Interplay of Optical,Morphological, and Electronic Effects of ZnO Optical Spacers in Highly Efficient Polymer Solar Cells

Optical spacers based on metal oxide layers have been intensively studied in poly(3‐hexylthiophene) (P3HT) based polymer solar cells for optimizing light distribution inside the device, but to date, the potential of such a metal oxide spacer to improve the electronic performance of the polymer solar cells simultaneously has not yet be investigated. Here, a detailed study of performance improvement in high efficient polymer solar cells by insertion of solution‐processed ZnO optical spacer using ethanolamine surface modification is reported. Insertion of the modified ZnO optical spacer strongly improves the performance of polymer solar cells even in the absence of an increase in light absorption. The electric improvements of the device are related to improved electron extraction, reduced contact barrier, and reduced recombination at the cathode. Importantly, it is shown for the first time that the morphology of optical spacer layer is a crucial parameter to obtain highly efficient solar cells in normal device structures. By optimizing optical spacer effects, contact resistance, and morphology of ZnO optical spacers, poly[[4,8‐bis[(2‐ethylhexyl)oxy]benzo[1,2‐b:4,5‐b′]dithiophene‐2,6diyl] [3‐fluoro‐2‐[(2‐ethylhexyl)carbonyl] thieno[3,4‐b]thiophenediyl]] (PTB7):[6,6]‐phenyl‐C71‐butyric acid (PC₇₀BM) bulk heterojunction solar cells with conversion efficiency of 7.6% are obtained in normal device structures with all‐solution‐processed interlayers.     

Identification and characterization of a novel peritrophic matrix protein, Ae-Aper50, and the microvillar membrane protein, AEG12, from the mosquito, Aedes aegypti

Immuno-screening of an adult Aedes aegypti midgut cDNA expression library with anti-peritrophic matrix antibodies identified cDNAs encoding a novel peritrophic matrix protein, termed Ae. aegypti Adult Peritrophin 50 (Ae-Aper50), and the epithelial cell-surface membrane protein, AEG12. Both genes are expressed exclusively in the midguts of adult female mosquitoes and their expression is strongly induced by blood feeding. Ae-Aper50 has a predicted secretory signal peptide and five chitin-binding domains with intervening mucin-like domains. Localization of Ae-Aper50 to the peritrophic matrix was demonstrated by immuno-electron microscopy. Recombinant Ae-Aper50 expressed in baculovirus-infected insect cells binds chitin in vitro. Site-directed mutagenesis was used to study the role that cysteine residues from a single chitin-binding domain play in the binding to a chitin substrate. Most of the cysteine residues proved to be critical for binding. AEG12 has a putative secretory signal peptide at the amino-terminus and a putative glycosyl-phosphatidylinositol (GPI) anchor signal at its carboxyl-terminus and the protein was localized by immuno-electron microscopy to the midgut epithelial cell microvilli.     

Development and validation of a fluorogenic assay to measure separase enzyme activity

Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples.     

Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.GroEL, an essential chaperonin, is known to form a ring-shaped structure for sequestering substrate proteins from the crowded cellular milieu and is responsible for the occurrence of various cellular processes, such as de novo folding, transport, and macromolecular assembly, within a biologically relevant time scale (7, 26, 48, 53). In Escherichia coli, GroEL, along with its cofactor GroES, assists the folding of about 10 to 30% of cytosolic proteins, among which some are known to be essential for cell viability (15, 26, 27, 31). GroEL was originally identified as the host factor responsible for phage λ and T4 capsid protein assembly and was subsequently shown to be essential for cell viability (17, 20). E. coli groEL is found in an operonic arrangement with groES (groESL), and its expression is regulated by multiple promoter elements.GroEL function has been shown to be a complex interplay between its interaction with and encapsulation of substrate proteins, with concomitant conformational changes induced by ATP binding, hydrolysis, and GroES binding (24, 56, 62). E. coli GroEL exists as a homotetradecamer forming two isologous rings of seven identical subunits each. Crystallographic analyses have delineated the three-domain architecture of GroEL monomers and the GroES-GroEL interactions (4, 63). The central region of the GroEL polypeptide, spanning amino acid residues 191 to 376, constitutes the GroES and substrate polypeptide-binding apical domain. The equatorial ATPase domain spanning two extremities of the GroEL polypeptide, that is, residues 6 to 133 and 409 to 523, is responsible for the ATPase activity and the bulk of intersubunit interactions. The hinge-forming intermediate domain, spanning two regions on the polypeptide, namely, residues 134 to 190 and 377 to 408, connects the said two domains in the tertiary structure. The conformational changes resulting from ATP binding and hydrolysis at the equatorial domain are coupled to those occurring at the apical domain via this hinge region (4, 63).The usual size limit for the substrate proteins, as shown by both in vitro and in vivo studies, is around 57 kDa, although the cis cavity is reported to theoretically accommodate larger proteins, on the order of 104 kDa (10, 27, 35, 46). Productive in vivo folding of the proteins larger than the usual size limit, such as the 86-kDa maltose binding protein fusion and 82-kDa mitochondrial aconitase, has also been reported (9, 29). Since such large substrates are difficult to accommodate in the central cavity, it has been suggested that their productive folding might occur outside the cis cavity. These studies therefore indicate that the substrate recognition patterns of GroEL may be more diverse than initially thought.Recent genome annotation studies of various bacteria have revealed that a few bacterial genomes possess multiple copies of groEL genes (2, 18, 30). The Mycobacterium tuberculosis genome bears two copies of groEL genes (groELs). One of these, groEL1, is arranged in an operon, with the cognate cochaperonin groES being the first gene, while the second copy, groEL2, exists separately on the genome (13). Recombinant mycobacterial GroELs were shown to possess biochemical features that deviated significantly from the trademark properties of E. coli GroEL. The most striking feature of M. tuberculosis GroELs, however, was their oligomeric state, where contrary to expectations, in vitro they did not form the canonical tetradecameric assembly when purified from E. coli. The proteins instead existed as lower oligomers (dimers) irrespective of the presence or absence of cofactors, such as the cognate GroES or ATP (40, 41). Furthermore, they displayed weak ATPase activities and GroES independence in preventing aggregation of the denatured polypeptides.Evolutionary studies of M. tuberculosis groEL sequences have suggested rapid evolution of the groEL1 gene, yet without turning these into pseudogenes (21). The other hypothesis suggests that M. tuberculosis, being an organism that grows slowly, might require GroEL function that does not utilize ATP rapidly but, rather, with a slow turnover rate. Alternately, additional mechanisms might exist in M. tuberculosis which could mediate regulated oligomerization of M. tuberculosis chaperonins. Such regulation might help in the controlled utilization of ATP in nutrient-deprived M. tuberculosis, as observed for other chaperones, such as small heat shock proteins (23).In the present study, we have exploited the unusual oligomeric status of the recombinant M. tuberculosis GroELs to study the significance of oligomer formation for GroEL''s function as a molecular chaperone. Furthermore, we have explored the possibility of the existence of regulated oligomerization for native M. tuberculosis GroELs in their natural setting. We first show that M. tuberculosis groEL genes are not capable of complementing a conditional allele of E. coli groEL, namely, groEL44. The results of phenotypic and biochemical analyses of GroEL variants obtained by gene shuffling and domain swapping suggest that the impaired chaperoning ability of recombinant M. tuberculosis GroELs is a consequence of their inability to form higher-order oligomers in E. coli and that oligomerization is the prelude to the formation of an active GroEL chaperonin. Further, by immunochemical and mass spectrometric (MS) analysis of native mycobacterial GroELs, we show that M. tuberculosis GroEL1 exists in multiple oligomeric forms, viz., monomeric, dimeric, heptameric (single ring), and tetradecameric (double ring) forms, and that the switch between single-ring and double-ring variants is operated by phosphorylation on a serine residue. These observations suggest that the determinants of oligomerization for M. tuberculosis GroEL1 are distinct from those of its E. coli counterpart and that it does oligomerize in M. tuberculosis (its native environment), whereas it loses its oligomerization capability when expressed in E. coli. It could thus be possible that M. tuberculosis GroEL1 requires a certain native M. tuberculosis protein, probably a eukaryotic-like Ser-Thr protein kinase, to oligomerize properly, though the precise reason cannot be discerned by these observations.     

A novel nucleoid-associated protein of Mycobacterium tuberculosis is a sequence homolog of GroEL

The Mycobacterium tuberculosis genome sequence reveals remarkable absence of many nucleoid-associated proteins (NAPs), such as HNS, Hfq or DPS. In order to characterize the nucleoids of M. tuberculosis, we have attempted to identify NAPs, and report an interesting finding that a chaperonin-homolog, GroEL1, is nucleoid associated. We report that M. tuberculosis GroEL1 binds DNA with low specificity but high affinity, suggesting that it might have naturally evolved to bind DNA. We are able to demonstrate that GroEL1 can effectively function as a DNA-protecting agent against DNase I or hydroxyl-radicals. Moreover, Atomic Force Microscopic studies reveal that GroEL1 can condense a large DNA into a compact structure. We also provide in vivo evidences that include presence of GroEL1 in purified nucleoids, in vivo crosslinking followed by Southern hybridizations and immunofluorescence imaging in M. tuberculosis confirming that GroEL1: DNA interactions occur in natural biological settings. These findings therefore reveal that M. tuberculosis GroEL1 has evolved to be associated with nucleoids.     

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.     

Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-Å-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.Flaviviruses such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) belong to the family Flaviviridae and are the causative agents of a range of serious human diseases including hemorrhagic fever, meningitis, and encephalitis (37). They remain a global health priority, as many viruses are endemic in large parts of the Americas, Africa, Australia, and Asia, and vaccines remain unavailable for most members (31, 46, 57).Flaviviruses have a positive-sense single-stranded RNA (ssRNA) genome (approximately 11 kb) that encodes one large open reading frame containing a 5′ type 1 cap and conserved RNA structures at both the 5′ and 3′ untranslated regions that are important for viral genome translation and replication. The genomic RNA is translated into a single polyprotein precursor (11) consisting of three structural (C [capsid], prM [membrane], and E [envelope]) and seven nonstructural (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) proteins arranged in the order C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5 (reviewed in reference 33) (Fig. ​(Fig.1).1). Only the structural proteins become part of the mature, infectious virion, whereas the nonstructural proteins are involved in polyprotein processing, viral RNA synthesis, and virus morphogenesis (33, 43). The precursor protein is directed by signal sequences into the host endoplasmic reticulum (ER), where NS1 and the exogenous domains of prM and E face the lumen, while C, NS3, and NS5 are cytoplasmic. NS2A, NS2B, NS4A, and NS4B are largely hydrophobic transmembrane proteins with small hydrophilic segments (Fig. ​(Fig.1).1). The post- and cotranslational cleavage of the polyprotein is performed by NS3 in the cytoplasm and by host proteases in the ER lumen to yield the mature proteins (Fig. ​(Fig.1)1) (33, 43). Of the nonstructural proteins, NS3 and NS5 are the best characterized, and both are essential for viral replication (23, 27, 41). Both proteins are multifunctional. The N-terminal one-third of NS3 contains the viral protease (NS3pro), which requires a portion of NS2B for its activity, while the remaining portion codes for the RNA helicase/NTPase/RTPase domain (NS3hel) (21, 22, 32, 55). NS5, however, contains both an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (16, 51). The functions of NS1, NS2A, NS4A, and NS4B are not well understood, but they appear to play important roles in replication and virus assembly/maturation and have been found to bind to NS3 and NS5, possibly modulating their activity (33, 36).Open in a separate windowFIG. 1.Schematic diagram of flavivirus polyprotein organization and processing. (Top) Linear organization of the structural and nonstructural proteins within the polyprotein. (Middle) Putative membrane topology of the polyprotein predicted from biochemical and cellular analyses, which is then processed by cellular and viral proteases (indicated by arrows). (Bottom) Different complexes that are thought to arise in different cellular compartments during and following polyprotein processing.Because of its enzymatic activities and its critical role in viral replication and polyprotein processing, NS3 constitutes a promising drug target for antiviral therapy (31). NS3pro (residues 1 to 169) is a trypsin-like serine protease with the characteristic catalytic triad (Asp-His-Ser) and a highly specific substrate recognition sequence, conserved in all flaviviruses, consisting of two basic residues in P2 and P1 followed by a small unbranched amino acid in P1′ (11). NS3pro has an aberrant fold compared to the canonical trypsin structure, and its folding and protease activity are dependent on a noncovalent association with a central 47-amino-acid hydrophilic domain of NS2B (19, 21). The remainder of NS2B contains three transmembrane helices involved in membrane associations. NS3 mediates cleavages at the C-terminal side of the highly conserved dibasic residue located at the coding junctions NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 and also between the C terminus of C and NS4A (11, 33) (Fig. ​(Fig.11).The C-terminal portion of NS3 (NS3hel, residues 170 to 619) performs several catalytically related activities, namely, RNA strand separation and (poly)nucleotide hydrolysis (5, 22, 32, 55) at a common, RecA-like NTPase catalytic center that couples the energy released from the hydrolysis of the triphosphate moieties of nucleotides to RNA unwinding. Although the precise role of NS3 in replication has not been established, its helicase activity is thought to separate nascent RNA strands from the template strands and to assist replication initiation by unwinding RNA secondary structure in the 3′ untranslated region (11, 13, 15, 33). NS3 is a member of the DEAH/D box family within helicase superfamily 2 (SF2) and is characterized by seven conserved sequence motifs involved in nucleic acid binding and hydrolysis (45). In addition, its RNA triphosphatase activity is thought to be involved in the capping of the viral RNA. In the process of replication, NS3 interacts, most likely via its C-terminal domain, with NS5 (13, 15, 24, 26, 58, 62). The NS3 5′ triphosphatase and NS5 methyltransferase activities probably cooperate in cap formation by removing the terminal γ-phosphate and performing sequential N7 and 2′ O methylations, respectively (16, 28, 46, 56). The guanylyltransferase activity required for cap formation remains elusive at present, although recent evidence suggests that it may be present in NS5 (8, 17). In addition, the interaction between NS3 and NS5 can stimulate NS3 helicase/NTPase activity (15, 62).The atomic structures of NS3pro in the presence and absence of ligands and/or the NS2B activating domain (2, 19, 47) and NS3hel (35, 38, 39, 49, 58-60) are known, and recently, the structure of full-length DENV4 (one of four dengue virus serotypes) NS3 fused to an 18-residue NS2B cofactor (NS2B₁₈NS3) was reported (34). This structure revealed an elongated conformation, with the protease domain interfacing with the NTP binding pocket and being separated from NS3hel by a relatively flexible linker, which suggested that the protease domain may have a positive effect on the activity of the NTPase/helicase domain. However, other reports suggested that NS3pro has no or a very limited effect on the activity of NS3hel (32, 62). In addition, since current evidence suggests that NS2B is not part of the replication complex (Fig. ​(Fig.1)1) (36), and it is known that in the absence of the NS2B cofactor, NS3pro is unfolded and inactive, it becomes hard to envisage what effect the NS3 protease domain may have on the helicase domain in a biologically relevant context. Equally, it is still not clear what role the helicase domain plays during polyprotein processing by NS3pro and, in general, why these two apparently distinct and unrelated catalytic activities are harbored within a single polypeptide.In order to gain further insights into these questions, we report the biochemical analysis and crystallographic structure at a 2.75-Å resolution of full-length NS3 from Murray Valley encephalitis virus (MVEV), a member of the JEV group of flaviviruses, fused to the entire protease activation peptide of the NS2B cofactor (NS2B₄₅NS3). The structure reveals the protease and helicase domains to be structurally independent and differs dramatically from the structure observed for DENV4 NS2B₁₈NS3. We discuss the implications of this unexpectedly different configuration of the NS3 protein and argue that the structural flexibility observed is likely to be crucial for its multifunctional nature.