RESUMO
SUMMARY: Although translation initiation has been well studied, many questions remain in elucidating its mechanisms. An ongoing challenge is to understand how ribosomes choose a translation initiation site (TIS). To gain new insights, we analyzed large sets of TISs with the aim of identifying common characteristics that are potentially of functional importance. Nucleotide sequence context has previously been demonstrated to play an important role in the ribosome's selection of a TIS, and mRNA secondary structure is also emerging as a contributing factor. Here, we analyze mRNA secondary structure using the folding predictions of the RNAfold algorithm. We present a method for analyzing these results using a rank-ordering approach to assess the overall degree of predicted secondary structure in a given region of mRNA. In addition, we used a modified version of the algorithm that makes use of only a subset of the standard version's output to incorporate base-pairing polarity constraints suggested by the ribosome scanning process. These methods were employed to study the TISs of 1735 genes in Saccharomyces cerevisiae. Trends in base composition and base-pairing probabilities suggest that efficient translation initiation and high protein expression are aided by reduced secondary structure upstream and downstream of the TIS. However, the downstream reduction is not observed for sets of TISs with nucleotide sequence contexts unfavorable for translation initiation, consistent with previous suggestions that secondary structure downstream of the ribosome can facilitate TIS recognition.
Assuntos
Biologia Computacional/métodos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Algoritmos , Genes Fúngicos , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/fisiologiaRESUMO
Optical microscopy and atomic force microscopy were used to study a mechanically induced wrinkling instability in thin film poly(caprolactone)/polystyrene and poly(ethylene oxide)/poly(methyl methacrylate) bilayers. The instability in these samples was shown to be driven by changes in the interfacial area between a semicrystalline polymer underlayer and a glassy polymer capping layer that occurred when the underlayers were melted. The wrinkling instability resulted in the formation of one-dimensional corrugations at the surface of the bilayer samples that had a well-defined wavelength on the micrometer length scale. A linear stability analysis was used to derive a simple model of the wrinkling process in these samples. This model considered the flow and deformation of material in the molten underlayer as well as the balance of stresses in the glassy polymer capping layers. Rheological data were also obtained from polymers similar to those used to form the bilayers. These data were used to show that the model is capable of quantitatively predicting the capping layer and underlayer thickness dependencies of the characteristic wrinkling wavelengths, if the mechanical properties of the two layers and the strain in the capping layers can be determined.
RESUMO
We study the conformations of polymer chains in polymer-graphene oxide nanocomposites. We show that the chains have a reduced radius of gyration that is consistent with confinement at a solid interface in the melt, as is expected for well-dispersed, high aspect ratio nanoparticles that are much larger than the polymer coil size. We show that confinement of the polymer chains causes a corresponding reduction in interchain entanglements, and we calculate a contribution to the plateau modulus from the distorted polymer network via a simple scaling argument. Our results are a significant step forward in understanding how two-dimensional nanoparticles affect global material properties at low loadings.
RESUMO
The effect of low pH on the secondary and tertiary structure of the monomeric single-disulphide protein interleukin-2 (IL2) was monitored by fluorescence and circular dichroism spectroscopy. Between pH 4 and pH 2 there is a gradual loosening of the tertiary structure as revealed by changes in tyrosine and tryptophan fluorescence emission, tryptophan fluorescence anisotropy, accessibility to the fluorescence quencher acrylamide and aromatic circular dichroism. The overall molecular size and secondary structure content are not significantly changed by acidification. These data are consistent with a 'molten globule' state for IL2 at low pH, in which the hydrophobic core/secondary structure is largely intact but the tertiary structure is flexible. Similar effects to low pH are seen at sub-denaturing concentrations of guanidine hydrochloride. Analysis of fluorescence lifetimes and derivative emission spectra of the single tryptophan, Trp-121, shows the existence of two distinct orientations for this side-chain, one of which is affected by a quenching group (the effect of which diminishes upon acidification) and another which is essentially unquenched. The identity of the quenching group is unclear but may well be Cys-125. The formation of the molten globule titrates with a pKa of about 2.3; this is unusually low for the acidic groups in proteins and indicates a perturbed pKa of a residue involved in a structurally important interaction such as a salt bridge. Candidate residues are Glu-15 or Asp-20, close to His-16 on the N-terminal helix of IL-2.
Assuntos
Interleucina-2/química , Ácidos , Dicroísmo Circular , Polarização de Fluorescência , Mutagênese Sítio-Dirigida , Conformação Proteica , Espectrometria de Fluorescência , Triptofano/análise , Tirosina/análiseRESUMO
Preparations of haemosiderin and ferritin isolated from iron-loaded human spleens were studied by electron spin resonance (ESR) spectroscopy at X-band (approx. 9.2 GHz). The spectra were mainly composed of two overlapping, broad features, one extremely anisotropic with its major component occurring at 0.1-0.2 T (feature A), the other nearly isotropic and occurring at around g = 2 (feature B). There is relatively more feature A and less feature B in ferritin than in haemosiderin. Both features originate from the iron oxyhydroxide crystallites of these iron proteins which, due to their small size, are superparamagnetic. Feature B is maximal in small cores or at high temperatures, where superparamagnetic fluctuations average out anisotropic magnetic interactions; feature A is greatest at low temperatures or in large cores, for which such fluctuations are blocked and an ESR spectrum characteristic of a magnetically ordered system is observed. It is concluded that there is no evidence in the ESR spectra for 'loose' protein-bound Fe3+ in ferritin or haemosiderin, and that haemosiderin cores are on average smaller than those of ferritin. The relationship of the ESR spectra between these two proteins supports the view that haemosiderin is derived from ferritin.
Assuntos
Ferritinas/análise , Hemossiderina/análise , Baço/análise , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Cavalos , Humanos , TemperaturaRESUMO
Ferritin and haemosiderin isolated from iron-overloaded human spleens have been investigated by 57Fe Mössbauer spectroscopy at temperatures between 1.3 and 200 K and also in applied magnetic fields. Virtually identical spectra were obtained from both materials at the high and low-temperature ends of this range, and also at 4.2 K in an applied magnetic field of 10 T; this indicates that both must contain iron in a closely similar chemical form. The difference between the two materials lies in the temperature dependence of their Mössbauer spectra in the intermediate temperature range, between 10 and 100 K. The temperature dependence of the Mössbauer spectra is characteristic of superparamagnetic behaviour, which occurs when a magnetically ordered material is present in the form of small particles. The details of this temperature dependence are related to the distribution of particle sizes and the magnetic anisotropy constant of each substance. Electron microscopy shows the haemosiderin cores to be markedly smaller on average than those of ferritin. Combining the Mössbauer spectroscopy and electron microscopy data we have shown that the magnetic anistropy constant of haemosiderin is considerably larger than that of ferritin. This is thought to result from the smaller core size and less symmetrical protein shell of the former. These data are consistent with the proposal that haemosiderin is derived from ferritin.
Assuntos
Ferritinas , Hemossiderina , Humanos , Magnetismo , Microscopia Eletrônica , Espectrofotometria , Baço/análise , TemperaturaRESUMO
Interleukin 2 (IL-2) is one of the major cytokines produced by T lymphocytes in response to antigen. It is a potent growth and differentiation factor for several cell-types and is structurally related to the four-helix bundle family of cytokines. Mutation of residue Phe42 to Ala abolishes binding to the alpha chain of the tri-partite IL-2 receptor. The three-dimensional structure of the F42A mutant IL-2 has been calculated by two dimensional NMR methods and compared to a structure of wild-type IL-2 determined by X-ray crystallography. The overall topology of the two structures is the same. The main differences between the structures are within the ill-defined loops connecting the helices and the region of the protein that is believed to interact with the alpha-chain of the receptor. Thus, the mutation of Phe42 to Ala does not perturb the overall three-dimensional structure of IL-2, and does not appear to change the putative binding sites for the beta and gamma chains of the receptor. The structural differences observed in this mutant suggest that the replacement of Phe42 with Ala causes the re-orientation of neighbouring side-chains that are also involved in binding the alpha-chain of the receptor.
Assuntos
Interleucina-2/química , Alanina/química , Alanina/genética , Sequência de Aminoácidos , Simulação por Computador , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Interleucina-2/genética , Interleucina-2/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fenilalanina/química , Fenilalanina/genética , Conformação Proteica , Estrutura Secundária de Proteína , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes/química , Soluções/químicaRESUMO
Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function. Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process. The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level. Proteomics can be divided into expression proteomics, the study of global changes in protein expression, and cell-map proteomics, the systematic study of protein-protein interactions through the isolation of protein complexes.
Assuntos
Biotecnologia/tendências , Espectrometria de Massas/tendências , Mapeamento de Peptídeos/tendências , Genoma , Proteínas/análise , Proteínas/genética , Proteínas/isolamento & purificaçãoRESUMO
The binding modes of a series of penicillin-derived C2 symmetric dimer inhibitors of HIV-1 proteinase were investigated by NMR, protein crystallography, and molecular modeling. The compounds were found to bind in a symmetrical fashion, tracing and S-shaped course through the active site, with good hydrophobic interactions in the S1/S1' and S2/S2' pockets and hydrogen bonding of inhibitor amide groups. Interactions with the catalytic aspartates appeared poor and the protein conformation was very similar to that seen in complexes with peptidomimetics, in spite of the major differences in ligand structure.
Assuntos
Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/farmacologia , Penicilinas/síntese química , Penicilinas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia , Protease de HIV/química , Inibidores da Protease de HIV/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Penicilinas/química , Relação Estrutura-AtividadeRESUMO
Ferritin and haemosiderin were isolated from fresh frozen human spleens that had been removed from patients with secondary iron overload due to multiple transfusions. Haemosiderin was solubilised by a novel technique that maintains its integrity. Unstained preparations of haemosiderin and ferritin were visualised and quantitative measurements made of the volumes of iron core. The mean diameter of the ferritin core (6.4 nm) was larger than that of haemosiderin (5.7 nm). In addition, haemosiderin, in contrast to ferritin, showed a large number of cores of less than 5 nm in diameter. Negatively stained preparations of haemosiderin and ferritin were visualised, confirming the small core size of the haemosiderin. The protein shell of haemosiderin, unlike that of ferritin, was thinner and irregular. These findings are consistent with the suggestion that haemosiderin is derived from ferritin by partial proteolysis and partial solubilisation of the iron core, presumably by lysosomal action.
Assuntos
Ferritinas , Hemossiderina , Humanos , Membranas Intracelulares/ultraestrutura , Lisossomos/ultraestrutura , Microscopia Eletrônica , Tamanho da Partícula , Baço/ultraestruturaRESUMO
The availability of target proteins in sufficient quantity is a limiting factor in crystallographic studies and therefore in rational drug design. Even after optimisation, expression of recombinant proteins may be low and the only way to produce enough protein is by large scale cell growth/purification. HIV-1 proteinase in Escherichia coli, which due to its toxicity is expressed as a soluble protein only at around 0.1% of total protein, is a paradigm for this. In this paper a detailed process for large scale expression and purification of HIV-1 proteinase which delivers material of suitable quantity (30 mg from 500 g of wet weight of cells) and quality for crystallographic studies is described.
Assuntos
Escherichia coli/genética , Protease de HIV/biossíntese , Escherichia coli/metabolismo , Protease de HIV/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificaçãoAssuntos
Antivirais/farmacologia , Dioxóis/farmacologia , Inibidores da Protease de HIV , HIV-1/efeitos dos fármacos , Penicilinas/farmacologia , Linhagem Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Dioxóis/uso terapêutico , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , Replicação Viral/efeitos dos fármacos , Difração de Raios XRESUMO
Our previous studies have suggested that all the known lineage compartment borders in the wing imaginal disc of Drosophila are coincident with boundaries of reduced gap junctional communication (communication compartment borders). Since engrailed discs have a disrupted anterior/posterior (A/P) lineage border (G. Morata and P. A. Lawrence, 1975, Nature (London) 255, 614-617), it was of great interest to determine if their A/P communication restriction boundary is similarly disrupted. Examination of gap-junction-mediated exchange of small fluorescent molecules between cells in the engrailed wing disc revealed a boundary of restricted communication that appeared to be identical to the wild-type A/P communication restriction boundary. This result suggests that lineage compartments are not required for the formation of A/P communication restrictions. Furthermore, we suggest that perhaps communication compartments are the domains within which information is provided for specifying the formation of lineage compartments.
Assuntos
Comunicação Celular , Asas de Animais/citologia , Animais , DrosophilaRESUMO
Transcripts of the engrailed and fushi tarazu genes in young Drosophila embryos are initially patterned in intervals larger than a single segment. The segmental pattern evolves in a stepwise sequence through successively smaller spatial units.
Assuntos
Drosophila/genética , Transcrição Gênica , Animais , Autorradiografia , Divisão Celular , Mapeamento Cromossômico , Drosophila/embriologiaRESUMO
Recombinant human interleukin-2 (IL-2) expressed as Escherichia coli was isolated as insoluble aggregates of protein (inclusion bodies) after cell breakage. IL-2 and contaminants were dissolved in 6 M-guanidinium chloride/10 mM-dithiothreitol, pH 8.5, and further purified in reduced and denatured form by gel-permeation chromatography in the same solvent. Renaturation was effected by dilution and autoxidation; IL-2 of native specific activity was isolated at over 95% purity by reversed-phase h.p.l.c.; an additional peak of reduced protein was also observed. Most losses of native IL-2 occurred on refolding, probably because of an aggregation process; concentrations around 1 microgram/ml were necessary to achieve 30% recovery. It was essential to maintain the denatured protein in reduced form before renaturation and autoxidation, which was most efficient at pH 8.5 with 1.5 microM-CuSO4. A procedure based on these observations has been used to prepare IL-2 on the 50 micrograms scale.
Assuntos
Interleucina-2/isolamento & purificação , Interleucina-2/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Substâncias Macromoleculares , Oxirredução , Desnaturação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
We have examined the gap junctional communication properties of cells in the wing imaginal disk of Drosophila, using intracellular injection of the fluorescent dye tracer Lucifer Yellow. The cell-to-cell passage of Lucifer Yellow is restricted at a boundary line that divides the wing disk into halves. We refer to each half as a "communication compartment" because there is a high level of gap junctional exchange within a compartment and much lower exchange between compartments. Comparison of the positions of the compartments with developmental fate maps suggests that cells in one communication compartment give rise to cuticular structures in the anterior half of the adult dorsal mesothorax, and cells in the other compartment contribute to posterior structures. This communication-restriction line appears to be coincident with the line between the anterior and posterior developmental compartments observed in studies of cell lineage. We propose that gap junctional communication restrictions may play a general role in generating or maintaining developmental compartments.
Assuntos
Comunicação Celular , Drosophila/citologia , Junções Intercelulares/fisiologia , Animais , Diferenciação Celular , Eletrofisiologia , Isoquinolinas , Asas de Animais/anatomia & histologia , Asas de Animais/embriologiaRESUMO
Using intracellular injection of small fluorescent molecules, we examined the gap-junctional communication properties of cells in the wing imaginal disk of Drosophila. We observed extensive spread of the injected dye between cells in all parts of the wing disk epithelium, but we found that this spread is not uniform. Instead, the cell-to-cell movement of dye is partially restricted at several defined boundaries such that cells on one side of the boundary have a low level of communication with cells on the other side. These communication-restriction boundaries are delineated by bands of cells with a low level of communication, and they subdivide the disk epithelium into a number of "communication compartments." Interestingly, the boundaries of some of these communication compartments appear to be coincident with compartment borders identified in cell lineage studies. Given this striking coincidence, we suggest that gap-junctional communication may play a role in compartment formation, and in controlling the appropriate differentiation of cells within compartments.
Assuntos
Comunicação Celular , Drosophila/fisiologia , Junções Intercelulares/fisiologia , Animais , Condutividade Elétrica , Larva/fisiologia , Microeletrodos , Microscopia de FluorescênciaRESUMO
We wish to identify genes involved in mediating early lineage decisions in the mouse embryo. F9 teratocarcinoma cells treated with retinoic acid (RA) in suspension culture develop into embryoid bodies (EBs) with an outer layer of visceral endoderm. In order to identify genes that are involved in establishing this extraembryonic endoderm lineage we have employed a PCR-based approach using cDNAs from early EBs as templates. PCR reactions were performed with degenerate oligonucleotide primers coding for the highly conserved regions of the homeodomains of the Drosophila Antennapedia, bicoid, and zerknüllt proteins. Among the PCR products were representatives of previously identified mouse genes, including Hox-A5 (1.3), A1 (1.6), A9 (1.7), B8 (2.4), B2 (2.8), C8 (3.1), and D12 (4.7). Whole mount in situ hybridization analysis, performed to examine the temporal and spatial distribution of transcripts, suggests a possible role for the Hox-D12 gene during endoderm differentiation in F9 EBs. Whereas the expression patterns of several other homeobox genes are essentially uniform throughout the aggregates, Hox-D12 expression is restricted to the outer surface of early EBs at a time when lineage decisions may be occurring. In order to establish the relationship between the Hox-D12 expression pattern and the role of RA in inducing F9 EB differentiation, we examined PSA-1 EBs that differentiate in the absence of added RA. PSA-1 EBs show similar temporal and spatial localization of Hox-D12 when compared to F9 EBs. These data suggest that the pattern of Hox-D12 expression correlates with endoderm differentiation and not with RA treatment and point to a possible role for homeobox-containing genes during the early stages of mouse embryogenesis.
Assuntos
Diferenciação Celular/genética , Endoderma/citologia , Genes Homeobox , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Teratoma , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
Recombinant Interleukin-2 (IL-2) is expressed in E. coli as insoluble aggregates; a protocol has been developed for solubilization, renaturation and purification of IL-2 from such aggregates at the 5-10-mg level. IL-2 aggregates were isolated from soluble proteins by centrifugation, subjected to a 1 M guanidine hydrochloride wash and a butan-1-ol wash (the latter to remove lipid), dissolved in 8 M guanidine hydrochloride-10 mM dithiothreitol and partly purified by gel permeation chromatography. Refolding/oxidation was then performed by dilution into Tris-HCl, pH 8.5 containing 1.5 microM copper sulphate to accelerate autoxidation. Final purification was by successive cation-exchange and reversed-phase high-performance liquid chromatographic steps, yielding over 99.5% pure IL-2 with an overall recovery of 20%.
Assuntos
Interleucina-2/isolamento & purificação , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Proteínas Recombinantes/isolamento & purificação , Dodecilsulfato de SódioRESUMO
High levels of haemosiderin occur in iron overload syndromes such as idiopathic haemochromatosis or secondary iron overload in thalassaemic patients; haemosiderin is the predominant iron-storage compound in such cases. It consists of a large aggregate of FeOOH cores, many of which have an incomplete shell of protein, and is probably derived from ferritin by lysosomal proteolysis. In addition, some chemical degradation of the ferritin cores appears to occur on conversion to haemosiderin. Other biochemical components are phosphate and magnesium, which may be adsorbed to the core surface, and perhaps certain lipids. Haemosiderin may have a central role, either directly or indirectly, in iron cytotoxicity and therefore the chemistry and biochemistry of this material warrants further study.