH levels without affecting pool turnover or the SAM/SAH ratio. A decrease in SAM concentration could also be observed in a variant cell line resistant to differentiation with DMSO. DMSO inhibited SAM-synthase in cell-free extracts. This inhibition was noncompetitive with respect to l-methionine. Inhibition of SAM-synthase by cycloleucine lowered SAM levels in intact cells, but resulted in differentiation of only a minor percentage of cells. These data indicate that changes in SAM and SAH levels in HL-60 cells seem to be a consequence rather than a cause of differentiation.     

1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis

1-Aminocyclopropanecarboxylate (ACC) synthase, which catalyzes the conversion of S-adenosylmethionine (SAM) to ACC and methylthioadenosine, was demonstrated in tomato extract. Methylthioadenosine was then rapidly hydrolyzed to methylthioribose by a nucleosidase present in the extract. ACC synthase had an optimum pH of 8.5, and a K_m of 20 μm with respect to SAM. S-Adenosylethionine also served as a substrate for ACC synthase, but at a lower efficiency than that of SAM. Since S-adenosylethionine had a higher affinity for the enzyme than SAM, it inhibited the reaction of SAM when both were present. S-Adenosylhomocysteine was, however, an inactive substrate. The enzyme was activated by pyridoxal phosphate at a concentration of 0.1 μm or higher, and competitively inhibited by aminoethoxyvinylglycine and aminooxyacetic acid, which are known to inhibit pyridoxal phosphate-mediated enzymic reactions. These results support the view that ACC synthase is a pyridoxal enzyme. The biochemical role of pyridoxal phosphate is catalyzing the formation of ACC by α,γ-elimination of SAM is discussed.     

Electron tunneling through Pseudomonas aeruginosa azurins on SAM gold electrodes

Robust voltammetric responses were obtained for wild-type and Y72F/H83Q/Q107H/Y108F azurins adsorbed on CH₃(CH₂)_nSH:HO(CH₂)_mSH (n = m = 4, 6, 8, 11; n = 13, 15 m = 11) self-assembled-monolayer (SAM) gold electrodes in acidic solution (pH 4.6) at high ionic strengths. Electron-transfer (ET) rates do not vary substantially with ionic strength, suggesting that the SAM methyl headgroup binds to azurin by hydrophobic interactions. The voltammetric responses for both proteins at higher pH values (>4.6-11) also were strong. A binding model in which the SAM hydroxyl headgroup interacts with the Asn47 carboxamide accounts for the relatively strong coupling to the copper center that can be inferred from the ET rates. Of particular interest is the finding that rate constants for electron tunneling through n = 8, 13 SAMs are higher at pH 11 than those at pH 4.6, possibly owing to enhanced coupling of the SAM to Asn47 caused by deprotonation of nearby surface residues.     

Structural insights into auxiliary cofactor usage by radical S-adenosylmethionine enzymes

Radical S-adenosylmethionine (SAM) enzymes use a common catalytic core for diverse transformations. While all radical SAM enzymes bind a Fe₄S₄ cluster via a characteristic tri-cysteine motif, many bind additional metal cofactors. Recently reported structures of radical SAM enzymes that use methylcobalamin or additional iron-sulfur clusters as cosubstrates show that these auxiliary units are anchored by N- and C-terminal domains that vary significantly in size and topology. Despite this architectural diversity, all use a common surface for auxiliary cofactor docking. In the sulfur insertion and metallocofactor assembly systems evaluated here, interaction with iron-sulfur cluster assembly proteins or downstream scaffold proteins is an important component of catalysis. Structures of these complexes represent important new frontiers in structural analysis of radical SAM enzymes.     

Conversion of the g = 4.1 EPR signal to the multiline conformation during the S2 to S3 transition of the oxygen evolving complex of Photosystem II

The oxygen evolving complex of Photosystem II undergoes four light-induced oxidation transitions, S₀-S₁,…,S₃-(S₄)S₀ during its catalytic cycle. The oxidizing equivalents are stored at a (Mn)₄Ca cluster, the site of water oxidation. EPR spectroscopy has yielded valuable information on the S states. S₂ shows a notable heterogeneity with two spectral forms; a g = 2 (S = 1/2) multiline, and a g = 4.1 (S = 5/2) signal. These oscillate in parallel during the period-four cycle. Cyanobacteria show only the multiline signal, but upon advancement to S₃ they exhibit the same characteristic g = 10 (S = 3) absorption with plant preparations, implying that this latter signal results from the multiline configuration. The fate of the g = 4.1 conformation during advancement to S₃ is accordingly unknown. We searched for light-induced transient changes in the EPR spectra at temperatures below and above the half-inhibition temperature for the S₂ to S₃ transition (ca 230 K). We observed that, above about 220 K the g = 4.1 signal converts to a multiline form prior to advancement to S₃. We cannot exclude that the conversion results from visible-light excitation of the Mn cluster itself. The fact however, that the conversion coincides with the onset of the S₂ to S₃ transition, suggests that it is triggered by the charge-separation process, possibly the oxidation of tyr Z and the accompanying proton relocations. It therefore appears that a configuration of (Mn)₄Ca with a low-spin ground state advances to S₃.     

Two-Electron Reactions S2QB → S0QB and S3QB → S1QB are Involved in Deactivation of Higher S States of the Oxygen-Evolving Complex of Photosystem II

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S₀-S₄), and dark incubation leads to 25% S₀ and 75% S₁. This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor Q_B⁻. We measured flash-induced oxygen evolution to understand how S₃ and S₂ are converted to lower S states when the electron required to reduce the manganese cluster does not come from Q_B⁻. Thylakoid samples preconditioned to make the concentration of the S₁ state 100% and to oxidize tyrosine Y_D were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S₂ and S₃ are converted to lower S states via recombination from S₂Q_B⁻ and S₃Q_B⁻ and by a slow change of the state of oxygen-evolving complex from S₃ and S₂ to S₁ and S₀ in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S₂Q_B → S₀Q_B, and S₃Q_B → S₁Q_B. The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S₃Q_B → S₂Q_B → S₁Q_B in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H₂O₂ acts as an electron donor for the reaction S₂Q_B → S₀Q_B but added H₂O₂ did not enhance this reaction.     

Structures and excitation energies of ozone-water clusters O3(H2O)n (n = 1-4)

Hybrid density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations have been carried out for ozone-water clusters O₃(H₂O)_n (n = 1-4) in order to obtain hydration effects on the absorption spectrum of ozone. The first water molecule in n = 1 is bound to the ozone molecule by an oxygen orientation form in which the oxygen atom of H₂O orients the central oxygen atom of O₃. In n = 2, the water dimer is bound to O₃ and then the cyclic structure is formed as the most stable structure. For n = 3 (or n = 4), the cyclic water trimer (or tetramer) is bound by a hydrogen bond to the ozone molecule. The TD-DFT calculations of O₃(H₂O)_n (n = 0-4) show that the first and second excitation energies of O₃ are blue-shifted by the interaction with the water clusters. The magnitude of the spectral shift is largest in n = 2, and the shifts of the excitation energies are +0.07 eV for S₁ and +0.13 eV for S₂ states. In addition to the spectral shifts (S₁ and S₂ states), it is suggested that a charge-transfer band is appeared as a low-lying excited state above the S₁ and S₂ states. The origin of the spectrum shifts was discussed on the basis of theoretical results.     

Synthesis, structure, and catalytic activity of chiral Cu(II) and Ag(I) complexes with (S,S)-1,2-diaminocyclohexane-based N4-donor ligands

Condensation of (S,S)-1,2-cyclohexanediamine with 2 equiv. of 2-pyridine carboxaldehyde in toluene in the presence of molecular sieves at 70 °C gives N,N′-bis(pyridin-2-ylmethylene)-(S,S)-1,2-cyclohexanediamine (S,S-1) in 95% yield. Reduction of 1 with an excess of NaBH₄ in MeOH at 50 °C gives N,N′-bis(pyridin-2-ylmethyl)-(S,S)-1,2-cyclohexanediamine (S,S-2) in 90% yield. Reaction of 1 or 2 with 1 equiv. of CuCl₂ · 2H₂O in methanol gives complexes [N-(pyridin-2-ylmethylene)-(S,S)-1,2-cyclohexanediamine]CuCl₂ (3) and [Cu(S,S-2)(H₂O)]Cl₂ · H₂O (4), respectively, in good yields. Complex 4 can further react with 1 equiv. of CuCl₂ · 2H₂O in methanol to give [Cu(S,S-2)][CuCl₄] (5) in 75% yield. The rigidity of the ligand coupled with the steric effect of the free anion plays an important role in the formation of the helicates. Treatment of ligand S,S-1 with AgNO₃ induces a polymer helicate {[Ag(S,S-1)][NO₃]}_n (6), while reaction of ligand 2 with AgPF₆ or AgNO₃ in methanol affords a mononuclear single helicate [Ag(S,S-2)][PF₆] (7) or a dinuclear double helicate [Ag₂(S,S-2)₂][NO₃]₂ · 2CH₃OH (8) in good yields, respectively. All compounds have been characterized by various spectroscopic data and elemental analyses. Compounds 1, 3-5, 7 and 8 have been further subjected to single-crystal X-ray diffraction analyses. The Cu(II) complexes do not show catalytic activity for allylation reaction, in contrast to Ag(I) complexes, but they do show catalytic activity for Henry reaction (nitroaldol reaction) that Ag(I) complexes do not.     

The Endosymbiont Amoebophilus asiaticus Encodes an S-Adenosylmethionine Carrier That Compensates for Its Missing Methylation Cycle

All organisms require S-adenosylmethionine (SAM) as a methyl group donor and cofactor for various biologically important processes. However, certain obligate intracellular parasitic bacteria and also the amoeba symbiont Amoebophilus asiaticus have lost the capacity to synthesize this cofactor and hence rely on its uptake from host cells. Genome analyses revealed that A. asiaticus encodes a putative SAM transporter. The corresponding protein was functionally characterized in Escherichia coli: import studies demonstrated that it is specific for SAM and S-adenosylhomocysteine (SAH), the end product of methylation. SAM transport activity was shown to be highly dependent on the presence of a membrane potential, and by targeted analyses, we obtained direct evidence for a proton-driven SAM/SAH antiport mechanism. Sequence analyses suggest that SAM carriers from Rickettsiales might operate in a similar way, in contrast to chlamydial SAM transporters. SAM/SAH antiport is of high physiological importance, as it allows for compensation for the missing methylation cycle. The identification of a SAM transporter in A. asiaticus belonging to the Bacteroidetes phylum demonstrates that SAM transport is more widely spread than previously assumed and occurs in bacteria belonging to three different phyla (Proteobacteria, Chlamydiae, and Bacteroidetes).     

Electrochemical behavior of S,S-bridged adducts of square planar metalladithiolene complexes [M(S2C2Ph2)2](M=Ni, Pd, and Pt)

The electrochemical behavior of the S,S^′-bridged adducts of square planar metalladithiolene complexes was investigated by using cyclic voltammetry and electrochemical spectroscopies (visible, near-IR, and ESR). The norbornene-bridged S,S^′-adduct [Ni(S₂C₂Ph₂)₂(C₇H₈)] (2a; C₇H₈=norbornene) formed by [Ni(S₂C₂Ph₂)₂] (1a) and quadricyclane (Q) was dissociated by an electrochemical reduction, and anion 1a⁻ and norbornadiene (NBD) were formed. Q was isomerized to NBD in the overall reaction. The o-xylyl-bridged S,S^′-adduct [Ni(S₂C₂Ph₂)₂(CH₂)₂(C₆H₄)] (3a; (CH₂)₂(C₆H₄)=o-xylyl) was also dissociated by an electrochemical reduction, and this reaction gave the o-xylyl radical (o-quinodimethane). The reduction of complex 3a in the presence of excess o-xylylene dibromide underwent the catalytic formation of o-quinodimethane. The butylene-bridged S,S^′-adduct [Ni(S₂C₂Ph₂)₂(CH₂)₄] (4a; (CH₂)₄=butylene) was stable on an electrochemical reduction. The lifetimes of reduced species of these adducts 2a-4a were influenced by the stability of the eliminated group (stability: NBD > o-xylyl radical (o-quinodimethane) > butylene radical). Therefore, the reduced species are stable in the sequence 4a⁻ > 3a⁻ > 2a⁻. Although the palladium complex [Pd(S₂C₂Ph₂)₂] (1b) was easier to reduce than the nickel complex 1a or the platinum complex [Pt(S₂C₂Ph₂)₂] (1c), their S,S^′-adducts were easier to reduce in the order of Ni adduct > Pd adduct > Pt adduct.     

Introduction to the Thematic Minireview Series on Radical S-Adenosylmethionine (SAM) Enzymes

In the early days, radical enzyme reactions that use S-adenosylmethionine (SAM) coordinated to an Fe-S cluster, which Perry Frey described as a “poor man''s coenzyme B₁₂,” were believed to be relatively rare chemical curiosities. Today, bioinformatics analyses have revealed the wide prevalence and sheer numbers of radical SAM enzymes, conferring superfamily status. In this thematic minireview series, the JBC presents six articles on radical SAM enzymes that accomplish wide-ranging chemical transformations. We learn that despite the diversity of the reactions catalyzed, family members share some common structural and mechanistic themes. Still in its infancy, continued explorations promise to be fertile grounds for discoveries that will undoubtedly further broaden our understanding of the catalytic repertoire and deepen our understanding of the chemical strategies used by radical SAM enzymes.     

S-adenosylmethionine decarboxylase and spermidine synthase from chinese cabbage

The enzyme, S-adenosylmethionine (SAM) decarboxylase (EC 4.1.1.50), has been demonstrated in leaves of Chinese cabbage, (Brassica pekinensis var Pak Choy). All of the enzyme can be found in extracts of the protoplasts obtained from the leaves of growing healthy or virus-infected cabbage. The protein has been purified approximately 1500-fold in several steps involving ammonium sulfate precipitation, affinity chromatography, and Sephacryl S-300 filtration. The reaction catalyzed by the purified enzyme has been shown to lead to the equimolar production of CO₂ and of decarboxylated S-adenosylmethionine (dSAM). The K_m for SAM is 38 micromolar. The reaction is not stimulated by Mg⁺⁺ or putrescine, and is inhibited by dSAM competitively with SAM. It is also inhibited strongly by methylglyoxal bis(guanylhydrazone). The enzyme, spermidine synthase (EC 2.5.1.16), present in leaf or protoplast extracts in many fold excess over SAM decarboxylase, has been purified approximately 1900-fold in steps involving ammonium sulfate precipitation, affinity chromatography, and gel filtration on Sephacryl S-300. Standardization of the Sephacryl column by proteins of known molecular weight yielded values of 35,000 and 81,000 for the decarboxylase and synthase, respectively.     

Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis

EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.     

S-sulfonation of transthyretin is an important trigger step in the formation of transthyretin-related amyloid fibril

Senile systemic amyloidosis and familial amyloid polyneuropathy are caused by oxidative deposition of conformationally altered transthyretin (TTR). We identified oxidative modification of the 10th cysteine of TTR through S-sulfonation in vitro. Based on mass spectrometric analysis, we determined the spectrophotometric, western blotting, and fluororescent microscopic properties of TTR incubated with and without cysteine-S-sulfonate in acidic (pH 4) and alkaline (pH 8) conditions at 37°. The absorption of the aggregated TTR molecules increased more with incubation time and the concentration of cysteine-S-sulfonate at pH 4 than at pH 8. The Congo red binding to the S-sulfonated TTR at pH 4 was saturated with an apparent Bmax of 2.01 mol per mole of the S-sulfonated TTR and apparent K_D of 7.75 × 10⁻⁶ M. On the other hand, the Bmax of cysteinyl TTR was 1.38, and its K_D was 3.52 × 10⁻⁶ M while the Bmax of reduced TTR was 0.86, and its K_D was 2.86 × 10⁻⁶ M. Moreover, we detected positive amyloid fibril staining using Thioflavin T and Congo red with the S-sulfonated TTR but not with untreated or reduced TTR by microscopic fluororescent analysis. After modification of TTR in vitro, oligomers resisted reduction and denaturation was irreversibly induced, and which contributed differences in the Western blotting patterns obtained with four anti-TTR antibodies. In conclusion, this study showed that the formation of S-sulfonation of TTR through oxidative modifications of the thiol residue on the 10th cysteine of TTR is an important trigger step in the formation of transthyretin-related amyloid fibril.     

Mesolamellar thiogermanates [CnH2n + 1NH3]4Ge4S10

The mesostructured lamellar phases with the general formula [C_nH_2n + 1NH₃]₄Ge₄S₁₀ (n = 12, 14, 16, 18) were synthesized by metathesis of alkyl ammonium chloride surfactants and Na₄Ge₄S₁₀ in aqueous medium. The crystal structures of the phases with n = 12 and 14 were determined by single-crystal X-ray diffraction; [C₁₂H₁₃NH₃]₄Ge₄S₁₀ crystallizes in monoclinic C2/c space group (a = 16.149(3), b = 46.576(9) c = 9.147(2) Å, β = 97.13(3)° and Z = 4) and [C₁₄H₂₉NH₃]₄Ge₄S₁₀ in the triclinic space group (a = 9.1280(6), b = 16.1992(1), c = 26.971(2) Å, α =73.370(1)°, β = 88.307(1)°, γ = 82.825(1)° and Z = 2). These compounds possess layers of adamantane [Ge₄S₁₀]⁴⁻ anions separated by layers of deeply interpenetrated long chain alkylammonium molecules. Strong hydrogen bonding is observed between the terminal sulfur atoms of the [Ge₄S₁₀]⁴⁻ clusters and H atoms of the NH₃ groups of the surfactant molecules. Spectroscopic and thermal characterization of these compounds is reported.