Unusual Affinity Research Articles

Regio- and sequence-controlled conjugated topological oligomers and polymers via boronate-tag assisted solution-phase strategy

The regulation of polymer topology and the precise control over the monomer sequence is crucial and challenging in polymer science. Herein, we report an efficient solution-phase synthetic strategy to prepare regio- and sequence-controlled conjugated polymers with topological variations via the usage of methyliminodiacetic acid (MIDA) boronates. Based on the solubility of MIDA boronates and their unusual binary affinity for silica gel, the synthesized regio- and sequence-defined conjugated oligomers can be rapidly purified via precipitation or automatic liquid chromatography. These synthesized discrete oligomers can be used for iterative exponential and sequential growth to obtain linear and dendrimer-like star polymers. Moreover, different topological sequence-controlled conjugated polymers are conveniently prepared from these discrete oligomers via condensation polymerization. By investigating the structure-property relationship of these polymers, we find that the optical properties are strongly influenced by the regiochemistry, which may give inspiration to the design of optoelectronic polymeric materials.

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy.

Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.

Correction: Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications.

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

The Story of Alice: Lewis Carroll and the Secret History of Wonderland by Robert Douglas-Fairhurst

Reviewed by: The Story of Alice: Lewis Carroll and the Secret History of Wonderland by Robert Douglas-Fairhurst James Hamby (bio) Robert Douglas-Fairhurst. The Story of Alice: Lewis Carroll and the Secret History of Wonderland. Cambridge: Belknap P of Harvard U P, 2015. Print. Lewis Carroll's Alice books have always been curiosities. For generations, readers and scholars have debated their depth and literary merit. While some scholars look for allegorical interpretations of Carroll's characters and narratives, Robert Douglas-Fairhurst takes a different approach by examining how the Alice books are a product of the rich tapestry of ideas and experiences of Carroll's entire life. In many ways, Carroll is as enigmatic as the works themselves, and this literary biography elucidates the connections between Carroll's brilliant, quirky personality and the wonderful world he created. The word "story" aptly appears in the title of Douglas-Fairhurst's work because it truly does read as engagingly as a novel. Douglas-Fairhurst begins the work describing Alice Liddell Hargreaves's trip to New York City in 1932, noting how the press insisted on creating the narrative that the literary Alice had strayed into modernity from her mid-Victorian world, and was once again off on a strange adventure. The aging Alice Hargreaves seems to have played along by giving interviews and posing for pictures. At any rate, the media's attention to Hargreaves's visit and their need to conflate her with the literary Alice demonstrate just how deeply ingrained the Alice books had become in popular imagination, a popularity that continues to this day as movies and television still produce adaptations of Alice fairly regularly. [End Page 132] Like any gifted novelist who begins at the end, Douglas-Fairhurst then shifts back to the very beginning of the story of Alice and examines the life of the man who created her. In many ways, Douglas-Fairhurst's text reads like a kunstlerroman as he traces the development of Carroll's personality. Even as a child, Carroll exhibited many idiosyncratic traits. He had unusual affinities for everyday items such as thimbles or gloves. He loved word games and was fascinated by language's ability to bring together dissimilar things and to create absurd situations. As a school child, he came to be especially proficient at mathematics, seemingly drawn to that discipline by its ability to impose order upon a chaotic world. In fact, the idea of bringing order from chaos seems to have been a driving force for Carroll throughout his entire life. Douglas-Fairhurst notes that many of Carroll's other intellectual and artistic pursuits, such as theology, journal-writing, and photography, often aided Carroll in his need to exert control on the world around him. Douglas-Fairhurst observes that, when Carroll had taken up photography, he "had chosen a pastime that was measured in equally uncompromising terms. It left little room for creative accidents; like religion or mathematics, it was a matter of all or nothing" (79). Ultimately, the desire for such absolutes is destined to fail, and the world around us will always remain a strange admixture of order and chaos. Carroll shaped his Alice books from these various ideas, producing a world where nonsense and rationality maintain an uneasy tension with one another. Douglas-Fairhurst also pays particular attention to the emotional matrix from which the Alice books evolved. Not only was control over chaos an integral component of Carroll's intellectual pursuits, but it also maintained a prominent place in his emotions as well. Control over time was particularly important to Carroll. Douglas-Fairhurst notes that Carroll held a fascination for incomplete things, such as unfinished paintings or unsolvable riddles, that force time into a state of perpetual suspended animation (17). Carroll found the idea of finality in anything to be depressing, and the passage of time painful. Perhaps worst of all was the loss of childhood. According to Douglas-Fairhurst, when Carroll befriended the Liddell sisters and began taking their photographs, he was fixing them forever in their childhoods, which he highly idealized. Carroll did the same when he began writing down what had begun as an ephemeral tale told to pass the...

Differential Water Thermodynamics Determine PI3K-Beta/Delta Selectivity for Solvent-Exposed Ligand Modifications

Phosphoinositide 3-kinases (PI3Ks) are involved in important cellular functions and represent desirable targets for drug discovery efforts, especially related to oncology; however, the four PI3K subtypes (α, β, γ, and δ) have highly similar binding sites, making the design of selective inhibitors challenging. A series of inhibitors with selectivity toward the β subtype over δ resulted in compound 3(S), which has entered a phase I/Ib clinical trial for patients with advanced PTEN-deficient cancer. Interestingly, X-ray crystallography revealed that the modifications making inhibitor 3(S) and related compounds selective toward the β-isoform do not interact directly with either PI3Kβ or PI3Kδ, thereby confounding rationalization of the SAR. Here, we apply explicit solvent molecular dynamics and solvent thermodynamic analysis using WaterMap in an effort to understand the unusual affinity and selectivity trends. We find that differences in solvent energetics and water networks, which are modulated upon binding of different ligands, explain the experimental affinity and selectivity trends. This study highlights the critical role of water molecules in molecular recognition and the importance of considering water networks in drug discovery efforts to rationalize and improve selectivity.

Sterically expanded CGC catalysts: substituent effects on ethylene and α-olefin polymerization

Several analogues of the sterically expanded constrained geometry catalyst Me2Si(η(1)-C29H36)(η(1)-N-tBu)ZrCl2·OEt2 (2) were synthesized to assess the effect on branching and molecular weight for ethylene homopolymerization. Catalysts based on tetramethyltetrahydrobenzofluorene (TetH), ethylTetH, t-butylTetH, and octamethyloctahydrodibenzofluorenyl (OctH) bearing a diphenylsilyl bridge were prepared and characterized: Me2Si(η(5)-C21H22)(η(1)-N-tBu)ZrCl2 (3); Me2Si(η(5)-C23H26)(η(1)-N-tBu)ZrCl2 (4); and Me2Si(η(5)-C25H30)(η(1)-N-tBu)ZrCl2 (5); Me2Si(η(5)-C21H22)(η(1)-N-tBu)ZrMe2 (6); and Ph2Si(η(5)-C29H36)(η(1)-N-tBu)ZrCl2 (7). Complexes 4, 5, 6, and 7 were characterized by X-ray crystallography and displayed η(5) hapticity to the carbon ring in each case, in contrast to 2. In comparison to 2, complexes 3, 4, 5, and 7 (in combination with methylaluminoxane = MAO) showed diminished branching, higher molecular weight, and higher polydispersity indices for obtained ethylene homopolymers. While 4/MAO produced the greatest molecular weight polymers, no branching was observed. Reactivity ratios were determined for the copolymerization of ethylene and 1-decene with 2/MAO. A value of r(ethylene) = 14.9 and an exceedingly high value of r(1-decene) = 0.49 were found--in line with previous reports of this catalyst's unusual affinity for α-olefins.

A fluorous copper(II)–carboxylate complex which magnetically and reversibly responds to humidity in the solid state

Reacting the fluorous carboxylate C 8F 17CO 2·HNEt 3 with Cu(OTf) 2 in acetone leads, depending on the carboxylate/copper ratio, either to the neutral dimeric complex [Cu 2(C 8F 17CO 2) 4(acetone) 2] ( 2), or to the dianionic monomeric complex [Cu(C 8F 17CO 2) 4](HNEt 3) 2 ( 4). Both complexes were characterized by IR, UV–vis and EPR spectroscopies, magnetic susceptibility measurements and X-ray diffraction analysis. The complex 2 displays the classical dimeric “paddlewheel” structure with four carboxylates bridging the two copper(II) ions while the complex 4 displays an unusual monomeric structure with four monodentate carboxylates bounded to the copper(II) in a square planar geometry. In contrast to 4, the complex 2 shows a very high affinity for water vapors in the solid state and behaves as a magnetic sponge. When exposed to air, ground crystals of 2 rapidly trap up to 6 water molecules per copper ion leading to a decrease of the magnetic coupling from 2 J = −480 cm −1 to 2 J = −7 cm −1. The hydration process is accompanied by drastic changes of the EPR and FTIR spectra, revealing that the binding of the water molecules to the copper atoms leads to the release of the acetone ligands from the solid and to the shifting of two carboxylates from bridging bidentate to non-bridging monodentate coordination mode. When hydrated 2 is exposed to high vacuum, the water molecules are removed from the solid. The dehydration process leads to the partial recovery of the antiferromagnetic coupling between the copper(II) ions as revealed by EPR and magnetic susceptibility measurements. These data show that the hydration/dehydration process is not accompanied by a fully reversible structural rearrangement in the solid state. The unusual affinity for water and consequently, the modification of the magnetic properties, is observed neither with the fluorous dimeric complex 1, nor with the dianionic monomeric complex 4.

The Intertwining of Structure and Function: Proposed Helix-Swapping of the SH2 Domain of Grb7, A Regulatory Protein Implicated in Cancer Progression and Inflammation

Grb7 is a multidomain intracellular signaling protein that links activated tyrosine kinases with downstream signaling targets. Best known for its regulatory role in cell migration and tumor metastasis, Grb7 also regulates inflammation by coupling NF-kappaB-inducing kinase with erbB/EGFR family receptors. The "adaptor" role of Grb7 in these processes depends upon binding to membrane-associated tyrosine kinases through its C-terminal SH2 domain. The Grb7-SH2 domain shares structural and functional similarity with the SH2 domain of Grb2, a constituent of the MAP kinase pathway. Both domains show unusual affinity for cyclic (beta-turn) ligands. The Grb2-SH2 domain also shows distinctive self-association behavior, forming intertwined ("swapped") dimers. While Grb7 and its SH2 domain are each known to dimerize, the mechanisms and functional significance of this self-association are incompletely understood. Additional residues in the Grb7-SH2 domain effectively lengthen its "EF loop" and render the domain a good candidate for swapped dimerization, through exchange of a C-terminal helix. We propose the existence of a swapped dimeric form of the Grb7-SH2 domain and offer a structural model derived through novel application of nuclear magnetic resonance-derived restraints.

Replacement of a Nitrogen by a Phosphorus Donor in Biomimetic Copper Complexes: a Surprising and Informative Case Study with Calix[6]arene-Based Cryptands

The aim of the paper is to characterize Cu complexes in the P(Ar)N(3) environment provided by ligands derived from triphenylphosphine P(C(6)H(4)CH(2)NHR)(3) and compare their coordination behavior and reactivity with those obtained with all-nitrogen ligands such as tren. It is shown that coordination of the PN(3) ligand (R = iPr) to Cu(I) and Cu(II) leads to complexes whose coordination sphere is hardly controlled as they readily undergo decoordination of either one N or the P donor together with oxidation of the latter. In strong contrast, when grafted on the small rim of a calix[6]arene, the P(Ar)N(3) is geometrically constrained into a tripod that enforces the metal center to remain in the same environment with a P-Cu bond for both oxidation states. These calix[6]PN(3)-based Cu(I) and Cu(II) complexes react readily with exogenous ligands, making a comparison with calix[6]tren-based copper complexes possible. Indeed, reactivity studies in solution highlight very different behaviors. The complex [Cu(calix[6]PN(3))](2+) shows an unusual affinity for weak sigma-donors (e.g., MeCN > EtOH), while the analogous cuprous complex, [Cu(calix[6]PN(3))](+), displays a surprising affinity for hard O-donor ligands (EtOH, DMF), which has never been observed for the tren analogues. Even more surprising is the lack of reactivity of [Cu(calix[6]PN(3))](+) toward dioxygen, which contrasts strongly with the high reactivity of the [Cu(calix[6]tren)](+) complex. In an attempt to explain the observed differences in binding properties and reactivity, Density Functional Theory calculations and electronic spectra simulations were undertaken. They suggest that coordination of the soft P(Ar)(3) center allows to tune the metal ion properties, either by absorbing excess electron density from Cu(I), or by increasing the electronic density of Cu(II). This is due to the simultaneous presence of the phosphorus atom (sigma-donor) in apical position and the aromatic groups (pi-acceptors) bound to the P-atom.

Affinity adsorption mechanism studies of adsorbents for oligopeptides using model polymer

To understand the principle of designing efficient adsorbents toward specific peptides, adsorption mechanism between oligopeptide VVRGCTWW (VW-8) and alkylamine modified polyacrylamide adsorbents was studied in this article. Linear models of the adsorbents were used to facilitate various measurements except for adsorption experiment. 2D 1H NMR experiments reveal the main binding site of VW-8 to the adsorbents is C-terminal Trp. When peptide CTWW (CW-4) was introduced as the reference, the same binding site was found. However, in adsorption experiments, a remarkable difference was observed with the adsorption capacity of the two peptides. According to isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) experiments, which are in good agreement with the adsorption results, VW-8 has much higher affinity to the adsorbents than CW-4 and the main driving force in adsorption process is considered to be electrostatic force. However, the unusual affinity adsorption of two oligopeptides on the adsorbents contrasted to the traditional view of electrostatic interaction. With a comprehensive understanding of above experimental results along with computer-aided analysis, we conclude that the difference in conformation of the two peptides has a great impact on the interaction mode and further affects the affinity between the peptides and the adsorbents. This provides valuable information for the further design of synthetic affinity ligand for VW-8.

Structure of Aart, a Designed Six-finger Zinc Finger Peptide, Bound to DNA

Cys2-His2 zinc fingers are one of the most common types of DNA-binding domains. Modifications to zinc-finger binding specificity have recently enabled custom DNA-binding proteins to be designed to a wide array of target sequences. We present here a 1.96 Å structure of Aart, a designed six-zinc finger protein, bound to a consensus DNA target site. This is the first structure of a designed protein with six fingers, and was intended to provide insights into the unusual affinity and specificity characteristics of this protein. Most protein−DNA contacts were found to be consistent with expectations, while others were unanticipated or insufficient to explain specificity. Several were unexpectedly mediated by glycerol, water molecules or amino acid−base stacking interactions. These results challenge some conventional concepts of recognition, particularly the finding that triplets containing 5′A, C, or T are typically not specified by direct interaction with the amino acid in position 6 of the recognition helix.

Monster Blood Tattoo: Foundling (review)

Reviewed by: Monster Blood Tattoo: Foundling April Spisak Cornish, D. M. Monster Blood Tattoo: Foundling; written and illus. by D. M. Cornish. Putnam, 2006 [434p] ISBN 0-399-24638-X$18.99 Reviewed from galleys Ad Gr. 5-7 Rossamund has had a difficult childhood living in an orphanage and struggling with bullies because of his female name. Now, armed with his quick wit and sharp mind, along with a few coins and a new outfit, he's ready to take up an apprenticeship, but his carefully planned future as a lamplighter falls apart on his first day out in the world. After Rossamund boards the wrong boat, he finds himself thrown into a dangerous world filled with deadly monsters and even more threatening humans, and he must sort out his allies from his enemies in order to survive. This is clearly the first book in a series: there are far more questions than answers, and several important elements, such as Rossamund's feminine name and unusual affinity for monsters, are left unaddressed, hopefully to be revisited in future installments. The fact that the end matter constitutes over a quarter of the pages additionally foregrounds world creation over plotting, though readers may enjoy the additional illustrations, definitions, maps, and diagrams. Nonetheless, the book's blend of horror, fantasy, and science fiction could appeal to a broad audience, who will appreciate the heroism of a young man facing frightening peril. Annotated, sharply drawn illustrations scattered throughout the book will help readers keep track of the large cast of characters. Copyright © 2006 The Board of Trustees of the University of Illinois

Endonuclease-like activity of heme proteins

Heme proteins, metmyoglobin, methemoglobin, and metcytochrome c showed unusual affinity for double-stranded DNA. Calorimetric studies show that binding of methemoglobin to calf thymus DNA (CTDNA) is weakly endothermic, and the binding constant is 4.9+/-0.7x10(5) M(-1). The Soret absorption bands of the heme proteins remained unchanged, in the presence of excess CTDNA, but a new circular dichroic band appeared at 210 nm. Helix melting studies indicated that the protein-DNA mixture denatures at a lower temperature than the individual components. Thermograms obtained by differential scanning calorimetry of the mixture indicated two distinct transitions, which are comparable to the thermograms obtained for individual components, but there was a reduction in the excess heat capacity. Activation of heme proteins by hydrogen peroxide resulted in the formation of high valent Fe(IV) oxo intermediates, and CTDNA reacted rapidly under these conditions. The rate was first-order in DNA concentration, and this reactivity resulted in DNA strand cleavage. Upon activation with hydrogen peroxide, for example, the heme proteins converted the supercoiled pUC18 DNA into nicked circular and linear DNA. No reaction occurred in the absence of the heme protein, or hydrogen peroxide. These data clearly indicate a novel property of several heme proteins, and this is first report of the endonuclease-like activity of the heme proteins.

Peripheral Benzodiazepine Receptor: Characterization in Human T-Lymphoma Jurkat Cells

Peripheral benzodiazepine receptor (PBR) has been considered a promising drug target for cancer therapy, and several ligands have been developed for this purpose. Human T-lymphoma Jurkat cells have been considered as lacking PBR and are often used as negative control to prove the specificity of PBR ligands effects. It is surprising that we evidenced PBR protein expression in this cell line by means of Western blotting and immunocytochemistry assays using specific anti-PBR antibodies. PBR intracellular localization was evidenced in mitochondria and nuclei, as demonstrated by confocal and electron microscopy. The binding of the [(3)H]4'-chloro derivative of diazepam [(3)H]7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and the isoquinoline carboxamide derivative [(3)H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3 isoquinolinecarboxamide (PK11195) evidenced a single class of binding sites with an unusual affinity constant (K(d)) of 1.77 +/- 0.30 and 2.20 +/- 0.20 microM, respectively. The pharmacological profile of the classic ligands showed that PK11195 was the most potent inhibitor in the radioligand binding assays followed by Ro5-4864 and diazepam, whereas clonazepam, a specific ligand for the central-type receptor, showed a K(i) >1.0 x 10(-4) M. By a combined strategy of reverse transcriptase-polymerase chain reaction and Southern blot experiments, we succeeded in isolating and cloning the full-length Jurkat PBR cDNA, called JuPBR. The JuPBR gene showed two single-nucleotide polymorphisms resulting in the two substitutions, Ala147 --> threonine and His162 --> arginine, of PBR amino acidic sequence. In conclusion, for the first time, we demonstrated PBR expression in Jurkat cells: the protein bound classic PBR ligands with micromolar affinity constants and presented a modified amino acidic sequence consequent to the detection of two gene polymorphisms.

The phaseolin vacuolar sorting signal promotes transient, strong membrane association and aggregation of the bean storage protein in transgenic tobacco

Vacuolar storage proteins of the 7S class are co-translationally introduced into the endoplasmic reticulum and reach storage vacuoles via the Golgi complex and dense vesicles. The signal for vacuolar sorting of one of these proteins, phaseolin of Phaseolus vulgaris, consists of a four-amino acid hydrophobic propeptide at the C-terminus. When this sequence is deleted, phaseolin is secreted instead of being sorted to vacuoles. It is shown here that in transgenic tobacco plants newly-synthesized phaseolin has unusual affinity to membranes and forms SDS-resistant aggregates, but mutated phaseolin polypeptides that are either secreted or defective in assembly do not have these characteristics. Association to membranes and aggregation are transient events: phaseolin accumulated in vacuoles is soluble in the absence of detergents and is not aggregated. Association to membranes starts before the phaseolin glycan acquires a complex structure and therefore before the protein reaches the medial or trans-cisternae of the Golgi complex. These results support the hypothesis of a relationship between aggregation and vacuolar sorting of phaseolin and indicate that sorting may start in early compartments of the secretory pathway.

Purification and biochemical characterization of native ERp29 from rat liver

ERp29 is a recently characterized resident of the ER (endoplasmic reticulum) lumen that has broad biological significance, being expressed ubiquitously and abundantly in animal cells. As an apparent housekeeper, ERp29 is thought to be a general folding assistant for secretory proteins and to probably function as a PDI (protein disulphide isomerase)-like molecular chaperone. In the present paper, we report the first purification to homogeneity and direct functional analysis of native ERp29, which has led to the unexpected finding that ERp29 lacks PDI-like folding activities. ERp29 was purified 4800-fold in non-denaturing conditions exploiting an unusual affinity for heparin. Two additional biochemical hallmarks that will assist the classification of ERp29 homologues were identified, namely the idiosyncratic behaviours of ERp29 on size-exclusion chromatography (M(r)<globular homodimer) and SDS/PAGE (M(r)>monomeric mass). In contrast with PDI and parallel-purified co-residents (calreticulin, ERp60), native ERp29 lacked classical chaperone, disulphide reductase and isomerase, and calcium-binding activities. In the chaperone assays, ERp29 neither protected substrate proteins against thermal aggregation nor interacted stably with chemically denatured proteins as detected by cross-linking. ERp29 also did not exhibit helper activity toward calreticulin (chaperone) or PDI and ERp60 (disulphide reductase). By refuting long-standing predictions about chaperone activity, these results expose ERp29 as a functionally distinct member of the ER machinery and prompt a revised hypothesis that ERp29 acts as a non-classical folding assistant. The native preparation and biochemical hallmarks established here provide a useful foundation for ongoing efforts to resolve the functional orphan status of ERp29.

Revisiting Paternal Care in the Assassin Bug, Atopozelus pallens (Heteroptera: Reduviidae)

Exclusive paternal care is exceptionally rare among arthropods, with reports of this behavior in only 13 phylogenetically independent lineages (Tallamy, 2000, 2001). Scott Ralston (1977) described males of an unidentified Zelus species aggressively defending aggregations of egg clutches from predators and parasites. This was a particularly interesting and important observation because it was (and remains) the only record of paternal care in New World Reduviidae and suggested remarkable convergence between Neotropical Zelus and several paternal Rhynocoris species from Africa (Odhiambo, 1959; Thomas and Manica, 2003). As part of a larger effort to test hypotheses regarding the evolution of exclusive paternal care, we have reexamined the reproductive behavior of Ralston’s “Zelus” in both lab and field. We conclude that his descriptions of paternal care were accurate with one important caveat: we believe he reversed the sexes! Ralston’s study was conducted in Cali, Colombia, on the campus of the Universidad del Valle. One of the similarities he noted between “Zelus” and paternal Rhynocoris species is an unusual affinity these predators share for a particular host plant species. Paternal Rhynocoris are restricted to Stylosanthes, a forage legume; and Ralston found populations of “Zelus” only on Pithecelobium dulce, a common leguminous shade tree in Central America and northern South America. In 2000 and 2001 we visited the same Pithecelobium trees studied by Ralston and found large populations of a reduviid

Remarkable N 2 affinity of a steam-activated FeZSM-5 catalyst: a 57Fe Mössbauer study

57Fe Mössbauer spectroscopy was used to investigate the redox behavior of iron species in steam-treated FeZSM-5, a catalyst material with outstanding properties in benzene-to-phenol oxidation as well as nitrous oxide decomposition. It was found that Fe 3+ ions in isolated extraframework sites and in extremely small oxo–iron clusters are easily converted into Fe 2+ species using a thermal treatment at 623 K. Fe 3+ ions in larger iron oxide particles of 2 nm in diameter located at the outer surface of the zeolite are less prone to reduction. Some of the iron species in the FeZSM-5 catalyst show an unusual affinity for N 2 leading to redox changes (Fe 3+→Fe 2+) at temperatures as low as 300 K. In addition, it has been observed that some of the Fe 2+ species, which are present in the catalyst after thermal reduction, reversibly adsorb N 2.

Intermolecular Dimerization within Pillared, Layered Clay Templates

Solutions of pyrene in the presence of a pillared, layered montmorillonite clay produce hybrid organic-inorganic materials with substantial molecular loading in the gallery regions between the clay layers. The results are in sharp contrast to other aromatics, such as benzene, naphthalene, or perylene, which show minimal incorporation of the molecules into the gallery regions of the clay. We present evidence that the unusual affinity for pyrene to form intermolecular dimers is the reason for the high loading. Pyrene monomers are easily introduced to the layers. Through steric hindrance, subsequent intermolecular dimer formation is allowed, and they are captured by the pillared, layered structure. CW and time-resolved emission spectra strongly indicate the presence of face-to-face intermolecular dimers (excimers) within the clay galleries. The combination of the ease of high molecular loading into an inorganic, high aspect ratio template and the collective optical properties of the organic layer may be useful as a new means to create hybrid structures.