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Full-length Protein (full-length + protein)
Selected AbstractsPeptides corresponding to helices 5 and 6 of Bax can independently form large lipid poresFEBS JOURNAL, Issue 5 2006Ana J. García-Sáez Proteins of the B-cell lymphoma protein 2 (Bcl2) family are key regulators of the apoptotic cascade, controlling the release of apoptotic factors from the mitochondrial intermembrane space. A helical hairpin found in the core of water-soluble folds of these proteins has been reported to be the pore-forming domain. Here we show that peptides including any of the two ,-helix fragments of the hairpin of Bcl2 associated protein X (Bax) can independently induce release of large labelled dextrans from synthetic lipid vesicles. The permeability promoted by these peptides is influenced by intrinsic monolayer curvature and accompanied by fast transbilayer redistribution of lipids, supporting a toroidal pore mechanism as in the case of the full-length protein. However, compared with the pores made by complete Bax, the pores made by the Bax peptides are smaller and do not need the concerted action of tBid. These data indicate that the sequences of both fragments of the hairpin contain the principal physicochemical requirements for pore formation, showing a parallel between the permeabilization mechanism of a complex regulated protein system, such as Bax, and the much simpler pore-forming antibiotic peptides. [source] Pleiotropic phenotypes caused by an opal nonsense mutation in an essential gene encoding HMG-CoA reductase in fission yeastGENES TO CELLS, Issue 6 2009Yue Fang Schizosaccharomyces pombe genome contains an essential gene hmg1+ encoding the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Here, we isolated an allele of the hmg1+ gene, hmg1-1/its12, as a mutant that showed sensitivities to high temperature and to FK506, a calcineurin inhibitor. The hmg1-1 allele contained an opal nonsense mutation in its N-terminal transmembrane domain, yet in spite of the mutation a full-length protein was produced, suggesting a read-through termination codon. Consistently, overexpression of the hmg1-1 mutant gene suppressed the mutant phenotypes. The hmg1-1 mutant showed hypersensitivity to pravastatin, an HMGR inhibitor, suggesting a defective HMGR activity. The mutant treated with FK506 caused dramatic morphological changes and showed defects in cell wall integrity, as well as displayed synthetic growth phenotypes with the mutant alleles of genes involved in cytokinesis and cell wall integrity. The mutant exhibited different phenotypes from those of the disruption mutants of ergosterol biosynthesis genes, and it showed normal filipin staining as well as showed normal subcellular localization of small GTPases. These data suggest that the pleiotropic phenotypes reflect the integrated effects of the reduced availability of ergosterol and various intermediates of the mevalonate pathway. [source] Molecular characterization of a peroxiredoxin from the hard tick Haemaphysalis longicornisINSECT MOLECULAR BIOLOGY, Issue 2 2001N. Tsuji Abstract Antioxidant enzymes in eukaryotes play an important role in protection against the oxygen radicals generated during aerobic metabolism. Here we report the cloning and characterization of a cDNA encoding the antioxidant enzyme peroxiredoxin from the hard tick Haemaphysalis longicornis (HlPrx). HlPrx is 939 bp long and contains a 101 bp non-translated sequence at the 5, end and a polyadenylation singnal followed by a poly(A) tail at the 3, end. HlPrx encodes a full-length protein with a predicted molecular mass of 26 kDa that possesses one cysteine residue at amino acid 49 that is conserved among Prx proteins of various species. GenBankÔ analysis showed that the deduced amino acid sequence had significant similarity to mammalian and plant Prxs at the amino acid level. A DNA-nicking assay revealed that Escherichia coli,expressed recombinant HlPrx (rHlPrx) inhibited oxidative-nicking of supercoiled plasmid DNA. Two-dimensional immunoblot analysis with mouse antirHlPrx serum showed reaction with a major constituent protein spot in extracts of adult ticks. In addition, immunoblot analysis showed that rHlPrx was immunoreacted with serum from rabbits repeatedly infested with H. longicornis. Localization analysis using mouse antirHlPrx serum revealed that native HlPrx was highly expressed in the salivary gland of the tick. Moreover, Northern blot analysis showed that the level of HlPrx transcripts was increased during blood sucking. The present results indicate that HlPrx may be an important detoxifying enzyme during the normal life span as well as during blood sucking in ticks. [source] Aminoglycoside-mediated partial suppression of MECP2 nonsense mutations responsible for Rett syndrome in vitroJOURNAL OF NEUROSCIENCE RESEARCH, Issue 11 2010Andreea C. Popescu Abstract Rett syndrome is a pediatric neurological condition that affects primarily girls. Approximately 30% of Rett syndrome cases arise from point mutations that introduce a premature stop codon into the MECP2 gene. Several studies have now shown that certain aminoglycosides can facilitate read-through of some types of nonsense mutations in a context-dependent manner and allow the generation of a full-length protein. It remains mostly unclear whether different nonsense mutations of MECP2 will be responsive to aminoglycoside treatment. In this study, we tested whether the common premature terminating mutations of MECP2 seen in Rett syndrome cases can be partially suppressed by aminoglycoside administration. Our results show that aminoglycosides allow different mutant forms of MECP2 to be overcome in transiently transfected HEK293 cells, but with differing levels of efficiency. In addition, we also show that aminoglycosides increased the prevalence of full-length MeCP2 protein in a dose-dependent manner in a lymphocyte cell line derived from a Rett syndrome girl with the R255X mutation. This study helps to establish the "proof of principle" that some nonsense mutations causing Rett syndrome can be at least partially suppressed by drug treatment. © 2010 Wiley-Liss, Inc. [source] Role of side chains in collagen triple helix stabilization and partner recognition,JOURNAL OF PEPTIDE SCIENCE, Issue 3 2009Rita Berisio Abstract Collagen is a widespread protein family involved in a variety of biological processes. The complexity of collagen and its fibrous nature prevent detailed investigations on the full-length protein. Reductionist approaches conducted by dissecting the protein complexity through the use of model peptides have proved to be quite effective. There are, however, several issues regarding structure,stability relationships, aggregation in higher-order assemblies, and partner recognition that are still extensively investigated. In this review, we discuss the role that side chains play in triple helix stabilization and in partner recognition. On the basis of recent literature data, we show that collagen triple helix stability is the result of the interplay of different factors. As a general trend, interactions established by amino/imino acid side chains within the triple helix scaffold effectively modulate the intrinsic residue propensity for this common structural motif. The use of peptide models has also highlighted the role that side chains play in collagen self-association and in its interactions with receptors. Valuable examples in these fields are illustrated. Finally, future actions required to obtain more detailed information on the structure and the function of this complex protein are also delineated. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Intracellular readthrough of nonsense mutations by aminoglycosides in coagulation factor VIIJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2006M. PINOTTI Summary.,Background: Nonsense mutations in coagulation factor (F) VII potentially cause a lethal hemorrhagic diathesis. Readthrough of nonsense mutations by aminoglycosides has been studied in a few human disease models with variable results. Objectives: We investigated the K316X and W364X FVII mutations, associated with intracranial hemorrhage, and their correction by aminoglycosides. The rare nonsense mutations in FVII represent favorite models to test this strategy, because even tiny increases in the amount of functional full-length protein in patients could ameliorate hemorrhagic phenotypes. Results: A FVII,green fluorescent protein (GFP) chimaera provided us with a fluorescent model of FVII expression in living cells. Appreciable fluorescence in cells transfected with nonsense FVII,GFP mutants was detected upon geneticin treatment, thus demonstrating suppression of premature translation termination. To investigate the rescue of FVII function, nonsense variants of the native FVII without GFP (p316X,FVII and p364X,FVII) were transfected and found to secrete low amounts of FVII (,1% of Wt,FVII activity), thus suggesting a spontaneous stop codon readthrough. Geneticin treatment of cells resulted in a significant and dose-dependent increase of secreted FVII molecules (p316X,FVII, 24 ± 12 ng mL,1, 3.6 ± 0.8% of Wt,FVII activity; p364X,FVII, 26 ± 10 ng mL,1, 3.7±0.6%) characterized by reduced specific activity, thus indicating the synthesis of dysfunctional proteins. Similar results were observed with gentamicin, a commonly used aminoglycoside of potential interest for patient treatment. Conclusions: Our approach, extendable to other coagulation factors, represents an effective tool for a systematic study of the effects of aminoglycosides and neighboring sequences on nonsense codon readthrough. These results provide the rationale for a mutation-specific therapeutic approach in FVII deficiency. [source] Current trends in the structure,activity relationship studies of the endogenous agouti-related protein (AGRP) melanocortin receptor antagonistMEDICINAL RESEARCH REVIEWS, Issue 5 2005Andrzej M. Wilczynski Abstract Agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin-3 and -4 (MC3R and MC4) G-protein coupled receptors. The 87,132 amino acid C-terminal domain of hAGRP possesses five disulfide bridges and a well-defined three-dimensional structure that displays full biological activity as compared to the full-length protein. Based on the NMR structure of the C-terminal AGRP(87,132), a novel mini-protein, referred to as "Mini-AGRP" was designed that exhibited receptor binding affinity and antagonism similar to that of the parent hAGRP(87,132) protein. It was demonstrated that this new-engineered protein autonomously folds to the inhibitor cystine knot (ICK) motif. As this AGRP is a novel mammalian protein involved in energy homeostasis and possibly other physiological functions remaining to be identified, structure-function studies are starting to emerge toward the understanding of how this unique protein putatively interacts with the melanocortin receptors with the objective of designing potential therapeutic agents for in vivo physiological studies. This article summarizes the progress to date of AGRP-based structure,activity relationships and putative ligand,receptor interactions. © 2005 Wiley Periodicals, Inc. [source] Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001Aikaterini Kontrogianni-Konstantopoulos Abstract SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (,P) and the other a truncation of the C-terminal F-domain (,F), revealed that ,P is localized similarly to full-length receptor, whereas ,F is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas ,P does not bind DNA and ,F binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development. Mol. Reprod. Dev. 60: 147,157, 2001. © 2001 Wiley-Liss, Inc. [source] Structural characterization of unphosphorylated STAT5a oligomerization equilibrium in solution by small-angle X-ray scatteringPROTEIN SCIENCE, Issue 4 2009Pau Bernadó Abstract Signal transducer and activator of transcription (STAT) proteins play a crucial role in the activation of gene transcription in response to extracellular stimuli. The regulation and activity of these proteins require a complex rearrangement of the domains. According to the established models, based on crystallographic data, STATs convert from a basal antiparallel inactive dimer into a parallel active one following phosphorylation. The simultaneous analysis of small-angle X-ray scattering data measured at different concentrations of unphosphorylated human STAT5a core domain unambiguously identifies the simultaneous presence of a monomer and a dimer. The dimer is the minor species but could be structurally characterized by SAXS in the presence of the monomer using appropriate computational tools and shown to correspond to the antiparallel assembly. The equilibrium is governed by a moderate dissociation constant of Kd , 90 ,M. Integration of these results with previous knowledge of the N-terminal domain structure and dissociation constants allows the modeling of the full-length protein. A complex network of intermolecular interactions of low or medium affinity is suggested. These contacts can be eventually formed or broken to trigger the dramatic modifications in the dimeric arrangement needed for STAT regulation and activity. [source] Solution structure of HI1506, a novel two-domain protein from Haemophilus influenzaePROTEIN SCIENCE, Issue 5 2007Nese Sari Abstract HI1506 is a 128-residue hypothetical protein of unknown function from Haemophilus influenzae. It was originally annotated as a shorter 85-residue protein, but a more detailed sequence analysis conducted in our laboratory revealed that the full-length protein has an additional 43 residues on the C terminus, corresponding with a region initially ascribed to HI1507. As part of a larger effort to understand the functions of hypothetical proteins from Gram-negative bacteria, and H. influenzae in particular, we report here the three-dimensional solution NMR structure for the corrected full-length HI1506 protein. The structure consists of two well-defined domains, an ,/, 50-residue N-domain and a 3-, 32-residue C-domain, separated by an unstructured 30-residue linker. Both domains have positively charged surface patches and weak structural homology with folds that are associated with RNA binding, suggesting a possible functional role in binding distal nucleic acid sites. [source] Atomic model of human Rcd-1 reveals an armadillo -like-repeat protein with in vitro nucleic acid binding propertiesPROTEIN SCIENCE, Issue 2 2007Robert G. Garces Abstract Rcd-1, a protein highly conserved across eukaryotes, was initially identified as a factor essential for nitrogen starvation-invoked differentiation in fission yeast, and its Saccharomyces cerevisiae homolog, CAF40, has been identified as part of the CCR4,NOT transcription complex, where it interacts with the NOT1 protein. Mammalian homologs are involved in various cellular differentiation processes including retinoic acid-induced differentiation and hematopoetic cell development. Here, we present the 2.2 Å X-ray structure of the highly conserved region of human Rcd-1 and investigate possible functional abilities of this and the full-length protein. The monomer is made up of six armadillo repeats forming a solvent-accessible, positively-charged cleft 21,22 Å wide that, in contrast to other armadillo proteins, stays fully exposed in the dimer. Prompted by this finding, we established that Rcd-1 can bind to single- and double-stranded oligonucleotides in vitro with the affinity of G/C/T , A. Mutation of an arginine residue within the cleft strongly reduced or abolished oligonucleotide binding. Rcd-1's ability to bind to nucleic acids, in addition to the previously reported protein,protein interaction with NOT1, suggests a new feature in Rcd-1's role in regulation of overall cellular differentiation processes. [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 Å and 2.3 Å 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] Plasmodium falciparum Rab6 GTPase: expression, purification, crystallization and preliminary crystallographic studiesACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2000Debasish Chattopadhyay The Plasmodium falciparumrab6 gene encodes a 208 amino-acid polypeptide. Two recombinant versions of P. falciparum Rab6 protein were expressed in Escherichia coli: the full-length protein and a truncated form containing residues 1,175. Both forms were purified from the soluble fraction of bacterial extract and were purified by ion-exchange chromatography and size-exclusion chromatography. Purified proteins were crystallized at pH 6.5 using the hanging-drop vapor-diffusion technique at room temperature. The full-length protein diffracted to 2.4,Å and belongs to the tetragonal space group P43212 or P41212, with unit-cell parameters a = b = 80.6, c = 90.4,Å. The crystals of the truncated protein were isomorphous with those of the full-length construct and diffracted X-rays to 2.2,Å resolution. [source] Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus' molecule 1 proteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Tom J. Petty Of the known epigenetic control regulators found in plants, the Morpheus' molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to ,3.2,Å resolution. They belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = 85.64, c = 292.74,Å. Structure determination is ongoing. [source] Structure of pig heart citrate synthase at 1.78,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Steven B. Larson Pig heart citrate synthase was crystallized from a small-molecule cocktail containing cystamine dihydrochloride, aspartame and benzamidine hydrochloride. The structure was refined to an R factor of 0.179 (Rfree = 0.222) using synchrotron data to a resolution of 1.78,Å. The model includes the full-length protein, a chloride ion, a sulfate ion, 305 water molecules and an unexpected moiety attached through a disulfide linkage to Cys184, which was modeled as a half-cystamine molecule generated by disulfide exchange with the cystamine in the small-molecule cocktail. [source] Ion channel activity of transmembrane segment 6 of Escherichia coli proton-dependent manganese transporterBIOPOLYMERS, Issue 8 2010uková Abstract Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure,function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His211 were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure,function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 718,726, 2010. 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] The cytochrome P450 aromatase lacking exon 5 is associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissuesCLINICAL ENDOCRINOLOGY, Issue 5 2007Carolina M. Pepe Summary Objective, The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated. Design To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (,Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) and by splicing assays performed in Y1 cells transfected with a P450aro ,Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the ,Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues. Patients An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8·4 years, spontaneous breast development and a 194·6 pmol/l serum oestradiol level was observed. Results The ,Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, ,Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the ,Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues. Conclusions Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full-length protein might explain the phenotype of partial aromatase deficiency. [source] Identification of two cDNAs encoding synaptic vesicle protein 2 (SV2)-like proteins from epithelial tissues in the cat flea, Ctenocephalides felisINSECT MOLECULAR BIOLOGY, Issue 3 2004S. J. Walmsley Abstract Two distinct cDNAs that appear to encode proteins in the synaptic vesicle-2 (SV2) family were identified as expressed sequence tags from a Ctenocephalides felis hindgut and Malpighian tubule (HMT) cDNA library. To date, SV2 proteins have been described only in vertebrates, and have been detected only in synaptic vesicles in neuronal and endocrine tissues, where they are thought to regulate synaptic vesicle exocytosis. The cDNAs for the C. felis SV2-like proteins SVLP-1 and SVLP-2 encode predicted full-length proteins of 530 and 726 amino acids, respectively. Of characterized proteins, the SVLP protein sequences were most similar to rat SV2B. Northern blot analysis revealed that both mRNAs were up-regulated in larval stages that feed and in adults after feeding, and were expressed primarily or exclusively in the HMT tissues in adult fleas. These results suggest that the flea SVLP-1 and SVLP-2 gene products may have roles that are specific for the HMT tissues, and may differ in function from vertebrate SV2 proteins. [source] Cone arrestin binding to JNK3 and Mdm2: conformational preference and localization of interaction sitesJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Xiufeng Song Abstract Arrestins are multi-functional regulators of G protein-coupled receptors. Receptor-bound arrestins interact with >30 remarkably diverse proteins and redirect the signaling to G protein-independent pathways. The functions of free arrestins are poorly understood, and the interaction sites of the non-receptor arrestin partners are largely unknown. In this study, we show that cone arrestin, the least studied member of the family, binds c-Jun N-terminal kinase (JNK3) and Mdm2 and regulates their subcellular distribution. Using arrestin mutants with increased or reduced structural flexibility, we demonstrate that arrestin in all conformations binds JNK3 comparably, whereas Mdm2 preferentially binds cone arrestin ,frozen' in the basal state. To localize the interaction sites, we expressed separate N- and C-domains of cone and rod arrestins and found that individual domains bind JNK3 and remove it from the nucleus as efficiently as full-length proteins. Thus, the arrestin binding site for JNK3 includes elements in both domains with the affinity of partial sites on individual domains sufficient for JNK3 relocalization. N-domain of rod arrestin binds Mdm2, which localizes its main interaction site to this region. Comparable binding of JNK3 and Mdm2 to four arrestin subtypes allowed us to identify conserved residues likely involved in these interactions. [source] Lipid bilayers: an essential environment for the understanding of membrane proteinsMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2007Richard C. Page Abstract Membrane protein structure and function is critically dependent on the surrounding environment. Consequently, utilizing a membrane mimetic that adequately models the native membrane environment is essential. A range of membrane mimetics are available but none generates a better model of native aqueous, interfacial, and hydrocarbon core environments than synthetic lipid bilayers. Transmembrane ,-helices are very stable in lipid bilayers because of the low water content and low dielectric environment within the bilayer hydrocarbon core that strengthens intrahelical hydrogen bonds and hinders structural rearrangements within the transmembrane helices. Recent evidence from solid-state NMR spectroscopy illustrates that transmembrane ,-helices, both in peptides and full-length proteins, appear to be highly uniform based on the observation of resonance patterns in PISEMA spectra. Here, we quantitate for the first time through simulations what we mean by highly uniform structures. Indeed, helices in transmembrane peptides appear to have backbone torsion angles that are uniform within ± 4° . While individual helices can be structurally stable due to intrahelical hydrogen bonds, interhelical interactions within helical bundles can be weak and nonspecific, resulting in multiple packing arrangements. Some helical bundles have the capacity through their amino acid composition for hydrogen bonding and electrostatic interactions to stabilize the interhelical conformations and solid-state NMR data is shown here for both of these situations. Solid-state NMR spectroscopy is unique among the techniques capable of determining three-dimensional structures of proteins in that it provides the ability to characterize structurally the membrane proteins at very high resolution in liquid crystalline lipid bilayers. Copyright © 2007 John Wiley & Sons, Ltd. [source] Structure of the B3 domain from Arabidopsis thaliana protein At1g16640PROTEIN SCIENCE, Issue 9 2005Jeanette K. Waltner Abstract A novel DNA binding motif, the B3 domain, has been identified in a number of transcription factors specific to higher plant species, and was recently found to define a new protein fold. Here we report the second structure of a B3 domain, that of the Arabidopsis thaliana protein, At1g16640. As part of an effort to ,rescue' structural genomics targets deemed unsuitable for structure determination as full-length proteins, we applied a combined bioinformatic and experimental strategy to identify an optimal construct containing a predicted conserved domain. By screening a series of N- and C-terminally truncated At1g16640 fragments, we isolated a stable folded domain that met our criteria for structural analysis by NMR spectroscopy. The structure of the B3 domain of At1g16640 consists of a seven-stranded ,-sheet arranged in an open barrel and two short ,-helices, one at each end of the barrel. While At1g16640 is quite distinct from previously characterized B3 domain proteins in terms of amino acid sequence similarity, it adopts the same novel fold that was recently revealed by the RAV1 B3 domain structure. However, putative DNA-binding elements conserved in B3 domains from the RAV, ARF, and ABI3/VP1 subfamilies are largely absent in At1g16640, perhaps suggesting that B3 domains could function in contexts other than transcriptional regulation. [source] Removal of the N-terminal hexapeptide from human ,2-microglobulin facilitates protein aggregation and fibril formationPROTEIN SCIENCE, Issue 5 2000G. Esposito Abstract The solution structure and stability of N-terminally truncated ,2-microglobulin (,N6,2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that ,N6,2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at ,M concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that ,N6,2-m is significantly less protected than its wild-type counterpart. Analysis of ,N6,2-m by NMR shows that this loss of protection occurs in , strands I, III, and part of II. At mM concentration gel filtration analysis shows that ,N6,2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of ,2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that ,N6,2-m could be a key intermediate of a proteolytic pathway of ,2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of ,2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between ,-strands far removed from this constrain is greatly perturbed. [source] Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissuesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2009Maria Filippa Addis Abstract A wealth of information on proteins involved in many aspects of disease is encased within formalin-fixed paraffin-embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this "hidden treasure" is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full-length proteins. Here, we describe an optimised method for extraction of full-length proteins from FFPE tissues. This method builds on the classical "antigen retrieval" technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3,,g and 86.8,,g of proteins were obtained per 80,mm2 tissue slice of formalin-fixed paraffin-embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS-PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC-nanoESI-Q-TOF MS of FFPE proteins resolved by SDS-PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported. [source] | |||||||||||||||||||||||||||||||