Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕ_c) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕ_c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin''s time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Differences in β-strand Populations of Monomeric Aβ40 and Aβ42

Using homonuclear ¹H NOESY spectra, with chemical shifts, ³J_H^N_H^α scalar couplings, residual dipolar couplings, and ¹H-¹⁵N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1–40 (Aβ40) and amyloid-β 1–42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16–21) with residues 29–36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9–13, that sometimes forms a β-sheet by association with residues 35–37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Xanthonolignoids from Kielmeyera and Caraipa species—13C NMR spectroscopy of xanthones

Kielcorin and the cadensins A and B, isolated respectively from Kielmeyera coriacea and Caraipa densiflora (Guttiferae), were shown to be xanthonolignoids. The structure of (5S,6S)-6(or 5)-hydroxymethyl-5(or 6)-(4″-hydroxy-3″-methoxyphenyl)-2,3:3′,4′-(2′-methoxyxanthono)-1,4-dioxane was proposed for kielcorin by analysis of high resolution MS and PMR spectra. The carbon shifts of xanthone were assigned and used in the ¹³C NMR spectral confirmation of the proposed structure.     

1,3-Bis(α-aminoisopropyl)benzene, meta-C6H4(CMe2NH2)2: An N,N-bridging and N,C,N-cyclometalating ligand

Saponification of the bis(carbamic acid ester) 1,3-C₆H₄(CMe₂NHCO₂Me)₂ (1), made by the addition of methanol to commercial 1,3-C₆H₄(CMe₂NCO)₂, yielded the meta-phenylene-based bis(tertiary carbinamine) 1,3-C₆H₄(CMe₂NH₂)₂ (2). Dinuclear [{(η⁴-1,5-C₈H₁₂)RhCl}₂{μ-1,3-C₆H₄(CMe₂NH₂)₂}] (3) resulted from the action of 2 on [{(η⁴-1,5-C₈H₁₂)Rh(μ-Cl)}₂] in toluene. Combination of 2 with PdCl₂ or K₂[PdCl₄] gave the dipalladium macrocycle trans,trans-[{μ-1,3-C₆H₄(CMe₂NH₂)₂}₂(PdCl₂)₂] (4) along with cyclometalated [{2,6-C₆H₃(CMe₂NH₂)₂-κN,κC¹,κN′}PdCl] (5). Substitution of PEt₃ for the labile chlorido ligand of 5 afforded [{2,6-C₆H₃(CMe₂NH₂)₂-κN,κC¹, κN′}Pd(PEt₃)]Cl (6). The crystal structures of the following compounds were determined: bis(carbamic acid ester) 1, ligand 2 as its bis(trifluoroacetate) salt [1,3-C₆H₄(CMe₂NH₃)₂](O₂CCF₃)₂, 2 · (HAc_f)₂, complexes 3 and 6, as well as 1,3-C₆H₄(CMe₂OH)₂ (the diol analogue of 2).     

Visual learning and memory of Drosophila melanogaster wild type CS and the mutants dunce1, amnesiac, turnip and rutabaga

Dunce¹, amnesiac, turnip and rutabaga, mutants of Drosophila melanogaster deficient in olfactory learning and/or memory, were tested for visual learning ability and memory. All these mutants are able to learn conditioned visual information, but not as well as the corresponding wildtype CS. Memory of all four mutants is reduced during the first 30 min after training, but indistinguishable from that of the wildtype two hours after conditioning.     

Novel homozygous mutations in the genes ARL6 and BBS10 underlying Bardet–Biedl syndrome

Bardet–Biedl syndrome (BBS) is an autosomal recessive disorder resulting from structural and functional defects in numerous organs. Frequent manifestations reported in the syndrome include obesity, renal dysplasia, cognitive impairment, postaxial polydactyly, pigmentary retinal degeneration and hypogonadism. To date, 17 genes causing BBS have been identified. Two of these BBS1 and BBS10 are the most frequently mutated genes.     

Mitochondrially-targeted bacterial phosphatidylethanolamine methyltransferase sustained phosphatidylcholine synthesis of a Saccharomyces cerevisiae Δpem1 Δpem2 double mutant without exogenous choline supply

In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3 × HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1Δpem2Δ) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1Δpem2Δ strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations.     

Synthesis and characterisation of κ-P and κ-P,N palladium(II) complexes of the open cage water soluble aminophosphine PTN

New palladium(II) complexes containing the water soluble aminophosphine PTN ligand (PTN = 7-phospha-3,7-dimethyl-1,3,5-triazabicyclo[3.3.1]nonane) in 1:1 and 1:2 ratio Pd/PTN ligand, respectively, were prepared and fully characterised by mono and bidimensional ³¹P, ¹H and ¹³C NMR techniques showing that PTN can adopt both κ¹-P and κ²-P,N coordination modes. The complexes with Pd/PTN ratio 1:2 are highly soluble in water at room temperature. Suitable crystals for X-ray structure determination were obtained for the neutral complex κ²-P,N-Pd(PTN)(OAc)₂ (1) and for the monocationic complex [Pd(κ²-P,N-PTN)(κ¹-P-PTN)Cl][PF₆] (5).     

Hidden diversity uncovered in Hygrophorus sect. Aurei (Hygrophoraceae), including the Mediterranean H. meridionalis and the North American H. boyeri, spp. nov.

For many years, the binomial Hygrophorus hypothejus was widely applied to collections from various geographical regions in different continents, assuming a circum-boreal and circum-mediterranean distribution for this species. This hypothesis, however, had never been put to the test. To assess the diversity and species-limits within this complex of yellow-coloured waxcaps, a phylogenetic, morphological and taxonomical investigation into Hygrophorus sect. Aurei and similar species in sect. Olivaceoumbrini was carried out, including material of pan-European origin, as well as the east and west coasts of North America. Following sequencing of the ITS rDNA locus, nine lineages are confirmed in sect. Aurei, most of them highly continentalised. Of these, two are new to science, introduced here as Hygrophorus boyeri sp. nov., from Pinus banksiana and P. rigida forests in eastern North America and from P. muricata and P. contorta forests in western North America, and Hygrophorus meridionalis sp. nov., from Pinus brutia and Pinus halepensis forests in the island of Cyprus and mainland Greece. H. hypothejus is lectotypified and epitypified, and here resolved as a strictly European species, with the old forgotten taxon Hygrophorus siccipes revived as its North American vicariant. The placement of Hygrophorus fuligineus in sect. Aurei is phylogenetically confirmed and detailed comparisons between morphologically similar and phylogenetically affiliated taxa in sect. Aurei and sect. Olivaceoumbrini are provided. The chronic confusion associated with Hygrophorus fuscoalbus, a highly controversial taxon described from Germany nearly two centuries ago and variously interpreted since, is discussed, concluding that this name is too ambiguous to be applied to any currently recognized species.     

Biosynthesis of α-spinasterol from (2- 14C (4R)-4-3H1) mevalonic acid by Spinacea oleracea and Medicago sativa

Leaves of Spinacea oleracea and Medicago sativa were incubated with (2-¹⁴C, (4R)-4³H₁ mevalonic acid and the sterols isolated. Cycloartenol had a ³H: ¹⁴C atomic ratio of 6:6 whilst oxidation to cycloartenone resulted in a ratio of 5:6 showing that tritium was present in the 3α-position and that the cycloartenol was symmetrically labelled. Separation of the 4-demethyl sterols gave α-spinasterol and a mixture of stigmast-7-enol and 24-methylcholest-7-enol, which had ³H: ¹⁴C atomic ratios of 3:5. Ozonolysis of α-spinastery] acetate gave the terminal side chain fragment as 2-ethyl-3-methyl butanoic acid. The acid contained ¹⁴C but no tritium thus showing that the C-24 hydrogen of cycloartenol is lost during the alkylation reactions leading to the C-24 ethyl group of α-spinasterol.     

New lignans attenuating cognitive deterioration of Aβ transgenic flies discovered in Acorus tatarinowii

Five new lignan racemates, (±)-tatarinoid D–H (1–5), and three known analogues (6–8) were isolated from the rhizomes of Acorus tatarinowii. Their structures were established by 1D, 2D-NMR, HR-MS, IR and optical spectral data. Among them, 1 and 6–8 can alleviate the cognitive deterioration of Aβ transgenic flies.