Major Structural Rearrangements Research Articles

Aberration Of SF3B1 Results In Deregulated Splicing Of Key Genes and Pathways In Myelodysplastic Syndromes

The recent discovery of a variety of somatic splicesomal mutations in the myelodysplastic syndromes (MDS) has revealed a new leukaemogenic pathway involving spliceosomal dysfunction. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous non-snRNP proteins. The snRNPs are involved in recognising short conserved sequences of the pre-mRNA, including the 5′ and 3′ splice sites and the branch site, and in positioning the reactive nucleotides for catalysis. The spliceosome is a dynamic molecular machine, undergoing several major structural rearrangements during its functional cycle. Mutation of the Splicing Factor 3B, subunit 1 (SF3B1) gene is common in MDS, occurring in over 70% of patients whose disease is characterised by ring sideroblasts (RARS). The close association between SF3B1 mutation and ring sideroblasts is consistent with a causal relationship, and makes this the first gene to be strongly associated with a specific feature of MDS.Sf3b1 heterozygous knockout mice show the presence of ringed sideroblasts. In order to investigate the role of SF3B1 haploinsufficiency in MDS we have silenced SF3B1 using siRNA in the myeloid cell lines K562, TF-1, SKM1, HeL and OCIM2. Cell growth was impaired in all the cell lines with SF3B1 knockdown. Using Flow Cytometry, cell cycle analysis showed a significant increase in cells in the sub-G0 phase as well as G2/M arrest in the cell lines. We also observed impaired erythroid differentiation in hemin treated K562 and TF-1 cell lines with SF3B1 knockdown.Gene expression profiling (GEP) was performed in two cell lines with SF3B1 knockdown (K562 and TF1). Deregulated pathways and gene ontology categories included cell cycle regulation and alternative splicing using Ingenuity Pathway Analysis. We next performed Gene Set Enrichment Analysis (GSEA). The GSEA showed a significant enrichment of nonsense-mediated mRNA decay (NMD) genes that were up-regulated in cells with SF3B1 knockdown, suggesting NMD activation following SF3B1 silencing. We used Human Exon-Junction arrays (Affymetrix) to evaluate global transcript exon usage in the K562 and TF1 cell lines with SF3B1 knockdown. We observed significant differential exon usage in genes involved in RNA degradation, spliceosome, cell cycle and apoptosis. We further observed aberrant splicing of the candidate gene ABCB7 showing exon skipping and TP53 gene showing exon skipping as well as intron retention.We have investigated the changes in the transcriptome in CD34+ cells from MDS patients with SF3B1 mutation by RNA sequencing and found many genes showing significant differential exon usage including CCND1, EIF3B, FKBP1A, BCL2 and RB1. Using Ingenuity Pathway Analysis we identified alternative splicing pattern of genes involved in cell cycle, RNA processing, mTOR signalling and P53 signalling pathways.We have studied CD34+ cells from MDS patients with SF3B1 mutation in vitro and observed impairment in cell growth compared to CD34+ cells from healthy controls or from MDS patients without splicing mutations. In colony forming assays we observed a decrease in the number of erythroid or myeloid colonies derived from CD34+ cells of patients with SF3B1 mutation compared to patient CD34+ cells without splicing factor mutation. The identification of SF3B1 downstream targets in SF3B1 mutant and wild-type erythroid and myeloid colonies from MDS patients is in progress using RNA sequencing.Our results show that knockdown of SF3B1 in haematopoietic cell lines results in impaired cell growth, deregulated global gene expression and aberrant splicing. Studies of the haematopoietic progenitor CD34+ cells of patients with SF3B1 mutation show impaired cell growth and erythroid differentiation as well as deregulation of many pathways including the cell cycle and RNA processing. The identification of the key target genes affected by the common splicing mutations in MDS is critical to our understanding of how the mutations contribute to the pathogenesis of this disorder. Disclosures:Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Ancient eudicot hexaploidy meets ancestral eurosid gene order.

BackgroundA hexaploidization event over 125 Mya underlies the evolutionary lineage of the majority of flowering plants, including very many species of agricultural importance. Half of these belong to the rosid subgrouping, containing severals whose genome sequences have been published. Although most duplicate and triplicate genes have been lost in all descendants, clear traces of the original chromosome triples can be discerned, their internal contiguity highly conserved in some genomes and very fragmented in others. To understand the particular evolutionary patterns of plant genomes, there is a need to systematically survey the fate of the subgenomes of polyploids, including the retention of a small proportion of the duplicate and triplicate genes and the reconstruction of putative ancestral intermediates between the original hexaploid and modern species, in this case the ancestor of the eurosid clade.ResultsWe quantitatively trace the fate of gene triples originating in the hexaploidy across seven core eudicot flowering plants, and fit this to a two-stage model, pre- and post-radiation. We also measure the simultaneous dynamics of duplicate orthologous gene loss in three rosids, as influenced by biological functional class. We propose a new protocol for reconstructing ancestral gene order using only gene adjacency data from pairwise genomic analyses, based on repeating MAXIMUM WEIGHT MATCHING at two levels of resolution, an approach designed to transcend limitations on reconstructed contig size, while still avoiding the ambiguities of a multiplicity of solutions. Applied to three high-quality rosid genomes without subsequent polyploidy events, our automated procedure reconstructs the ancestor of the eurosid clade.ConclusionsThe gene loss analysis and the ancestor reconstruction present complementary assessments of post-hexaploidization evolution, the first at the level of individual gene families within and across sister genomes and the second at the chromosome level. Despite the loss of more than 95% of gene duplicates and triplicates, and despite major structural rearrangement, our reconstructed eurosid ancestor clearly identifies the three regions corresponding to each of the seven original chromosomes of the earlier pre-hexaploid ancestor. Functional analysis confirmed trends reported for more recent plant polyploidy events: genes involved with regulation and responses were retained in multiple copies, while genes involved with metabolic processes were lost.

Cofactor Molecules Induce Structural Transformation during Infectious Prion Formation

The spread of misfolded proteins may occur in many neurodegenerative diseases. Mammalian prions arecurrently the only misfolded proteins in which high specific biological infectivity can be produced invitro. Using a system that generates infectious prions de novo from purified recombinant PrP and conversion cofactors palmitoyl-oleoyl-phosphatidylglycerol (POPG) and RNA, we examined by deuterium exchange mass spectrometry (DXMS) the stepwise protein conformational changes that occur during prion formation. We found that initial incubation with POPG causes major structural changes in PrP involving all three α helices and one β strand, with subsequent addition of RNA rendering the N terminus highly exposed. Final conversion into the infectious PrP(Sc) form was accompanied by globally decreased solvent exposure, with persistence of the major cofactor-induced conformational features. Thus, we report that cofactor molecules appear to induce major structural rearrangements during prion formation, initiating a dynamic sequence of conformational changes resulting in biologically active prions.

β2‐Adrenergic Receptor Activation by Agonists Studied with 19F NMR Spectroscopy

β₂‐Adrenergic Receptor Activation by Agonists Studied with ¹⁹F NMR Spectroscopy

Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes

Author SummaryEnzymes use a variety of tools and strategies to enhance (catalyze) biological reactions; these include the use of general acids and bases, cofactors, and the employment of remote binding interactions to position substrates near reactive chemical groups. Phosphatases are some of Nature's best enzymes, affording exceptional rate enhancements to the biologically ubiquitous removal of a phosphate group from a substrate (dephosphorylation). The apparent challenge faced by nonspecific phosphatases is that their wide substrate specificity precludes the efficient use of remote binding interactions. Previous work suggested that phosphatases could use negatively charged chemical groups (anionic nucleophiles) at the active site to destabilize substrate binding without simultaneously destabilizing the transition state barrier—an elusive catalytic strategy known as preferential ground state destabilization. In this work, we test this ground state destabilization model of catalysis by removing the anionic active site nucleophile of alkaline phosphatase and observing the effects on the enzyme's affinity for a phosphate ligand. We find that alkaline phosphatase has an exceptionally strong affinity for phosphate, and provide clear evidence for ground state destabilization by the anionic active site nucleophile that, when present, forestalls substrate saturation and product inhibition, and enhances catalysis by at least a thousand fold.

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ~2 μM and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.

Synthesis, Structural, Spectroscopic, Electrochemical, Magnetic, and Catalytic Properties of the Trinuclear MnIII Triplesalen Complex [(talen){Mn(OAc)}3] Exhibiting Three Salen‐Subunits in a β‐cis‐Conformation

AbstractReaction of the triplesalen ligand H6talen with three equivalents Mn(OAc)2·4H2O in MeOH results in the formation of a brown solid which upon recrystallization from CH3CN provides the trinuclear complex [(talen){Mn(OAc)}3]·7CH3CN as evidenced by single‐crystal X‐ray diffraction. The triple tetradentate ligand (talen)6– coordinates to three MnIII ions in the rare β‐cis‐conformation of the salen‐like ligand compartments with the central oxygen donor (Oc) being rotated out of the plane. This results in a longer Mn–Oc bond length of 2.00 Å compared to the mean Mn–Ot bond lengths of the terminal phenolates at 1.86 Å. The six‐coordination is saturated by bidentate OAc– ligands. The electronic absorption spectrum measured in MeOH appears to be almost identical to all other complexes already studied possessing a {(talen)MnIII3}3+ subunit (in the trans‐conformation). The spectra measured in CH2Cl2 and CH3CN exhibit significant variations of the absorption features in the CT region above 20000 cm–1 and a low‐energy shift of the d‐d transitions from a shoulder around 18000 cm–1 in CH3OH to maxima around 13000 cm–1 in CH2Cl2 and CH3CN. This indicates a physical dissolution of [(talen){Mn(OAc)}3] in CH2Cl2 and CH3CN solutions without major structural rearrangements, while in MeOH solution a structural rearrangement to the preferred trans‐conformation of the salen‐like coordination compartments occurs loosing the bidentate coordination mode of the OAc– ligands. Electrochemical measurements reveal unresolved irreversible processes in the range 0.9–1.4 V vs. Fc+/Fc corresponding to oxidations of the MnIII‐phenolate units, while irreversible reductive waves in the range –0.7–(–1.2) V vs. Fc+/Fc correspond to MnIII to MnII reductions. The analysis of the magnetic data reveals a weaker antiferromagnetic interaction of J = –0.067 cm–1 and a stronger zero‐field splitting of D = –5.57 cm–1 in comparison to the complexes with {(talen)MnIII3}3+ subunits in the trans‐conformation consistent with the longer Mn–Oc distances and the asymmetric coordination environment, respectively. The complex [(talen){Mn(OAc)}3] catalyzes the epoxidation of 1, 2‐dihydronaphthalene with iodosylbenzene with complete conversion at room temperature.

Activation and Inactivation Steps Altered by Proline Hinge Mutations in Kv1.4

Several voltage-gated channels share a Proline-Valine-Proline (“proline hinge”) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine (P558G) or alanine (P558A), and studied these mutations in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N and C-type inactivation.Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that the transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement plays a significant role in inactivation and recovery.To analyze the effect of these mutations in changing channel gating kinetics, we modified our previously published model of Kv1.4 by adding a primed but non-conducting state connected to the open state by a voltage insensitive step. In order to reproduce the experimental observation that N-type inactivation occurs at more positive potentials than C-type, the C-type inactivated state must be coupled to earlier voltage-dependent steps. Analyses of this model suggest that both P558G and P558A mutations not only modified early voltage dependent steps, but also made the voltage insensitive transition from the primed state to the open state very slow relative to activation.

The European Young Chemist Award 2012

The European Young Chemist Award 2012

Interaction of the S6 Proline Hinge with N-Type and C-Type Inactivation in Kv1.4 Channels

Several voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials.

Thermodynamics of the GTP-GDP-operated Conformational Switch of Selenocysteine-specific Translation Factor SelB

SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.

Endogenous and Psychostimulant Substrates but not Blockers Stimulate Dopamine Transporter Phosphorylation at a Proline‐Directed Site

The dopamine transporter (DAT) clears extraneuronal dopamine (DA) and thus controls the spatio‐temporal dynamics of dopaminergic neurotransmission. DAT is the major target for psychostimulants such as cocaine (COC) and amphetamine (AMPH). DAT has a serine cluster on the distal N‐terminus that is phosphorylated in a PKC‐dependent manner and a proline‐directed site, Thr (T) 53, that is phosphorylated in vitro by the MAP kinases. COC and AMPH impact DAT regulatory properties including uptake activity and surface expression. Although, the mechanism of drug action on DAT is unclear, DAT phosphorylation is associated with altered surface expression and DAT activity. In this study, we examined the effect of DA and several psychostimulant drugs on the phosphorylation of DAT using a newly developed phospho‐specific antibody that detects pT53. Immunoblotting revealed that DAT substrates led to increased DAT T53 phosphorylation. This increase was transport or binding dependent as COC blocked the AMPH effect. In rat striatal synaptosomes METH‐stimulated DAT T53 phosphorylation was rapid, occurring within 60 sec. In contrast, uptake blockers did not influence T53 phosphorylation. Because phosphorylation at proline‐directed sites causes cis‐isomerization of the protein backbone, our findings indicate that translocation or binding of substrates induces major structural rearrangements of the membrane‐proximal N‐terminus. We hypothesize that substrate‐mediated phosphorylation‐dependent structural rearrangement resulting from prolyl cis‐isomerization could thus act as a mechanism affecting DAT function.Supported by NIDA R01 DA 13147‐09

Genomic islands of divergence in hybridizing Heliconius butterflies identified by large-scale targeted sequencing

Heliconius butterflies represent a recent radiation of species, in which wing pattern divergence has been implicated in speciation. Several loci that control wing pattern phenotypes have been mapped and two were identified through sequencing. These same gene regions play a role in adaptation across the whole Heliconius radiation. Previous studies of population genetic patterns at these regions have sequenced small amplicons. Here, we use targeted next-generation sequence capture to survey patterns of divergence across these entire regions in divergent geographical races and species of Heliconius. This technique was successful both within and between species for obtaining high coverage of almost all coding regions and sufficient coverage of non-coding regions to perform population genetic analyses. We find major peaks of elevated population differentiation between races across hybrid zones, which indicate regions under strong divergent selection. These 'islands' of divergence appear to be more extensive between closely related species, but there is less clear evidence for such islands between more distantly related species at two further points along the 'speciation continuum'. We also sequence fosmid clones across these regions in different Heliconius melpomene races. We find no major structural rearrangements but many relatively large (greater than 1 kb) insertion/deletion events (including gain/loss of transposable elements) that are variable between races.

Crystal Structure, Energetics, And Electrochemistry of Li2FeSiO4 Polymorphs from First Principles Calculations

The influence of crystal structure and relative stability on the electrochemical properties of Li2FeSiO4 polymorphs as cathode materials for Li-ion batteries is investigated. Six Li2FeSiO4 forms related to the crystal structure of Li3PO4 have been considered: three known polymorphs crystallizing in layered structures (with space groups P21, Pmnb, and Pmn21) and three forms reported for other LiMSiO4 materials crystallizing in three-dimensional (3D) frameworks (with space groups Pmn21, Pbn21, and P21/n). While Li2FeSiO4 polymorphs are very close in energy, their energies begin to differ substantially upon removal of Li, rendering those with lower electrostatic energy more stable. The change in relative stability of polymorphs upon delithiation results in a driving force for a phase transformation of the exiting two-dimensional (2D)-Li2FeSiO4 polymorphs to a more stable structure having a 3D network of [SiO4] and [FeO4] tetrahedra. The resulting phase exhibits a voltage plateau that is predicted to be 0.3 V below that of the original phase for the first electron process (Li2FeSiO4/LiFeSiO4 couple). The calculated voltage–capacity curves for the first and second cycles of Li2FeSiO4 at room temperature, assuming transformation to the new polymorph occurs after the first cycle, are in excellent agreement with experiments. Independently of the polymorphs, removal of the second lithium ion occurs at too high of a voltage (above 4.7 V) and is accompanied by major structural rearrangements, precluding the utilization of any of the unmodified Li2FeSiO4 polymorph derived from Li3PO4 as high specific capacity material.

Crystal structures of the Chromobacterium violaceumω‐transaminase reveal major structural rearrangements upon binding of coenzyme PLP

The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof ' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form • -transaminases and -transaminases bind by dynamic light scattering (View interaction) • -transaminase and -transaminase bind by x-ray crystallography (View interaction) • -transaminase and -transaminase bind by x-ray crystallography (View interaction).

Alpha-Synuclein Amyloid Oligomers Exhibit Beta-Sheet Antiparallel Structure as Revealed by FTIR Spectroscopy

Parkinson's disease is an aged-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-aa protein α-synuclein (AS). AS amyloid fibrils consist of interwound protofilaments with a cross β-sheet conformation. Fibrillation of AS follows a nucleation polymerization pathway involving major structural rearrangements and the population of diverse transient prefibrillar species varying in size and morphology. It is currently believed that cytotoxicity relies on these prefibrillar species rather than on the insoluble aggregates, although the underlying molecular mechanism remains elusive. Structural studies may contribute to the understanding of both amyloid aggregation mechanism and oligomer-induced toxicity.It is already recognized that soluble oligomeric AS species contain β-sheet structural elements that differ from the typical fibrillar structure. However, more detailed information on the origin of this difference is still lacking. In the present work we used ATR-FTIR spectroscopy, a technique especially sensitive to β-sheet structure, to discriminate between the β-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel β-sheet structure, as opposed to the parallel arrangement present in fibrils. We discussed about the possible regions that may be involved in such early β-sheet interactions.We suggest that antiparallel β-sheet structure might represent a distinctive signature of amyloid oligomers (Cerf et al, Biochem J, 2009, 421:415-23) underlying their common pathogenic action.

Crystal structures of theChromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP

Crystal structures of the<i>Chromobacterium violaceum</i>ω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP

Structural changes in ethanol–water mixtures: Ultrasonics, Brillouin scattering and molecular dynamics studies

Mixing of two hydrogen-bonded liquids may lead to significant composition dependent changes of liquid microscopic structure, which in turn will be reflected by changes of the corresponding thermodynamic, thermophysical and spectroscopic properties. For the liquid system {xEtOHC2H5OH+(1−xEtOH)H2O} at ambient temperatures and pressures, we show that major structural rearrangements occur in the composition range of 0.15<xEtOH<0.20, where xEtOH denotes the ethanol mole fraction. Most interestingly, in this region, the excess enthalpy shows a minimum, the excess heat capacity at constant pressure shows a maximum, and both the ultrasonic speed and the hypersonic speed exhibit a maximum. In fact, molecular dynamics (MD) simulation results indicate the saturation of ethanol clustering in this low ethanol mole fraction region. The corresponding change of structural/vibrational dynamics is detected by Brillouin scattering, i.e. it is reflected by the observed sound dispersion in the composition region 0.15<xEtOH<0.65, where simulations show micro-segregated distributions of water and ethanol domains that form a bi-continuous mixture. At larger ethanol mole fractions, we found chain-like hydrogen-bonded water clusters.

PARG: A Macrodomain in Disguise

Our understanding of poly-ADP-ribosylation as a posttranslational modification was limited by the lack of structural information on poly-ADP-ribose (PAR) hydrolysing enzymes. A recent study in Nature (Slade etal., 2011) reports the structure of PAR glycohydrolase (PARG), revealing unexpected similarity to the ubiquitous ADP-ribose-binding macrodomains.

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.