Dissection of the amyloid formation pathway in AL amyloidosis

In antibody light chain (AL) amyloidosis, overproduced light chain (LC) fragments accumulate as fibrils in organs and tissues of patients. In vitro, AL fibril formation is a slow process, characterized by a pronounced lag phase. The events occurring during this lag phase are largely unknown. We have dissected the lag phase of a patient-derived LC truncation and identified structural transitions that precede fibril formation. The process starts with partial unfolding of the V-L domain and the formation of small amounts of dimers. This is a prerequisite for the formation of an ensemble of oligomers, which are the precursors of fibrils. During oligomerization, the hydrophobic core of the LC domain rearranges which leads to changes in solvent accessibility and rigidity. Structural transitions from an anti-parallel to a parallel beta-sheet secondary structure occur in the oligomers prior to amyloid formation. Together, our results reveal a rate-limiting multi-step mechanism of structural transitions prior to fibril formation in AL amyloidosis, which offers, in the long run, opportunities for therapeutic intervention. AL amyloidosis is caused by the accumulation of overproduced light chain (LC) fragments as fibrils in patient organs and it is the most prevalent systemic amyloidosis. Here, the authors combine biochemical and biophysical experiments to characterise the lag phase of a patient-derived truncated LC and they identify structural transitions that precede fibril formation

A noncanonical PWI domain in the N-terminal helicase-associated region of the spliceosomal Brr2 protein

The spliceosomal RNA helicase Brr2 is required for the assembly of a catalytically active spliceosome on a messenger RNA precursor. Brr2 exhibits an unusual organization with tandem helicase units, each comprising dual RecA-like domains and a Sec63 homology unit, preceded by a more than 400-residue N-terminal helicase-associated region. Whereas recent crystal structures have provided insights into the molecular architecture and regulation of the Brr2 helicase region, little is known about the structural organization and function of its N-terminal part. Here, a near-atomic resolution crystal structure of a PWI-like domain that resides in the N-terminal region of Chaetomium thermophilum Brr2 is presented. CD spectroscopic studies suggested that this domain is conserved in the yeast and human Brr2 orthologues. Although canonical PWI domains act as low-specificity nucleic acid-binding domains, no significant affinity of the unusual PWI domain of Brr2 for a broad spectrum of DNAs and RNAs was detected in band-shift assays. Consistently, the C. thermophilum Brr2 PWI-like domain, in the conformation seen in the present crystal structure, lacks an expanded positively charged surface patch as observed in at least one canonical, nucleic acid-binding PWI domain. Instead, in a comprehensive yeast two-hybrid screen against human spliceosomal proteins, fragments of the N-terminal region of human Brr2 were found to interact with several other spliceosomal proteins. At least one of these interactions, with the Prp19 complex protein SPF27, depended on the presence of the PWI-like domain. The results suggest that the N-terminal region of Brr2 serves as a versatile protein-protein interaction platform in the spliceosome and that some interactions require or are reinforced by the PWI-like domain

FUELCELL2006-97257 DEVELOPMENT OF AN AUTOMATED CONTROL SYSTEM VERIFICATION PLATFORM FOR A SOLID OXIDE FUEL CELL

ABSTRACT The fuel cell industry is currently undergoing rapid development, and applications of fuel cell based power sources are diversifying. The advent of new and more sophisticated application areas and the expanding market necessitates development of efficient and robust fuel cell based power supplies that are reliable in their performance. These demands are answered not only by improved plant designs and innovations, but also by developing high-quality control algorithms. Quality and reliability of the complete system are ensured through extensive and varied testing. To this end an automated Hardware-in-the-Loop based control code verification and validation platform for the Delphi Solid Oxide Fuel Cell plant and control system has been developed. Verification activities are managed using the System Verification Manager tool. This paper outlines the application of this platform for safety and diagnostics verification and validation for a Solid Oxide Fuel Cell system. INTRODUCTION Control algorithms for complex industrial systems require thorough verification and validation against a variety of different operating conditions to ensure robustness and reliability of the design. Further, algorithm verification and validation becomes more important in the context of safety critical features of a system. Manual comprehensive testing of a development control algorithm can be a tedious, human-errorprone, time and resource intensive process, which is often infeasible

A Snu114-GTP-Prp8 module forms a relay station for efficient splicing in yeast

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114-GTP-Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing

Poly[[bis­{μ3-tris­[2-(1H-tetra­zol-1-yl)eth­yl]amine}copper(II)] bis­(perchlorate)]

In the title compound, {[Cu(C9H15N13)2](ClO4)2}n, the Cu2+ cation lies on an inversion center and is coordinated by the tetra­zole N4 atoms of six symmetry-equivalent tris­[2-(1H-tetra­zol-1-yl)eth­yl]amine ligands (t 3 z) in the form of a Jahn–Teller-distorted octa­hedron with Cu—N bond distances of 2.0210 (8), 2.0259 (8) and 2.4098 (8) Å. The tertiary amine N atom is stereochemically inactive. The cationic part of the structure, viz. [Cu(t 3 z)2]2+, forms an infinite two-dimensional network parallel to (100), in pockets of which the perchlorate anions reside. The individual networks are partially inter­locked and held together by C—H⋯O inter­actions to the perchlorate anions and C—H⋯N inter­actions to tetra­zole N atoms

Interplay of cis and trans regulatory mechanisms in the spliceosomal RNA helicase Brr2

RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing

Long range allostery mediates cooperative adenine nucleotide binding by the Ski2 like RNA helicase Brr2

Brr2 is an essential Ski2 like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N terminal helicase cassette and a structurally similar but enzymatically inactive C terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C terminal cassette is more than two orders of magnitude higher than that of the active N terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C terminal cassette reduces nucleotide binding at the N terminal cassette 70 away. Molecular dynamics simulations identified structural communication lines that likely mediate these long range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicin

Supersymmetric Dark Matter in the Light of LEP 1.5

We discuss the lower limit on the mass of the neutralino $\chi$ that can be obtained by combining data from $e^+e^-$ annihilation at LEP and elsewhere with astrophysical and theoretical considerations. Loopholes in the purely experimental analysis of ALEPH data from the Z peak and LEP 1.5, which appear when $\mu<0$ for certain values of the sneutrino mass $m_{\tilde\nu}$ and the ratio $\tan\beta$ of supersymmetric Higgs vacuum expectation values, may be largely or totally excluded by data from lower-energy $e^+e^-$ data, the hypothesis that most of the cosmological dark matter consists of $\chi$ particles, and the assumption that electroweak symmetry breaking is triggered by radiative corrections due to a heavy top quark. The combination of these inputs imposes m_{\chi} \ge 21.4~\gev, if soft supersymmetry-breaking masses are assumed to be universal at the grand-unification scale.Comment: 22 pages. one Latex file + nine eps figure

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The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43ATP-analogRNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a ?-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases

Light excited spin state switching of spin crossover coordination polymers

Zsfassung in dt. SpracheDie Eigenschaften, verbunden mit dem Einfluss von nicht koordinierenden Anionen und der Kettenl¨ange der verbr¨uckenden Liganden, von Eisen(II) Spin Crossover Verbindungen wurden systematisch untersucht. Hierzu wurden Komplexe mit Tetrafluoroborat, Perchlorat bzw.Hexafluorophosphat als Gegenionen und Ditetrazolliganden mit unterschiedlicher Anzahl von C-Atomen (n = 4-10 und 12) innerhalb der Alkylenkette hergestellt und analysiert.Die sich daraus ergebenden Ver¨anderungen des strukturellen, optischen, magnetischen bzw.magneto-optischen Verhaltens wurden im Detail gemessen und werden in der vorliegenden Arbeit vergleichend diskutiert. Der Tetrafluoroboratkomplex mit 4 C-Atomen in der Kette, bildet ein 3 dimensionales Netzwerk und weist einen gemischten High Spin (HS) / Low Spin (LS) Zustand unterhalb von 70 K auf.Wird der Komplex innerhalb kurzer Zeit auf 4,2 K abgek¨uhlt (gequencht), bildet sich ein metastabiler Zustand aus 80% HS und 20% LS Anteil, der bei h¨oheren Temperaturen mit einer Halbwertszeit von 2 Tagen relaxiert. Den selben metastabilen HS Zustand kann man mit Licht geeigneterWellenl¨ange ( = 537 nm) bei 10 K anregen (LIESST-Experiment). Im Vergleich dazu zeigt der Perchloratkomplex einen 2 stufigen ¨ Ubergang, mit Spin-Crossovertemperaturen bei 84 K und 134 K. Im Falle des Komplexes mit Hexafluorophosphat als Anion, wurde die Induzierung des Spin¨ubergangs durch Magnetfelder ¨uber 100 T erstmals gemessen und pr¨asentiert.Komplexe mit l¨angeren Br¨uckenliganden (n = 5-10 und 12) kristallisieren kettenf¨ormig wobei die Spin¨ubergangskurven mit steigender Anzahl von C-Atomen in der Kette gradueller werden.Vergleicht man die Spin¨ubergangstemperaturen der Perchloratkomplexe mit jenen der Tetrafluoroboratkomplexe, zeigen letztere h¨ohere ¨ Ubergangstemperaturen f¨ur 5-7 C-Atome in der Alkylenkette.Weiters teilen sich die Komplexserien abh¨angig von der Kettenl¨ange in zwei Gruppen: Solche mit geradzahliger Anzahl von C-Atomen in der Kette zeigen h¨ohere ¨ Ubergangstemperaturen als jene mit ungeradzahligen C-Atomen. Dieser "Parit¨atseffekt" kehrt sich in der Tetrafluoroborat Serie ab n = 8 um.Wird die Anzahl der C-Atome weiter erh¨oht, n¨ahern sich die Spin¨ubergangstemperaturen der beiden Serien 160 K. Der Parit¨atseffekt findet sich in der Analyse der Absorptionsspektren wieder und kann als Jahn-Teller Verzerrung interpretiert werden.Die Ergebnisse gew¨ahren Einblick in die sensiblen Zusammenh¨ange zwischen strukturellen Ver¨anderungen und elektronischen Besetzungszust¨anden, welche sich in thermisch, optisch und magnetisch anregbaren Spin¨ubergangen zeigen.The influence of non-coordinating anions and spacer length of the ligand is analysed in extension of the systematic investigations into iron(II) spin crossover coordination polymers. Ditetrazole complexes of iron(II) perchlorate, tetrafluoroborate and hexafluorophosphate where the two tetrazole moieties are separated by alkylene spacers with different number of carbon atoms (n) are presented. Pronounced structural, optical, magnetic and magneto-optical changes are analysed and found in the comparison of the two series.When n = 4 a three dimensional network is formed that in the case of the tetrafluoroborate derivative stabilises a mixed high spin (HS)/low spin (LS) state below 70 K. If, however, the sample is rapadly cooled (quenched) to 4.2 K a metastable 80% HS / 20% LS state is produced, which has a t1/2 = 2 days at 44 K. This metastable HS state is also obtained when irradiating the sample at 530 nm at 10 K (LIESST effect). The perchlorate form shows a two-step transition at 84 K and 134 K. For the hexafluorophosphate complex the induction of the spin crossover at magnetic fields higher than 100 T are measured and presented for the first time.Complexes with longer spacers (n = 5-10 and 12) crystallise in a chain type arrangement and the spin transition curves become more gradual with increasing n. When n = 5-7 the smaller tetrafluoroborate causes spin transitions to occur at higher temperatures than the equivalent perchlorate series. Furthermore the two series can be divided into two groups: those with an even number of carbons in the spacer show higher spin transition temperatures than the odd ones. This parity effect, reverses at n = 8 for the tetrafluoroborates. With a further increase of n, however, the complexes approach a limiting value of 160 K. This parity effect is also reflected in the analysis of the absorption spectra of the two series and can be attributed to the Jahn-Teller distortion.The results allow a deeper insight in coherences between structural changes and electronic occupations, which can be detected as thermal, optical or magnetic spin transitions.12