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C-terminal Regions (c-terminal + regions)
Selected AbstractsA FERM domain in a class XIV myosin interacts with actin and tubulin and localizes to the cytoskeleton, phagosomes, and nucleus in Tetrahymena thermophila,CYTOSKELETON, Issue 2 2010Michael Gotesman Abstract Previous studies have shown that Myo1(myosin class XIV) localizes to the cytoskeleton and is involved in amitosis of the macronucleus and trafficking of phagosomes. Myo1 contains a FERM domain that could be a site for interaction between Myo1 and the cytoskeleton. Here, we explore the function of FERM by investigating its cytoskeleton binding partners and involvement in localization of Myo1. Alignment of Myo1 FERM with a talin actin-binding sequence, a MAP-2 tubulin-binding sequence, the radixin FERM dimerization motif, and the SV40 nuclear localization sequence (NLS) revealed putative actin- and tubulin-binding sequences, a putative FERM dimerization motif, and NLS-like sequences in both the N-terminal and C-terminal regions of Myo1 FERM. Alignment of Myo1 with an ERM C-terminal motif revealed a similar sequence in the Myo1 motor domain. GFP-FERM and two truncated FERM domains were separately expressed in Tetrahymena. GFP-FERM contained the entire Myo1 FERM. Truncated Myo1 FERM domains contained either the N-terminal or the C-terminal region of FERM and one putative sequence for actin-binding, one for tubulin-binding, a putative dimerization motif, and a NLS-like sequence. Actin antibody coprecipitated GFP-fusion polypeptides and tubulin from lysate of cells expressing GFP-fusions. Cosedimentation assays performed with either whole cell extracts or anti-actin immunoprecipitation pellets revealed that F-actin (independent of ATP) and microtubules cosedimented with GFP-fusion polypeptides. GFP-FERM localized to the cytoskeleton, phagosomes, and nucleus. Truncated GFP-FERM domains localized to phagosomes but not to the cytoskeleton or nucleus. © 2009 Wiley-Liss, Inc. [source] Structural features in the C-terminal region of the Sinorhizobium meliloti RmInt1 group II intron-encoded protein contribute to its maturase and intron DNA-insertion functionFEBS JOURNAL, Issue 1 2010María D. Molina-Sánchez Group II introns are both catalytic RNAs and mobile retroelements that move through a process catalyzed by a RNP complex consisting of an intron-encoded protein and the spliced intron lariat RNA. Group II intron-encoded proteins are multifunctional and contain an N-terminal reverse transcriptase domain, followed by a putative RNA-binding domain (domain X) associated with RNA splicing or maturase activity and a C-terminal DNA binding/DNA endonuclease region. The intron-encoded protein encoded by the mobile group II intron RmInt1, which lacks the DNA binding/DNA endonuclease region, has only a short C-terminal extension (C-tail) after a typical domain X, apparently unrelated to the C-terminal regions of other group II intron-encoded proteins. Multiple sequence alignments identified features of the C-terminal portion of the RmInt1 intron-encoded protein that are conserved throughout evolution in the bacterial ORF class D, suggesting a group-specific functionally important protein region. The functional importance of these features was demonstrated by analyses of deletions and mutations affecting conserved amino acid residues. We found that the C-tail of the RmInt1 intron-encoded protein contributes to the maturase function of this reverse transcriptase protein. Furthermore, within the C-terminal region, we identified, in a predicted ,-helical region and downstream, conserved residues that are specifically required for the insertion of the intron into DNA targets in the orientation that would make it possible to use the nascent leading strand as a primer. These findings suggest that these group II intron intron-encoded proteins may have adapted to function in mobility by different mechanisms to make use of either leading or lagging-oriented targets in the absence of an endonuclease domain. [source] Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-IFEBS JOURNAL, Issue 17 2005Hiroyuki Tanaka Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca2+ -dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akazara scallop TnI (ATnI232,292), which contains the inhibitory region (residues 104,115 of rabbit TnI) and the regulatory TnC-binding site (residues 116,131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase. However, it did not interact with TnC, even in the presence of Ca2+. These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI130,252, which contains the structural TnC-binding site (residues 1,30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca2+ -dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI-fragment (RTnI1,116) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca2+ -dependent activation. [source] Role of the N- and C-terminal regions of the PufX protein in the structural organization of the photosynthetic core complex of Rhodobacter sphaeroidesFEBS JOURNAL, Issue 7 2002Francesco Francia The core complex of Rhodobacter sphaeroides is formed by the association of the light-harvesting antenna 1 (LH1) and the reaction center (RC). The PufX protein is essential for photosynthetic growth; it is located within the core in a 1 : 1 stoichiometry with the RC. PufX is required for a fast ubiquinol exchange between the QB site of the RC and the Qo site of the cytochrome bc1 complex. In vivo the LH1,PufX,RC complex is assembled in a dimeric form, where PufX is involved as a structural organizer. We have modified the PufX protein at the N and the C-terminus with progressive deletions. The nine mutants obtained have been characterized for their ability for photosynthetic growth, the insertion of PufX in the core LH1,RC complex, the stability of the dimers and the kinetics of flash-induced reduction of cytochrome b561 of the cytochrome bc1 complex. Deletion of 18 residues at the N-terminus destabilizes the dimer in vitro without preventing photosynthetic growth. The dimer (or a stable dimer) does not seem to be a necessary requisite for the photosynthetic phenotype. Partial C-terminal deletions impede the insertion of PufX, while the complete absence of the C-terminus leads to the insertion of a PufX protein composed of only its first 53 residues and does not affect the photosynthetic growth of the bacterium. Overall, the results point to a complex role of the N and C domains in the structural organization of the core complex; the N-terminus is suggested to be responsible mainly for dimerization, while the C-terminus is thought to be involved mainly in PufX assembly. [source] Assembly of the Rieske iron,sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplastsFEBS JOURNAL, Issue 2 2000Aliki Kapazoglou The assembly of the Rieske iron,sulphur protein into the cytochrome bf complex was examined following import of 35S-labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe-2S] centre; the mutant ,45,52 which removed a glycine-rich region predicted to form a flexible hinge between the hydrophobic membrane-associated region and the hydrophilic lumenal domain; and mutants ,168,173 and ,177,179 which removed two C-terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe,2S] cluster, the glycine-rich region and the C-terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts. [source] Isolation and characterisation of a 13.8-kDa bacteriolytic enzyme from house dust mite extracts: homology with prokaryotic proteins suggests that the enzyme could be bacterially derivedFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2002Leslie T. Mathaba Abstract Bacteriolytic activity was detected in extracts of whole mite and spent growth medium (SGM) from the clinically important Dermatophagoides pteronyssinus and Dermatophagoides farinae mites and was most abundant in whole mite extract. Gram-positive organisms Micrococcus lysodeikticus, Bacillus megaterium and Listeria monocytogenes were preferentially lysed and the lytic activity was enhanced by thiols, destroyed by mite proteases, inhibited by HgCl2 and high concentrations of NaCl but was resistant to heat and acid treatment. Substrate SDS,PAGE analysis indicated the presence of several lytic enzymes, two of which were isolated from D. pteronyssinus spent growth medium extract by hydroxyapatite chromatography. The N-terminal amino acid sequence of one of them was then used in PCR-based cloning studies. The complete amino acid sequence of this protein was determined and cDNA found to encode a 130-amino acid residue mature protein with a 20-amino acid leader sequence. The deduced protein demonstrated sequence similarity with the C-terminal regions of a group of bacterial proteins belonging to the P60 superfamily. These data suggest that the enzyme is derived from bacteria within the mites rather than from mites per se. [source] Actin filaments-stabilizing and -bundling activities of cofilin-phosphatase Slingshot-1GENES TO CELLS, Issue 5 2007Souichi Kurita Slingshot-1 (SSH1) is known to regulate actin filament dynamics by dephosphorylating and activating cofilin, an actin-depolymerizing factor. SSH1 binds to filamentous (F-) actin through its multiple F-actin-binding sites and its cofilin-phosphatase activity is enhanced by binding to F-actin. In this study, we demonstrate that SSH1 has F-actin-stabilizing and -bundling activities. In vitro actin depolymerization assays revealed that SSH1 suppressed spontaneous and cofilin-induced actin depolymerization in a dose-dependent manner. SSH1 inhibited F-actin binding and severing activities of cofilin. Low-speed centrifugation assays combined with fluorescence and electron microscopic analysis revealed that SSH1 has F-actin-bundling activity, independently of its cofilin-phosphatase activity. Deletion of N- or C-terminal regions of SSH1 significantly reduced its F-actin-stabilizing and -bundling activities, indicating that both regions are critical for these functions. As SSH1 does not form a homodimer, it probably bundles F-actin through its multiple F-actin-binding sites. Knockdown of SSH1 expression by RNA interference significantly suppressed stress fiber formation in C2C12 myoblast cells, indicating a role for SSH1 in stress fiber formation or stabilization in cells. SSH1 thus has the potential to regulate actin filament dynamics and organization in cells via F-actin-stabilizing and -bundling activities, in addition to its ability to dephosphorylate cofilin. [source] Cold-adapted signal proteins: NMR structures of pheromones from the antarctic ciliate Euplotes nobiliiIUBMB LIFE, Issue 8-9 2007William J. Placzek Abstract Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear 1H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-,-helix bundle with an up-down-up topology and a 310-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters. [source] Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar "Xiaoyan 54"JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 2 2006Xin-Yu Wang Abstract The low molecular weight (LMW) glutenin subunits account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenin genes (designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34) were isolated from the Chinese elite wheat cultivar "Xiaoyan 54" by PCR amplification of genomic DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenin genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 in the repetitive domain of LMW-34, indicating that it is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned into pET-30a expression vector and successfully expressed in Escherichia coli. Protein sodium dodecyl sulfate-polyacrylamide gel electro-phoresis analysis showed that all proteins expressed in E. coli by the four genes could be related to B-group LMW glutenin subunits of wheat. (Managing editor: Li-Hui Zhao) [source] Cloning and characterization of a Chlamydomonas reinhardtii cDNA arylalkylamine N -acetyltransferase and its use in the genetic engineering of melatonin content in the Micro-Tom tomatoJOURNAL OF PINEAL RESEARCH, Issue 4 2009Masateru Okazaki Abstract:, Melatonin is found in a wide variety of plant species. Several investigators have studied the physiological roles of melatonin in plants. However, its role is not well understood because of the limited information on its biosynthetic pathway. To clarify melatonin biosynthesis in plants, we isolated a cDNA-coded arylalkylamine N -acetyltransferase (AANAT), a possible limiting enzyme for melatonin biosynthesis, from Chlamydomonas reinhardtii (designated as CrAANAT). The predicted amino acid sequence of CrAANAT shares 39.0% homology to AANAT from Ostreococcus tauri and lacks cAMP-dependent protein kinase phosphorylation sites in the N- and C-terminal regions that are conserved in vertebrates. The enzyme activity of CrAANAT was confirmed by in vitro assay using Escherichia coli. Transgenic plants constitutively expressing the CrAANAT were produced using Micro-Tom, a model cultivar of tomato (Solanum lycopersicum L.). The transgenic Micro-Tom exhibited higher melatonin content compared with wild type, suggesting that melatonin was synthesized from serotonin via N -acetylserotonin in plants. Moreover, the melatonin-rich transgenic Micro-Tom can be used to elucidate the role of melatonin in plant development. [source] On the use of DXMS to produce more crystallizable proteins: Structures of the T. maritima proteins TM0160 and TM1171PROTEIN SCIENCE, Issue 12 2004Glen Spraggon DXMS, deuterium exchange mass spectroscopy Abstract The structure of two Thermotoga maritima proteins, a conserved hypothetical protein (TM0160) and a transcriptional regulator (TM1171), have now been determined at 1.9 Ĺ and 2.3 Ĺ resolution, respectively, as part of a large-scale structural genomics project. Our first efforts to crystallize full-length versions of these targets were unsuccessful. However, analysis of the recombinant purified proteins using the technique of enhanced amide hydrogen/deuterium exchange mass spectroscopy (DXMS) revealed substantial regions of rapid amide deuterium hydrogen exchange, consistent with flexible regions of the structures. Based on these exchange data, truncations were designed to selectively remove the disordered C-terminal regions, and the resulting daughter proteins showed greatly enhanced crystallizability. Comparative DXMS analysis of full-length protein versus truncated forms demonstrated complete and exact preservation of the exchange rate profiles in the retained sequence, indicative of conservation of the native folded structure. This study presents the first structures produced with the aid of the DXMS method for salvaging intractable crystallization targets. The structure of TM0160 represents a new fold and highlights the use of this approach where any prior structural knowledge is absent. The structure of TM1171 represents an example where the lack of a substrate/cofactor may impair crystallization. The details of both structures are presented and discussed. [source] All-trans retinoic acid affects subcellular localization of a novel BmNIF3l protein: functional deduce and tissue distribution of NIF3l gene from silkworm (Bombyx mori),ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Jianqing Chen Abstract A novel cDNA sequence encoding a predicted protein of 271 amino acids containing a conserved NIF3 domain was found from a pupal cDNA library of silkworm. The corresponding gene was named BmNIF3l (Bombyx mori NGG1p interacting factor 3-like). It was found by bioinformatics that BmNIF3l gene consisted of five exons and four introns and BmNIF3l had a high degree of homology to other NIF3-like proteins, especially in the N-terminal and C-terminal regions. A His-tagged BmNIF3l fusion protein with a molecular weight of approximately 33.6,kDa was expressed and purified to homogeneity. We have used the purified fusion protein to produce polyclonal antibodies against BmNIF3l for histochemical analysis. Subcellular localization revealed that BmNIF3l is a cytoplasmic protein that responds to all-trans retinoic acid (ATRA). Western blotting and real-time reverse transcription polymerase chain reaction showed that the expression level of BmNIF3l is higher in tissues undergoing differentiation. Taken together, the results suggest that BmNIF3l functions in transcription. © 2010 Wiley Periodicals, Inc. [source] Structure of BthA-I complexed with p -bromophenacyl bromide: possible correlations with lack of pharmacological activityACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2005Angelo J. Magro The crystal structure of an acidic phospholipase A2 isolated from Bothrops jararacussu venom (BthA-I) chemically modified with p -bromophenacyl bromide (BPB) has been determined at 1.85,Ĺ resolution. The catalytic, platelet-aggregation inhibition, anticoagulant and hypotensive activities of BthA-I are abolished by ligand binding. Electron-density maps permitted unambiguous identification of inhibitor covalently bound to His48 in the substrate-binding cleft. The BthA-I,BPB complex contains three structural regions that are modified after inhibitor binding: the Ca2+ -binding loop, ,-wing and C-terminal regions. Comparison of BthA-I,BPB with two other BPB-inhibited PLA2 structures suggests that in the absence of Na+ ions at the Ca2+ -binding loop, this loop and other regions of the PLA2s undergo structural changes. The BthA-I,BPB structure reveals a novel oligomeric conformation. This conformation is more energetically and conformationally stable than the native structure and the abolition of pharmacological activities by the ligand may be related to the oligomeric structural changes. A residue of the `pancreatic' loop (Lys69), which is usually attributed as providing the anticoagulant effect, is in the dimeric interface of BthA-I,BPB, leading to a new hypothesis regarding the abolition of this activity by BPB. [source] Crystallization and preliminary crystallographic analysis of the central domain of Drosophila Dribble, a protein that is essential for ribosome biogenesisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Tat-Cheung Cheng Dribble (DBE) is a Drosophila protein that is essential for ribosome biogenesis. Bioinformatics analysis revealed a folded central domain of DBE which is flanked by structural disorder in the N- and C-terminal regions. The protein fragment spanning amino-acid residues 16,197 (DBE16,197) was produced for structural determination. In this report, the crystallization and preliminary X-ray diffraction data analysis of the DBE16,197 protein domain are described. Crystals of DBE16,197 were grown by the sitting-drop vapour-diffusion method at 289,K using ammonium phosphate as a precipitant. The crystals belonged to space group P212121. Data were collected that extended to beyond 2,Ĺ resolution. [source] Merozoite surface protein 2 of Plasmodium falciparum: Expression, structure, dynamics, and fibril formation of the conserved N-terminal domainBIOPOLYMERS, Issue 1 2007Andrew Low Abstract Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP21,25) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields ,10 mg of unlabeled or 15N-labeled peptide per litre of culture. Two recombinant versions of MSP21,25, wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone 15N relaxation data indicated that it contains ,-turn and nascent helical structures in the central and C-terminal regions. Residues 6,21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP27,16) (with two Tyr residues) was predicted to have a higher propensity for ,-aggregation than the 8-mer sequence (MSP28,15), but there was no significant difference in conformation between MSP21,25 and [Y7A,Y16A]MSP21,25 and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 12,22, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] | |||||||||||||||||||||||||