Assuntos
Magnoliopsida/crescimento & desenvolvimento , Scrophulariaceae/crescimento & desenvolvimento , Benzoquinonas/metabolismo , Interações Hospedeiro-Parasita , Peróxido de Hidrogênio/metabolismo , Magnoliopsida/metabolismo , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Scrophulariaceae/metabolismo , Transdução de SinaisRESUMO
The pi-helix is a secondary structure with 4.4 amino acids per helical turn. Although it was proposed in 1952, no experimental support for its existence was obtained until the mid-1980s. While short peptides are unlikely to assume a marginally stable secondary structure spontaneously, they might do so in the presence of appropriate structural constraints. In this paper, we describe a peptide that is designed to assume a pi-helical conformation when stabilized by cetyltrimethylammonium bromide (CTAB) micelles and Zn(2+). In the designed peptide, lipophilic amino acids are placed such that it would be amphiphilic in the pi-helical, but not in the alpha-helical, conformation. Also, two His residues are incorporated with i, i + 5 spacing, designed to allow binding of Zn(2+) in a pi-helical but not an alpha-helical conformation. The peptide was found to form moderately stable monolayers at the air-water interface, with a collapse pressure that almost doubled when there was Zn(2+) in the subphase. Also, CTAB micelles induced a marked increase in the helicity of the peptide. In 50% TFE, the peptide had a CD spectrum consistent with an alpha-helical structure. The addition of 1 mM Zn(2+) to this solvent caused a saturable decline in ellipticity to approximately half of its original value. The peptide also bound Zn(2+) when it was bound to CTAB micelles, with Zn(2+) again inducing a decrease in ellipticity. The peptide had slightly greater affinity for Zn(2+) in the presence of the CTAB than in a 50% TFE solution (K(d) = 3.1 x 10(-4) M in CTAB and 2.3 x 10(-4) M in TFE). van't Hoff analysis indicated that thermal denaturation of the peptide in 50% TFE containing 1 mM Zn(2+) was associated with both enthalpic and entropic changes that were greater than those in the absence of Zn(2+). These observations are all consistent with the proposal that the peptide assumed a pi-helical conformation in the presence of Zn(2+) and CTAB micelles, and has allowed the stability of this rare conformation to be assessed.
Assuntos
Peptídeos/síntese química , Peptídeos/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Apolipoproteínas E/síntese química , Apolipoproteínas E/metabolismo , Cetrimônio , Compostos de Cetrimônio/farmacologia , Dicroísmo Circular , Estabilidade Enzimática , Humanos , Dados de Sequência Molecular , Pressão , Ligação Proteica/efeitos dos fármacos , Engenharia de Proteínas , Estrutura Secundária de Proteína/efeitos dos fármacos , Propriedades de Superfície , TermodinâmicaRESUMO
A systematic approach evaluating template-directed ligation reactions has now resulted in a simple outline for a two-stage replication cycle. This cycle builds on an efficient method for reading the information encoded in DNA into an amplified translation product. It is further demonstrated that the translation product strand is capable of catalyzing the synthesis of the original DNA strand. We propose that this cycle represents just one of many possible solutions; other chemical ligation or polymerization reactions could be accommodated with different templates. In that context, a new template, derived by modest changes to the DNA backbone, has been developed and has been shown to hybridize under reaction conditions different than those accessible to DNA. Therefore, the conceptual groundwork has been laid for extending this approach to encoding and reading stored information in molecules other than the natural biopolymers at the densities found in biology.
Assuntos
Técnicas de Química Combinatória/métodos , DNA/química , Oligonucleotídeos/síntese química , DNA/genética , DNA/metabolismo , Moldes Genéticos , Transcrição GênicaRESUMO
BACKGROUND: Agrobacterium tumefaciens has been successfully harnessed as the only natural vector for the incorporation of foreign genes into higher plants, but its use in the grain crops is often limited. Low transformation efficiency has been partly attributed to a failure in the initial events in the transformation process, specifically in the capacity of the VirA/VirG two-component system to induce expression of the virulence genes. RESULTS: Here we show that the root exudate of Zea mays seedlings specifically inhibits virulence gene expression, determine that 2-hydroxy-4,7-dimethoxybenzoxazin-3-one (MDIBOA), which constitutes > 98% of the organic exudate of the roots of these seedlings, is the most potent and specific inhibitor of signal perception in A. tumefaciens-mediated gene transfer yet discovered, and develop a model that is able to predict the MDIBOA concentration at any distance from the root surface. Finally, variants of A. tumefaciens resistant to MDIBOA-mediated inhibition of vir gene expression have been selected and partially characterized. CONCLUSIONS: These results suggest a strategy in which a plant may resist pathogen invasion by specifically blocking virulence gene activation and yet ensure that the 'resistance factor' does not accumulate to levels sufficient to impose toxicity and selection pressure on the pathogen. The data further establish that naturally occurring inhibitors directed against signal perception by the VirA/VirG two-component regulatory system can play an important role in host defense. Finally, selected variants resistant to specific MDIBOA inhibition may now be used to extend the transformation efficiency of maize and possibly other cereals.
Assuntos
Agrobacterium tumefaciens/efeitos dos fármacos , Agrobacterium tumefaciens/patogenicidade , Oxazinas/farmacologia , Transformação Genética/efeitos dos fármacos , Fatores de Virulência , Zea mays/genética , Zea mays/microbiologia , Agrobacterium tumefaciens/genética , Antibacterianos/análise , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Benzoxazinas , Técnicas de Cocultura , Difusão , Relação Dose-Resposta a Droga , Resistência Microbiana a Medicamentos/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Mutação/genética , Oxazinas/análise , Oxazinas/química , Raízes de Plantas/química , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Ativação Transcricional , Virulência/efeitos dos fármacos , Virulência/genética , Zea mays/químicaRESUMO
beta-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of beta-amyloid fibrils poses a challenge because of the limited solubility of beta-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of beta-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as beta-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10-35 of human beta-amyloid and indicates that in fibrils, this peptide assumes a parallel beta-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Abeta peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos/química , Peptídeos beta-Amiloides/ultraestrutura , Sequência de Bases , Físico-Química/métodos , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação ProteicaRESUMO
Parasitic strategies are widely distributed across the angiosperms and are estimated to have evolved at least eight different times. Within the obligate hemiparasitic and holoparasitic members, elaborate strategies for host selection have emerged. Here, we demonstrate that in the parasitic Scrophulariceae Striga asiatica, for which signal-mediated host detection is critical, expansin mRNA provides a reliable and accurate downstream molecular marker for the transition to the parasitic mode. Three different expansin genes, saExp1, saExp2, and saExp3, are regulated by xenognostic quinones. saExp3 appears to function as a seedling expansin, and its mRNA is depleted within minutes after induction of the host attachment organ. saExp1 and saExp2 share less homology with the known expansins, and their transcripts accumulate linearly over a critical induction period. The regulation of these genes suggests that the resources for developmental commitment must accumulate to a defined threshold before commitment to organogenesis is terminal. When the induction signal is removed prematurely, the accumulated message decays with a time constant that correlates with the time required for additional signal exposures to reinduce parasitic development. These results suggest that sophisticated controls exist for the accumulation of the necessary components for terminal commitment to the parasitic mode. Furthermore, building on the redox dependence of the inducing signal, they suggest a model akin to a "molecular capacitor" for clocking organogenesis in S. asiatica.
Assuntos
Adenina/análogos & derivados , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , Proteínas de Plantas/genética , Adenina/farmacologia , Sequência de Aminoácidos , Benzoquinonas/farmacologia , Clonagem Molecular , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Parasita/genética , Cinetina , Magnoliopsida/fisiologia , Dados de Sequência Molecular , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/química , Quinonas/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alinhamento de Sequência , Fatores de TempoRESUMO
Beta-amyloid (Abeta) peptides are the main protein component of the pathognomonic plaques found in the brains of patients with Alzheimer's disease. These heterogeneous peptides adopt a highly organized fibril structure both in vivo and in vitro. Here we use solid-state NMR on stable, homogeneous fibrils of Abeta(10-35). Specific interpeptide distance constraints are determined with dipolar recoupling NMR on fibrils prepared from a series of singly labeled peptides containing (13)C-carbonyl-enriched amino acids, and skipping no more that three residues in the sequence. From these studies, we demonstrate that the peptide adopts the structure of an extended parallel beta-sheet in-register at pH 7.4. Analysis of DRAWS data indicates interstrand distances of 5.3 +/- 0.3 A (mean +/- standard deviation) throughout the entire length of the peptide, which is compatible only with a parallel beta-strand in-register. Intrastrand NMR constraints, obtained from peptides containing labels at two adjacent amino acids, confirm the secondary structural findings obtained using DRAWS. Using peptides with (13)C incorporated at the carbonyl position of adjacent amino acids, structural transitions from alpha-helix to beta-sheet were observed at residues 19 and 20, but using similar techniques, no evidence for a turn could be found in the putative turn region comprising residues 25-29. Implications of this extended parallel organization for Abeta(10-35) for overall fibril formation, stability, and morphology based upon specific amino acid contacts are discussed.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Aminoácidos/química , Peptídeos beta-Amiloides/ultraestrutura , Isótopos de Carbono , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/ultraestrutura , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
A versatile expression vector utilizing a promoter of coliphage T5, P(N25) (Gentz and Bujard, 1985. J. Bacteriol. 164, 70-77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78-89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (delta1-284, G665D) in Escherichia coli as well as in A. tumefaciens. The signal transduction proteins VirA (delta1-284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P(N25) driven expression overrides the complex transcriptional regulation present with the native promoters. This expression system will enable a more detailed analysis of the activation events in signal transduction in A. tumefaciens, and we expect it to be useful in other prokaryotes.
Assuntos
Agrobacterium tumefaciens/genética , Vetores Genéticos/genética , Regiões Promotoras Genéticas , Fagos T/genética , Fatores de Virulência , Acetofenonas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Recombinante , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Histidina/genética , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The mechanisms of signal perception and transmission in the 'two-component' autokinase transmitters/response regulators are poorly understood, especially considering the vast number of such systems now known. Virulence induction from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens represents one of the best understood systems with regard to the chemistry of the activating signal, and yet the existing data does not support a receptor-mediated perception event for the xenognostic phenols. RESULTS: Here we provide the first conclusive evidence that a specific receptor must be involved in xenognostic phenol perception, detail structural requirements of the xenognosins necessary for perception by this receptor, and develop a genetic strategy that demonstrates critical components of the phenol recognition system are not encoded on the Ti plasmid. CONCLUSIONS: Although the basic elements of the two-component system required for phenol-mediated induction of virulence gene expression are encoded on the Ti plasmid, they are dependent on the chromosomal background for even the very first stage of signal perception. This discovery suggests a curious evolutionary history, and also provides functional insight into the mechanisms of two-component signal detection and transmission in general.
Assuntos
Agrobacterium tumefaciens/metabolismo , Agrobacterium tumefaciens/patogenicidade , Fenóis/metabolismo , Proteínas de Plantas , Receptores de Superfície Celular/metabolismo , Fatores de Virulência , Virulência/genética , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/metabolismo , Carboidratos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Immunoblotting , Espectroscopia de Ressonância Magnética , Plasmídeos/genética , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Estereoisomerismo , Fatores de Transcrição/metabolismo , beta-Galactosidase/metabolismoRESUMO
Vidal et al. (1999. Nature 399: 776-778) discovered that the underlying genetic lesion in familial British dementia (FBD) is a T-A transversion at the termination codon of a membrane protein, termed BRI. The mutation creates an arginine codon; translational read-through generates a novel protein, termed BRI-L, that is extended by 11 amino acids at the carboxyl-terminus. BRI-L is the precursor of the ABri peptide, a component of amyloid deposits in FBD brain. We demonstrate that both BRI and its mutant counterpart are constitutively processed by furin, resulting in the secretion of carboxyl-terminal peptide derivatives that correspond to all, or part of, ABri. Notably, elevated levels of peptides are generated from the mutant BRI precursor, suggesting that subtle conformational alterations at the carboxyl-terminus may influence furin-mediated processing. We have examined BRI/BRI-L processing by other members of the prohormone convertase (PC) family (PACE4, LPC, PC 5/6) and found that these enzymes also process BRI, albeit inefficiently. Moreover, BRI-L processing by the other PC members is severely compromised. Finally, our electron microscopic studies reveal that synthetic ABri peptides assemble into insoluble beta-pleated fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates amyloidogenic peptides that initiate the pathogenesis of FBD.
Assuntos
Amiloide/genética , Amiloide/metabolismo , Encéfalo/metabolismo , Demência/genética , Fragmentos de Peptídeos/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Amiloide/química , Animais , Células Cultivadas , Códon de Terminação , Furina , Humanos , Mamíferos , Glicoproteínas de Membrana , Proteínas de Membrana , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Subtilisinas/metabolismo , Transfecção , Reino UnidoRESUMO
The genetic lesion underlying familial British dementia (FBD), an autosomal dominant neurodegenerative disorder, is a T-A transversion at the termination codon of the BRI gene. The mutant gene encodes BRI-L, the precursor of ABri peptides that accumulate in amyloid deposits in FBD brain. We now report that both BRI-L and its wild-type counterpart, BRI, were constitutively processed by the proprotein convertase, furin, resulting in the secretion of carboxyl-terminal peptides that encompass all or part of ABri. Elevated levels of peptides were generated from the mutant BRI precursor. Electron microscopic studies revealed that synthetic ABri peptides assembled into irregular, short fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates fibrillogenic peptides that initiate the pathogenesis of FBD.
Assuntos
Amiloide/biossíntese , Demência/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Neurofibrilas/metabolismo , Fragmentos de Peptídeos/biossíntese , Subtilisinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Furina , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana , Camundongos , Microscopia Eletrônica , Células Tumorais Cultivadas , Reino UnidoRESUMO
Product inhibition has provided the limiting barrier to efficient template-directed ligation and polymerization reactions. Here we review the attempts to circumvent this limitation and outline a translation strategy that does overcome the barrier and allows the information encoded in DNA to be read and amplified into backbone-modified oligonucleotides.
Assuntos
DNA/genética , Biossíntese de Proteínas , Catálise , Modelos Moleculares , Moldes GenéticosRESUMO
The pathognomonic plaques of Alzheimer's disease are composed primarily of the 39- to 43-aa beta-amyloid (Abeta) peptide. Crosslinking of Abeta peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Abeta. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Abeta peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Abeta. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 A. Analysis of these data establish that this central core of Abeta consists of a parallel beta-sheet structure in which identical residues on adjacent chains are aligned directly, i. e., in register. Our data, in conjunction with existing structural data, establish that the Abeta fibril is a hydrogen-bonded, parallel beta-sheet defining the long axis of the Abeta fibril propagation.
Assuntos
Peptídeos beta-Amiloides/química , Dobramento de Proteína , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Bioactive peptides have multiple conformations in solution but adopt well-defined conformations at lipid surfaces and in interactions with receptors. We have used side chain lactam cross-links to stabilize secondary structures in the following peptide models of a conserved N-terminal domain of apolipoprotein E (cross-link periodicity in parentheses): I, H2N-GQTLSEQVQEELLSSQVTQELRAG-COOH (none); III, [sequence; see text] (i to i + 3); IV,[sequence; see text] (i to i + 4); IVa, [sequence, see text] (i to i + 4) (lactams above the sequence, potential salt bridges below the sequence). We previously demonstrated [Luo et al. (1994) Biochemistry 33, 12367-12377; Braddock et al. (1996) Biochemistry 35, 13975-13984] that peptide III, containing lactam cross-links between the i and i + 3 side chains, enhances specific binding of LDL via a receptor other than the LDL-receptor. Peptide III in solution consists of two short alpha helices connected by a non alpha helical segment. Here we examine the hypothesis that the domain modeled by peptide III is one antipode of a conformational switch. To model another antipode of the switch, we introduced two strategic modifications into peptide III to examine structure-function relationships in this domain: (1) the spacing of the lactam cross-links was changed (i to i + 4 in peptides IV and IVa) and (2) peptides IV and IVa contain the two alternative sequences at a site of a possible end-capping interaction in peptide III. The structure of peptide IV, determined by 2D-NMR, is alpha helical across its entire length. Despite the remarkable degree of structural order, peptide IV is biologically inactive. In contrast, peptides III and possibly IVa contain a central interruption of the alpha helix, which appears necessary for biological activity. These and other studies support the hypothesis that this domain is a conformational switch which, to the extent that it models apolipoprotein E itself, may modulate interactions between apo E and its various receptors.
Assuntos
Apolipoproteínas E/química , Sequência Conservada , Lactamas/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Apolipoproteínas E/metabolismo , Linhagem Celular , Dicroísmo Circular , Embrião de Mamíferos , Fibroblastos , Radioisótopos do Iodo , Lactamas/metabolismo , Camundongos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Receptores de LDL/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The temporal and spatial control of the transition from vegetative to parasitic growth is critical to any parasite, but is essential to the sessile parasitic plants. It has been proposed that this transition in Striga spp. is controlled simply by an exuded oxidase that converts host cell-surface phenols into benzoquinones which act as developmental signals that mediate the transition. An understanding of this mechanism may identify the critical molecular events that made possible the evolution of parasitism in plants. RESULTS: PoxA and PoxB are identified as the only apoplastic phenol oxidases in Striga asiatica seedlings, and the genes encoding them have been cloned and sequenced. These peroxidase enzymes are capable of oxidizing the 60 known inducing phenols into a small set of benzoquinones, and it is these quinones that induce parasitic development. Analysis of the reaction requirements and comparisons to host enzymes, however, lead us to argue that PoxA and PoxB are not necessary for host recognition. CONCLUSIONS: A new model is proposed where constitutive production of an activated oxygen species (in the case of Striga, H2O2) mediates host recognition. This strategy would allow a parasite to exploit abundant host enzymes to produce the diffusible recognition signals by converting a standard host defense into a parasitic offense.
Assuntos
Parede Celular/enzimologia , Peroxidases/química , Fenóis/metabolismo , Plantas/parasitologia , Sequência de Aminoácidos , Sequência de Bases , Benzoquinonas/farmacologia , Parede Celular/química , Clonagem Molecular , Sequência Conservada/genética , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Histocitoquímica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , Plantas/química , RNA Mensageiro/análise , Sementes/citologia , Sementes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We demonstrate a new method for investigating the structure of self-associating biopolymers using dipolar recoupling NMR techniques. This approach was applied to the study of fibrillar beta-amyloid (Abeta) peptides (the primary component of the plaques of Alzheimer's disease) containing only a single isotopic spin label (13C), by employing the DRAWS (dipolar recoupling with a windowless sequence) technique to measure 13C-13C distances. The 'single-label' approach simplified analysis of DRAWS data, since only interstrand contacts are present, without the possibility of any intrastrand contacts. As previously reported [T.L.S. Benzinger, D.M. Gregory, T.S. Burkoth, H. Miller-Auer, D.G. Lynn, R.E. Botto, S.C. Meredith, Proc. Natl. Acad. Sci. 95 (1998) 13407.], contacts of approximately 5 A were observed at all residues studied, consistent with an extended parallel beta-sheet structure with each amino acid in exact register. Here, we propose that our strategy is completely generalizable, and provides a new approach for characterizing any iterative, self-associating biopolymer. Towards the end of generalizing and refining our approach, in this paper we evaluate several issues raised by our previous analyses. First, we consider the effects of double-quantum (DQ) transverse relaxation processes. Next, we discuss the effects of various multiple-spin geometries on modeling of DRAWS data. Several practical issues are also discussed: these include (1) the use of DQ filtering experiments, either to corroborate DRAWS data, or as a rapid screening assessment of the proper placement of isotopic spin labels; and (2) the comparison of solid samples prepared by either lyophilization or freezing. Finally, data obtained from the use of single labels is compared with that obtained in doubly 13C-labeled model compounds of known crystal structure. It is shown that such data are obtainable in far more complex peptide molecules. These data,taken together, refine the DRAWS method, and demonstrate its precision and utility in obtaining high resolution structural data in complex biomolecular aggregates such as Abeta.
Assuntos
Peptídeos beta-Amiloides/química , Espectroscopia de Ressonância Magnética/métodos , Peptídeos beta-Amiloides/síntese química , Estrutura Molecular , Conformação ProteicaRESUMO
Parasitic strategies are widely distributed in the plant kingdom and frequently involve coupling parasite organogenesis with cues from the host. In Striga asiatica, for example, the cues that initiate the development of the host attachment organ, the haustorium, originate in the host and trigger the transition from vegetative to parasitic mode in the root meristem. This system therefore offers a unique opportunity to study the signals and mechanisms that control plant cell morphogenesis. Here we establish that the biological activity of structural analogs of the natural inducer displays a marked dependence on redox potential and suggest the existence of a semiquinone intermediate. Building on chemistry that exploits the energetics of such an intermediate, cyclopropyl-p-benzoquinone (CPBQ) is shown to be a specific inhibitor of haustorial development. These data are consistent with a model where haustorial development is initiated by the completion of a redox circuit.
RESUMO
Apolipoprotein E plays a critical role in plasma lipoprotein clearance. A peptide model of a highly conserved domain of this protein has been shown to increase low-density lipoprotein binding to fibroblast cell surface receptors. To distinguish between two potential structures--one essentially alpha-helical and nonamphiphilic, the other an amphiphilic pi-helix--synthetic side-chain lactam constraints have been incorporated into model peptides in order to restrict conformational flexibility favoring either the alpha- or pi-helix. Here we provide CD and 1H NMR data suggesting that the more biologically active, putatively alpha-helical peptide indeed contains two alpha-helical domains separated by a central bend. Whereas previous studies (Osapay & Taylor, 1992; Felix et al., 1988) indicated stabilization of alpha-helices by cross-links between the i and i + 4 residues, the current paper demonstrates that cross-links between the i and i + 3 residues also stabilize the helix. Indeed, the stabilization afforded by these cross-links is approximately 1 kcal/mol, similar to that reported for peptides cross-linked between the i and i + 4 residues, and derives exclusively from a loss of entropy of the unfolded state. The presence of the alpha-helical structure appears to correlate well with biological activity. This study provides initial insight into the bioactive structure of this domain of apo E and suggests strategies as to how peptides can be conformationally constrained to enhance their stability and biological function.
Assuntos
Apolipoproteínas E/química , Lactamas/química , Modelos Moleculares , Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Simulação por Computador , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Mutations at the cytokinin biosynthesis locus (tmr) of Agrobacterium tumefaciens usually result in strains that induce tumors exhibiting the rooty phenotype associated with high auxin-to-cytokinin ratios. However, tobacco (Nicotiana tabacum cv Havana 425) leaf disc explants responded to tmr- mutant strain A356 by producing rapidly growing, unorganized tumors, indicating that these lines can grow in a cytokinin-independent fashion despite the absence of a functional tmr gene. Several methods have been used to characterize the physiological and cellular basis of this phenotype. The results indicate that tmr- tumors have a physiologically distinct mechanism for cytokinin-independent growth in comparison to tumors induced by wild-type bacteria. The cytokinin-independent phenotype of the tmr- transformants appears to be cell autonomous in nature: only the transformed cells and their progeny were capable of cytokinin-independent growth. Specifically, the tmr- tumors did not accumulate cytokinin, and clonal analysis indicated the tmr- transformed cells were not capable of stimulating the growth of neighboring nontransformed cells. Finally, the cytokinin-independent phenotype of the tmr- transformants was shown to be cold sensitive, whereas the wild-type tumors exhibited a cold-resistant cytokinin-independent phenotype. Potential mechanisms for this novel form of cytokinin-independent growth, including the role of the dehydrodiconiferyl alcohol glucosides found in both tumor types, are discussed.
Assuntos
Agrobacterium tumefaciens/genética , Citocininas/biossíntese , Citocininas/farmacologia , Nicotiana/citologia , Plantas Tóxicas , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Citocininas/genética , Deleção de Genes , Genes Bacterianos , Genótipo , Glucosídeos/análise , Glucosídeos/metabolismo , Modelos Biológicos , Mutagênese , Fenótipo , Nicotiana/efeitos dos fármacosRESUMO
The virulence genes of the plant pathogen Agrobacterium tumefaciens are induced by more than 40 low-molecular-weight phenolic compounds. The prevailing opinion is that (i) wound-derived phenols produced on breach of the integrity of the cell wall act as the initiating signal in a series of events which results in host cell transformation, and (ii) a classical membrane receptor, putatively VirA, is responsible for the recognition of all such phenolic inducers. Here, we argue that the discovery of the subset of inducers that are relatives of the dehydrodiconiferyl alcohol glucoside (DCG) growth factors redirects our attention to work on the plant wound as a site of cell division, and suggests that we further explore the implications of early work on the relationship between transformation efficiency and the status of the cell cycle of the host. In addition, we argue that the significant structural diversity allowed in the para position of the phenol ring of inducers suggests that a receptor-ligand interaction based solely on structural recognition is insufficient, but that recognition followed by a specific proton transfer event may be sufficient to explain vir induction activity. Hence, the specificity of the response of A. tumefaciens may be a consequence of the features required for a chemical reaction to occur on the receptor surface. Finally, we review affinity labelling studies which exploit this phenol detection mechanism and which provide evidence that the phenol receptor may be other than VirA, the sensory kinase of the two component regulatory system implicated in Agrobacterium virulence.(ABSTRACT TRUNCATED AT 250 WORDS)