Structural, Spectroscopic (NMR, IR, and Raman), and DFT Investigation of the Self-Assembled Nanostructure of Pravastatin-LDH (Layered Double Hydroxides) Systems

Layered double hydroxide (LDH) nanocontainers, suitable as carriers for anionic drugs, were intercalated with Pravastatin drug using magnesium-aluminum and zinc-aluminum in a M-II/Al molar ratio equal 2 and different Al3+/Pravastatin molar ratios. Postsynthesis treatments were used in order to increase the materials crystallinity. Hybrid materials were characterized by a set of physical chemical techniques: chemical elemental analysis, X-ray diffraction (XRD), mass coupled thermal analyses, vibrational infrared and Raman spectroscopies, and solid-state C-13 nuclear magnetic resonance (NMR). Results were interpreted in light of computational density functional theory (DFT) calculations performed for Sodium Pravastatin in order to assign the data obtained for the LDH intercalated materials. XRD peaks of LDH-Pravastatin material and the one-dimensional (1D) electron density map pointed out to a bilayer arrangement of Pravastatin in the interlayer region, where its associated carboxylate and vicinal hydroxyl groups are close to the positive LDH. The structural organization observed for the stacked assembly containing the unsymmetrical and bulky monoanion Pravastatin and LDH seems to be promoted by a self-assembling process, in which local interactions are maximized and chloride ion cointercalation is required. It is observed a high similarity among vibrational and C-13 NMR spectra of Na-Pravastatin and LDH-Pravastatin materials. Those features indicate that the intercalation preserves the drug structural integrity. Spectroscopic techniques corroborate the nature of the guest species and their arrangement between the inorganic layers. Changes related to carboxylate, alcohol, and olefinic moieties are observed in both vibrational Raman and C-13 NMR spectra after the drug intercalation. Thus, Pravastatin ions are forced to be arranged as head to tail through intermolecular hydrogen bonding between adjacent organic species. The thermal decomposition profile of the hybrid samples is distinct of that one observed for Na-Pravastatin salt, however, with no visible increase in the thermal behavior when the organic anion is sequestrated within LDH gap.

Synthetic and mechanistic investigation of new radical processes: reaction of organic azides with group-XIII Lewis acids

Dichloroindium hydride revealed to be a valid alternative to tributyltin hydride for radical reduction of organic (alkyl, aryl, acyl, solfonyl) azides. The new approach entails mild reaction conditions and provides high yields of the corresponding amines and amides, also showing high degrees of selectivity. The system dichloroindium hydride / azides can be utilised in fivemembered ring closures of g-azidonitriles, as a new source of aminyl radicals for the attractive synthesis of interesting amidine compounds in the absence of both toxic reagents and tedious purification procedures. Allylindium dichloride seems a good substitute for dichloroindium hydride for generation of indium centred radicals under photolytic conditions, since it allows allylation of electrophilic azides (e.g. phenylsulfonyl azide) and halogen or ester δ-substituted azides, the latter through a 1,5-H transfer rearrangement mechanism. Evidences of the radical nature of the reactions mechanism were provided by ESR spectroscopy, furthermore the same technique, allowed to discover that the reaction of azides with indium trichloride and other group XIII Lewis acids, in particular gallium trichloride, gives rise to strongly coloured, persistent paramagnetic species, whose structure is consistent with the radical cation of the head-to-tail dimer of the aniline corresponding to the starting azide.

Photochemie von ungesättigten Sternverbindungen

Photochemie von ungesättigten Sternverbindungen.Wirt-Gast-Systeme bei stilbenoiden Dendrimeren. Diese Arbeit beschäftigt sich mit der Synthese und denphotochemischen Eigenschaften stilbenoider und chalkonoiderSternverbindungen.Die Synthese der stilbenoiden Dendrimere gelingt über einekonvergente in einzelnen Generationen gekoppelteSynthesestrategie durch Wittig-Horner-Olefinierung inKombination mit einer Schutzgruppentechnik.Experimente mit den stibenoiden Dendrimeren 1. Generationhaben gezeigt, dass geänderte Belichtungsbedingungen(monochromatisches Licht in Gegenwart einesTriplett-Sensibilisators) die photochemische[2+2]Cycloaddition bzw. CC-Verknüpfung unterdrücken. Dabeiverläuft selektiv eine cis/trans-Isomerisierung, die füroptische Schaltung genutzt werden könnte.Mit Triplett-Sensibilisatoren wie Diacetyl oder Acetonkonnten auch Wirt-Gast-Systeme mit stilbenoiden Dendrimerengebildet werden. Das zeigt die Möglichkeit der Einlagerungvon Gastmolekülen in freie Kavitäten der Dendrimere.Die Synthese der chalkonoiden Sternverbindungen gelingt übereine dreifache Aldolkondensation des 1,3,5-Triacetylbenzolsmit unsubstituierten oder 3,4,5-trialkoxysubstituiertenBenzaldehyden. Mit einer geeigneten Verzweigungseinheitwurden auch Armbauteile zur Generationenerweiterungsynthetisiert.Die photochemischen Eigenschaften der chalkonoidenSternverbindungen in Lösung und im Festzustand wurdenstudiert und die entstandenen Produkte charakterisiert. BeiBelichtung im Festzustand läuft bei topochemischer Kontrolleeine Photodimerisierung unter Bildung eines Cyclobutanringsdurch Cycloaddition olefinischer Doppelbindungen ab, wobeiein Kopf-Schwanz-anti-Dimer erhalten wird. BeiPhotodimerisierung in Lösung entsteht einKopf-Kopf-anti-Dimer als Hauptprodukt. Das kann zur Syntheseder sonst schwer zugänglichen [4.4.4](1,3,5)Cyclophanegenutzt werden.

RNA-basierte Impfstoffe zur Induktion optimaler Immunantworten

Antigen-kodierende RNA wird als eine sichere und effiziente Alternative zu traditionellen Impfstoff-Formulierungen, wie Peptid-, Protein-, rekombinanten viralen oder DNA basierten Impfstoffen betrachtet. Der endgültige klinische Nutzen RNA-basierter Impfstoffe wird von der Optimierung verschiedener Parameter abhängig sein, die zur Induktion und effizienten Expansion der humoralen und zellvermittelten Immunantwort beitragen. Vor diesem Hintergrund war die Zielsetzung der vorliegenden Arbeit, die Etablierung pharmakologischer und immunologischer Parameter für die Generierung effektiver Immunantworten durch RNA-Impfstoffe sowie deren Wirksamkeit in vitro und im Mausmodell unter Nutzung von Modellantigenen zu testen. Zur Untersuchung und Optimierung der RNA-Pharmakokinetik, als einem Schlüsselaspekt der klinischen Medikamentenentwicklung, wurde der Einfluss von strukturellen Modifikationen auf die Transkriptstabilität und Translationseffizienz von Reporter-Proteinen in einer zeitabhängigen Kinetik evaluiert. Es wurde gezeigt, dass ein poly(A) Schwanz von 120 Adenosinen, verglichen mit einem kürzeren, ein freies 3´ poly(A) Ende, verglichen mit einem verdeckten und eine doppelte β-globin 3´ UTR, unabhängig voneinander zu einer Erhöhung der IVT-RNA Stabilität und zu einer Verbesserung der Translationseffizienz beitrugen und dadurch insgesamt zu einer erhöhten Proteinexpression führten. Antigen-kodierende IVT-RNA mit diesen molekularen Merkmalen in Kombination führte, im Vergleich zur Standard IVT-RNA, zu einer erhöhten Dichte und Stabilität von Peptid/MHC-Komplexen auf der Zelloberfläche transfizierter DCs und dadurch zu einer verbesserten Stimulation von CD4+ und CD8+ T-Zellen im murinen und humanen System. Mit dem Ziel, die RNA kodierte Antigenform für die Induktion einer verstärkten Antikörperantwort zu modifizieren, wurde im zweiten Teil der Arbeit ein Antigen-IgM Fusionskonstrukt hergestellt und hinsichtlich seiner Eignung als neues Impfstoff-Format untersucht. Die Ausgangshypothese, dass die RNA kodierten Antigen-IgM Fusionsproteine polymerisieren, von transfizierten Zellen sezerniert werden und aufgrund der repetitiven Antigenstruktur im Vergleich mit dem monomeren Antigen zu einer Verstärkung der Antikörperantwort führen, wurde in vitro und in vivo im Mausmodell bestätigt. Die Entwicklung und Evaluierung von Zytokinfusionsproteinen zur selektiven Verstärkung der antigenspezifischen Immunantworten bildeten den dritten Schwerpunkt der vorliegenden Arbeit. Zur weiteren Verstärkung der Antikörperantwort wurde basierend auf den Resultaten aus dem zweiten Teil ein IL2-IgM Fusionskonstrukt hergestellt. Die Ko-Transfektion von Antigen-IgM und IL2-IgM kodierender IVT-RNA führte zu einer signifikant stärkeren Antikörperantwort als die Ko-Transfektion von Antigen-IgM und IL2. Für die Initiierung einer erfolgreichen anti-Tumor-Immunantwort ist das Priming antigenspezifischer T-Zellen essentiell. Um die Effizienz dieses Prozesses zu steigern, wurde ein bifunktionelles IL2-mCD40L Fusionskonstrukt hergestellt und sein Einfluss auf die Effektorfunktion von DCs in vitro und in vivo untersucht. Es wurde gezeigt, dass ein RNA kodiertes IL2-mCD40L Fusionsprotein als genetisches Adjuvanz zu einer Effizienzsteigerung des Priming zytotoxischer T-Zellen führt. Somit wurden in dieser Arbeit durch die Optimierung der Pharmakokinetik, die Modifikation der Antigenform und die Herstellung und Evaluierung von Zytokinfusionskonstrukten als genetische Adjuvantien, RNA-basierte Impfstoffe für eine optimierte Induktion von antigenspezifischen Immunantworten weiter verbessert.

Loss of β1-integrin-deficient cells during the development of endoderm-derived epithelia

Beta1-integrins (beta1) represent cell surface receptors which mediate cell-matrix and cell-cell interactions. Fässler and Meyer described chimeric mice containing transgenic cells that express the LacZ gene instead of the beta1 gene. They observed beta1-negative cells in all germ layers at embryonic day E 8.5. Later in development, using a glucose phosphate isomerase assay of homogenized tissue samples, high levels of transgenic cells were found in skeletal muscle and gut, low levels in lung, heart, and kidney and none in the liver and spleen (Fässler and Meyer 1995). In order to study which cell types require beta1 during development of the primitive gut including its derivatives, chimeric fetuses containing 15 to 25% transgenic cells were obtained at days E 14.5 and E 15.5. They were LacZ (beta-galactosidase) stained "en bloc" and cross-sectioned head to tail. In esophagus, trachea, lung, stomach, hindgut, and the future urinary bladder, we observed various mesoderm-derived beta1-negative cells (e.g. fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells) but no beta1-negative epithelial cells. Since the epithelia of lung, esophagus, trachea, stomach, hindgut, and urinary bladder are derived from the endodermal gut tube, we hypothesize that beta1 is essential for the development and/or survival of the epithelia of the fore- and hindgut and its derivatives.

The 2.0-Å resolution crystal structure of a trimeric antibody fragment with noncognate VH–VL domain pairs shows a rearrangement of VH CDR3

The 2.0-Å resolution x-ray crystal structure of a novel trimeric antibody fragment, a “triabody,” has been determined. The trimer is made up of polypeptides constructed in a manner identical to that previously described for some “diabodies”: a VL domain directly fused to the C terminus of a VH domain—i.e., without any linker sequence. The trimer has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion. For the particular structure reported here, the polypeptide was constructed with a VH domain from one antibody fused to the VL domain from an unrelated antibody giving rise to “combinatorial” Fvs upon formation of the trimer. The structure shows that the exchange of the VL domain from antibody B1-8, a Vλ domain, with the VL domain from antibody NQ11, a Vκ domain, leads to a dramatic conformational change in the VH CDR3 loop of antibody B1-8. The magnitude of this change is similar to the largest of the conformational changes observed in antibody fragments in response to antigen binding. Combinatorial pairing of VH and VL domains constitutes a major component of antibody diversity. Conformationally flexible antigen-binding sites capable of adapting to the specific CDR3 loop context created upon VH–VL pairing may be employed by the immune system to maximize the structural diversity of the immune response.

Transgenic rats reveal functional conservation of regulatory controls between the Fugu isotocin and rat oxytocin genes

We have asked whether comparative genome analysis and rat transgenesis can be used to identify functional regulatory domains in the gene locus encoding the hypothalamic neuropeptides oxytocin (OT) and vasopressin. Isotocin (IT) and vasotocin (VT) are the teleost homologues of these genes. A contiguous stretch of 46 kb spanning the Fugu IT-VT locus has been sequenced, and nine putative genes were found. Unlike the OT and vasopressin genes, which are closely linked in the mammalian genome in a tail-to-tail orientation, Fugu IT and VT genes are linked head to tail and are separated by five genes. When a cosmid containing the Fugu IT-VT locus was introduced into the rat genome, we found that the Fugu IT gene was specifically expressed in rat hypothalamic oxytocinergic neurons and mimicked the response of the endogenous OT gene to an osmotic stimulus. These data show that cis-acting elements and trans-acting factors mediating the cell-specific and physiological regulation of the OT and IT genes are conserved between mammals and fish. The combination of Fugu genome analysis and transgenesis in a mammal is a powerful tool for identifying and analyzing conserved vertebrate regulatory elements.

Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

HCC-2, a human chemokine: Gene structure, expression pattern, and biological activity

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named “HCC-2.” The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKβ8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1γ, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1α. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.

An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and γ irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

A structural model for a homeotic protein-extradenticle-DNA complex accounts for the choice of HOX protein in the heterodimer.

The genes of the homeotic complex (HOX) encode DNA binding homeodomain proteins that control developmental fates by differentially regulating the transcription of downstream target genes. Despite their unique in vivo functions, disparate HOX proteins often bind to very similar DNA sequences in vitro. Thus, a critical question is how HOX proteins select the correct sets of target genes in vivo. The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. For example, the HOX protein labial cooperatively binds with extradenticle protein to a 20-bp oligonucleotide that is sufficient to direct a labial-like expression pattern in Drosophila embryos. Here we have analyzed the protein-DNA interactions that are important for forming the labial-extradenticle-DNA complex. The data suggest a model in which labial and extradenticle, separated by only 4 bp, bind this DNA as a heterodimer in a head-to-tail orientation. We have confirmed several aspects of this model by characterizing extradenticle-HOX binding to mutant oligonucleotides. Most importantly, mutations in base pairs predicted to contact the HOX N-terminal arm resulted in a change in HOX preference in the heterodimer, from labial to Ultrabithorax. These results demonstrate that extradenticle prefers to bind cooperatively with different HOX proteins depending on subtle differences in the heterodimer binding site.

Biochemical characterization and DNA binding properties of the MocR family member GabR, a PLP-dependent transcription factor

GabR è un fattore di trascrizione chimerico appartenente alla famiglia dei MocR/GabR, costituito da un dominio N-terminale elica-giro-elica di legame al DNA e un dominio effettore e/o di oligomerizzazione al C-terminale. I due domini sono connessi da un linker flessibile di 29 aminoacidi. Il dominio C-terminale è strutturalmente omologo agli enzimi aminotransferasici fold-type I, i quali, utilizzando il piridossal-5’-fosfato (PLP) come cofattore, sono direttamente coinvolti nel metabolismo degli aminoacidi. L’interazione contemporanea di PLP e acido γ-aminobutirrico (GABA) a GabR fa sì che questa promuova la trascrizione di due geni, gabT e gabD, implicati nel metabolismo del GABA. GabR cristallizza come un omodimero con una configurazione testa-coda. Il legame con la regione promotrice gabTD avviene attraverso il riconoscimento specifico di due sequenze dirette e ripetute (ATACCA), separate da uno spacer di 34 bp. In questo studio sono state indagate le proprietà biochimiche, strutturali e di legame al DNA della proteina GabR di Bacillus subtilis. L’analisi spettroscopica dimostra che GabR interagisce con il PLP formando l’aldimina interna, mentre in presenza di GABA si ottiene l’aldimina esterna. L’interazione fra il promotore gabTD e le forme holo e apo di GabR è stata monitorata mediante Microscopia a Forza atomica (AFM). In queste due condizioni di legame è stata stimata una Kd di circa 40 ηM. La presenza di GABA invece, determinava un incremento di circa due volte della Kd, variazioni strutturali nei complessi GabR-DNA e una riduzione del compattamento del DNA alla proteina, indipendentemente dalla sequenza del promotore in esame. Al fine di valutare il ruolo delle caratteristiche topologiche del promotore, sono state inserite cinque e dieci bp all’interno della regione spacer che separa le due sequenze ripetute dirette riconosciute da GabR. I significativi cambiamenti topologici riscontrati nel frammento aggiunto di cinque bp si riflettono anche sulla forte riduzione dell’affinità di legame verso la proteina. Al contrario, l’inserzione di 10 bp provoca solamente l’allontanamento delle sequenze ripetute dirette. L’assenza quindi di cambiamenti significativi nella topologia di questo promotore fa sì che l’affinità di legame per GabR rimanga pressoché inalterata rispetto al promotore non mutato. L’analisi del potenziale elettrostatico superficiale di GabR mostra la presenza di una fascia carica positivamente che si estende lungo un’intera faccia della proteina. Per verificare l’importanza di questa caratteristica di GabR nel meccanismo di interazione al DNA, sono stati preparati ed indagati i mutanti R129Q e K362-366Q, in cui la carica positiva superficiale risultava indebolita. L’affinità di legame dei mutanti di GabR per il DNA era inferiore rispetto alla proteina non mutata, in particolar modo nel mutante K362-366Q. Le evidenze acquisite suggeriscono che la curvatura intrinseca del promotore ed il corretto orientamento delle sequenze sulla doppia elica, più della distanza che le separa, siano critici per sostenere l’interazione con GabR. Oltre a questo, la superficie positiva di GabR è richiesta per accomodare la curvatura del DNA sul corpo della proteina. Alla luce di questo, l’interazione GabR-gabTD è un esempio di come il riconoscimento specifico di sequenze, la topologia del DNA e le caratteristiche strutturali della proteina siano contemporaneamente necessarie per sostenere un’interazione proteina-DNA stabile.

The regulation of Hox gene expression during animal development

Hox genes encode a family of transcriptional regulators that elicit distinct developmental programmes along the head-to-tail axis of animals. The specific regional functions of individual Hox genes largely reflect their restricted expression patterns, the disruption of which can lead to developmental defects and disease. Here, we examine the spectrum of molecular mechanisms controlling Hox gene expression in model vertebrates and invertebrates and find that a diverse range of mechanisms, including nuclear dynamics, RNA processing, microRNA and translational regulation, all concur to control Hox gene outputs. We propose that this complex multi-tiered regulation might contribute to the robustness of Hox expression during development.

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae)

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.