RESUMO
In this study, we have investigated the fluorescence properties of SYBR Green I (SG) dye and its interaction with double-stranded DNA (dsDNA). SG/dsDNA complexes were studied using various spectroscopic techniques, including fluorescence resonance energy transfer and time-resolved fluorescence techniques. It is shown that SG quenching in the free state has an intrinsic intramolecular origin; thus, the observed >1,000-fold SG fluorescence enhancement in complex with DNA can be explained by a dampening of its intra-molecular motions. Analysis of the obtained SG/DNA binding isotherms in solutions of different ionic strength and of SG/DNA association in the presence of a DNA minor groove binder, Hoechst 33258, revealed multiple modes of interaction of SG inner groups with DNA. In addition to interaction within the DNA minor groove, both intercalation between base pairs and stabilization of the electrostatic SG/DNA complex contributed to increased SG affinity to double-stranded DNA. We show that both fluorescence and the excited state lifetime of SG dramatically increase in viscous solvents, demonstrating an approximate 200-fold enhancement in 100 % glycerol, compared to water, which also makes SG a prospective fluorescent viscosity probe. A proposed structural model of the SG/DNA complex is compared and discussed with results recently reported for the closely related PicoGreen chromophore.
Assuntos
DNA/metabolismo , Fluorescência , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Benzotiazóis , DNA/química , Diaminas , Conformação de Ácido Nucleico , Quinolinas , Soluções , Espectrometria de Fluorescência , Eletricidade Estática , Termodinâmica , ViscosidadeRESUMO
PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.
Assuntos
DNA/química , Corantes Fluorescentes/química , Sítios de Ligação , Fenômenos Biofísicos , Bisbenzimidazol/química , Substâncias Intercalantes , Substâncias Macromoleculares/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Compostos Orgânicos/química , Espectrometria de Fluorescência , Eletricidade EstáticaRESUMO
PicoGreen (PG) is a fluorescent probe for both double-stranded DNA (dsDNA) detection and quantification based on its ability to form a luminescent complex with dsDNA as compared with the free dye in solution. To expand the sensitivity of PG detection, we have studied the spectral properties of PG, both free and in complex with DNA in solution, when the fluorophore is in proximity to silver nanoparticles. We show that for a broad range of PG concentrations (20 pM-3.5 microM), it does not form dimers/oligomers and it exists in a monomeric state. On binding to DNA in the absence of silver, PG fluorescence increases approximately 1100-fold. Deposition of PG/DNA complex onto silver island films (SiFs) increases fluorescence approximately 7-fold due to the metal-enhanced fluorescence (MEF) effect, yielding fluorescence enhancement of 7700-fold as compared with the free dye on glass. In contrast to PG in complex with DNA, the free dye on SiFs demonstrates a decrease in brightness approximately 5-fold. Therefore, the total enhancement of PG on binding to DNA on silver reaches a value of approximately 38,000 as compared with free PG on SiFs. Consequently, the metal-enhanced detection of PG fluorescence is likely to find important utility for amplified dsDNA quantification.
Assuntos
DNA/análise , Prata/química , Pareamento de Bases , Vidro/química , Compostos Orgânicos/química , Concentração Osmolar , Fotodegradação , Soluções , Espectrometria de Fluorescência , Fatores de TempoRESUMO
The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found.
Assuntos
Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Sítios de Ligação , Desoxirribonuclease EcoRI , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Ligação Proteica , Espectrometria de Fluorescência , TriptofanoRESUMO
Phosphorescence and optically detected triplet state magnetic resonance (ODMR) spectroscopy studies of recA protein and its complexes with poly(5-HgU) and poly(dA-5BrdU) show that the two tryptophan residues are not involved in stacking interactions with the nucleotide bases of either single- or double-stranded polynucleotides. Solvent conditions which induce preferential binding to single-stranded ligands result in a shortening of the tyrosine phosphorescence lifetime, which is further reduced upon binding to poly(5-HgU). This suggests a change in the global conformation or self-aggregation state of the protein. Binding to poly(dA-5BrdU) induces small changes in the tryptophan zero field splittings of recA, but significant changes on those of 5BrdU, which are consistent with recA binding to the minor groove of the polynucleotide.
Assuntos
DNA de Cadeia Simples , DNA , Fenilalanina , Recombinases Rec A , Tirosina , Fenômenos Químicos , Físico-Química , Medições Luminescentes , Espectroscopia de Ressonância Magnética , Análise Espectral , Relação Estrutura-AtividadeRESUMO
We used intrinsic tryptophan fluorescence to study the nucleocapsid protein from human T-cell leukemia virus-type one, HTLV-1 p15, an 85-amino-acid protein with two Trp-containing zinc-finger motifs. Fluorescence spectra suggested an interaction between the two zinc fingers and another interaction involving the C-terminal tail and the zinc fingers. Titrations with nucleic acid revealed similar, sub-micromolar affinity for poly(dT) and poly(U) in 1 mM sodium phosphate, pH 7. Double-stranded DNA bound an order of magnitude weaker, suggesting helix-destabilizing activity. Base preference of p15 was T approximately U>I approximately C approximately G>A; affinity spanned about one order of magnitude. HTLV-1 p15 bound weaker and with less variation than reported values for either human or simian immunodeficiency virus homologues. The low affinity of p15 for nonspecific nucleic acids distinguishes it from other nucleocapsid proteins, and may suggest its involvement in additional steps of the virus life cycle other than RNA packaging.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano , Ácidos Nucleicos/química , Proteínas do Nucleocapsídeo/química , Proteínas dos Retroviridae/química , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Cloreto de Magnésio , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/isolamento & purificação , Fosfatos , Espectrometria de Fluorescência , Triptofano/química , Dedos de ZincoRESUMO
The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Ribonucleoproteínas/química , Triptofano/química , Tirosina/química , Sequência de Aminoácidos , DNA Helicases/química , Transferência de Energia , Polarização de Fluorescência , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Poli U/química , Conformação Proteica , Desnaturação Proteica , Proteínas de Ligação a RNA/química , Espectrometria de Fluorescência , Hormônios do Timo/químicaRESUMO
With a view toward further understanding the structure-function relationships of the eukaryotic heterogeneous ribonucleoprotein (hnRNP) A1, and in particular its multiplicity of nucleic acid-interactive domains, we have studied the nucleic acid binding properties of the globular N-domain (UP1) and sequence-repetitive, flexible C-domain, the thermal denaturation of UP1 and the concomitant effects of binding polynucleotide, and the self-associative properties of the full-length protein. Utilizing protein tryptophan fluorescence as a probe, polynucleotide binding was shown to stabilize UP1 against thermal unfolding. The denaturation profile of UP1-poly(thymidylic acid) complexes was biphasic, suggesting that unfolding of the two subdomains of UP1 can occur independently. This is in agreement with a previously proposed structure in which only one of the two UP1 subdomains binds the nucleic acid. The subdomains of UP1 can be prepared by controlled proteolysis of A1, further indicating that these two globular segments within A1 are connected by an exposed, flexible linkage. Circular dichroism measurements on UP1 confirm previous data that this portion of A1 binds single-stranded nucleic acids non-co-operatively. UP1 clearly shows a preference for single-stranded nucleic acids with a 2'-OH, since its affinity for poly(U) is three times higher than for poly(dU), and five times higher than its affinity for poly(2'-OCH3U). The nucleic acid-interactive properties of the C-domain were further examined by preparing a synthetic peptide polymer (M(r) approximately 12,000) containing about seven repeats of a 16-residue sequence, GNFGGGRGGNYGGSRG, which in turn comprises two copies of the C-terminal consensus, GN(F/Y)GG(G/S)RG. The polymer of this sequence exhibited significant affinity for the fluorescent polyribonucleotide, poly(ethenoadenylic acid), binding stoichiometrically at < or = 0.2 M-Na+. Complex formation was accompanied by an increase in aggregate formation, as indicated by the appearance of scattering. For purposes of comparison, the data were analyzed via the linear co-operative model of McGhee and von Hippel, though this model may not be fully descriptive of the protein-nucleic acid complex(es) formed in this case. In contrast to the non-co-operative binding mode of the UP1 domain, the C-polymer exhibited moderate co-operativity, comparable to that seen with full-length A1. Although addition of sufficient NaCl reversed the interaction, a sigmoidal binding isotherm could still be observed (with sufficient added polymer) at 0.8 M-NaCl. This suggests that non-electrostatic interactions contribute significantly to the free energy of binding.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , RNA Nuclear Heterogêneo/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Fluorescência , Fluorometria , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Poli A/metabolismo , Polinucleotídeos/química , Ligação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , RNA Nuclear Heterogêneo/química , Ribonucleoproteínas/química , Soluções , Temperatura , TirosinaRESUMO
HIV-1 nucleocapsid protein (NCp7) is a double zinc-fingered protein that has been traditionally implicated in viral RNA recognition and packaging, in addition to its tight association with genomic RNA and tRNA primer within the virion nucleocapsid. The availability of large quantities of viral or recombinant wild-type NCp7 and mutant p7 has made possible the assignment of the different roles that structural motifs within the protein play during RNA binding. At low ionic strength binding to the homopolymeric fluorescent RNA, poly(epsilonA), is electrostatically driven and four sodium ions are displaced. Arg7 in the flanking N-terminal region, Lys20 and Lys26 in the first zinc finger and one positively charged residue (attributed to Lys41) in the second zinc finger are involved in electrostatic contacts with RNA. The p7 zinc fingers do not function independently but concomitantly. The first zinc finger (both isolated or in the context of the full-length protein) has a more prominent electrostatic interaction than the second one. The second zinc finger dominates the non-electrostatic stabilization of the binding to RNA due to stacking of its Trp residue with nucleic acid bases. Mutations in the highly conserved retroviral Zn-coordinating residues (CCHC) to steroid hormone receptor (CCCC) or transcription factor (CCHH) metal cluster types do not affect RNA binding. In spite of the limited impact in RNA binding affinity in vitro or RNA packaging in vivo that such mutations or structural alterations impart, they impair or abolish virus infectivity. It is likely that such an effect stems from the involvement of NCp7 in crucial steps of the virus life cycle other than RNA binding.
Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , HIV-1 , Poli A/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais , Dedos de Zinco , Sequência de Aminoácidos , Sítios de Ligação , Capsídeo/genética , Corantes Fluorescentes/metabolismo , Produtos do Gene gag/genética , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/genética , Sequências Repetitivas de Aminoácidos , Termodinâmica , Dedos de Zinco/genética , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
The three-dimensional solution structure of circulin A, a 30 residue polypeptide from the African plant Chassalia parvifolia, has been determined using two-dimensional 1H-NMR spectroscopy. Circulin A was originally identified based upon its inhibition of the cytopathic effects and replication of the human immunodeficiency virus. Structural restraints consisting of 369 interproton distances inferred from nuclear Overhauser effects, and 21 backbone dihedral and nine chi1 angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimisation in the program X-PLOR. The final set of 12 structures had mean pairwise rms differences over the whole molecule of 0.91 A for the backbone atom, and 1.68 A for all heavy atoms. For the well-defined region encompassing residues 2-12 and 18-27, the corresponding values were 0.71 and 1.66 A, respectively. Circulin A adopts a compact structure consisting of beta-turns and a distorted segment of triple-stranded beta-sheet. Fluorescence spectroscopy provided additional evidence for a solvent-exposed Trp residue. The molecule is stabilised by three disulfide bonds, two of which form an embedded loop completed by the backbone fragments connecting the cysteine residues. A third disulfide bond threads through the centre of this loop to form a "cystine-knot" motif. This motif is present in a range of other biologically active proteins, including omega-contoxin GVIA and Cucurbita maxima trypsin inhibitor. Circulin A belongs to a novel class of macrocyclic peptides which have been isolated from plants in the Rubiaceae family. The global fold of circulin A is similar to kalata B1, the only member of this class for which a structure has previously been determined.
Assuntos
Fármacos Anti-HIV/química , Ciclotídeos , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/química , Peptídeos/química , Conformação Proteica , Sequência de Aminoácidos , Dissulfetos , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Soluções , Espectrometria de FluorescênciaRESUMO
In this paper, the steps required to validate a liquid chromatography peptide mapping method with mass spectrometric detection (LC-MS) for use as an identity test and characterization tool are presented. All aspects of peptide mapping are evaluated and optimized, including protein sample preparation (protein reduction, alkylation and enzymatic digestion), high performance liquid chromatography (HPLC) separation of the resulting peptides, and the use of a mass spectrometric detection. In addition, the validation of a single quadruple MS detector is described and the implementation of on-line electrospray ionization MS (ESI-MS) as an adjunct detector to support the investigation of peak differences is presented. Applications of peptide mapping with tandem MS using an electrospray ion-trap instrument throughout the biopharmaceutical product development cycle are discussed, including assessing protein product heterogeneity derived from post-translational modifications (e.g. multiple N- or C-termini, deamidation, oxidation and glycosylation) and protein degradation.
Assuntos
Mapeamento de Peptídeos , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização por Electrospray , Preparações Farmacêuticas/química , Sensibilidade e EspecificidadeRESUMO
The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.
Assuntos
DNA/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , DNA/química , Eletroforese , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Mutação Puntual , Proteínas Recombinantes/metabolismo , Proteínas Oncogênicas de Retroviridae/química , Espectrometria de Fluorescência , TermodinâmicaRESUMO
A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV-1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole-killed virus vaccine formulation that exhibits unaffected surface antigenic properties.
Assuntos
Fármacos Anti-HIV/química , Proteínas do Capsídeo , Proteínas dos Retroviridae/antagonistas & inibidores , Proteínas Virais , Dedos de Zinco/efeitos dos fármacos , Fármacos Anti-HIV/farmacologia , Capsídeo/antagonistas & inibidores , Capsídeo/química , Capsídeo/metabolismo , Dissulfetos/química , Dissulfetos/farmacologia , Eletroquímica , Produtos do Gene gag/antagonistas & inibidores , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Humanos , Cinética , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Oxirredução , Relação Quantitativa Estrutura-Atividade , Proteínas dos Retroviridae/química , Proteínas dos Retroviridae/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
Analysis of fluorimetric equilibrium-binding isotherms of a proteolytic fragment of bacteriophage T4 gene 32 protein (g32P) lacking residues 1-9 shows that this region contains the site responsible for the function of the NH2-terminal 'B' domain (residues 1-21). The end codon of the frameshift mutant g32P-PR201 has been identified as TAG at nucleotide position 852. The PR201 gene 32 product ends at Ser283 and carries a truncated COOH-terminal 'A' domain (residues 253-301). Fluorimetric titrations of g32P-PR201 with double-stranded DNA show that the functional residues of the A domain are located within the region spanning residues 284-301.
Assuntos
Fagos T/genética , Proteínas Virais/metabolismo , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão , DNA Viral/metabolismo , Fluorescência , Espectrofotometria Ultravioleta , Fagos T/metabolismo , Proteínas Virais/genéticaRESUMO
A polypeptide containing the amino-terminal region of ACE1 (residues 1-122; 122*), the activator of yeast Cu-metallothionein gene transcription, shows charge-transfer and metal-centered UV absorption bands, and orange luminescence which are characteristic of Cu-cysteinyl thiolate cluster structures. These spectral features are abolished by the Cu(I) complexing agents CN- and diethyldithiocarbamate or exposure to acid, but not by the Cu(II) chelator, EDTA. Binding of the polypeptide to its specific DNA recognition site, but not to calf-thymus double-stranded DNA, induces quenching of its Tyr and Cu-S cluster luminescence emission. The CD spectrum is characteristic of a tightly folded structure that may be organized around the Cu cluster.
Assuntos
Cobre/análise , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Genes Fúngicos , Luminescência , Metalotioneína/genética , Dados de Sequência Molecular , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/análiseRESUMO
Zinc finger (ZF) domains in retroviral nucleocapsid proteins usually contain one histidine per metal ion coordination complex (Cys-X(2)-Cys-X(4)-His-X(4)-Cys). Visna virus nucleocapsid protein, p8, has two additional histidines (in the second of its two ZFs) that could potentially bind metal ions. Absorption spectra of cobalt-bound ZF2 peptides were altered by Cys alkylation and mutation, but not by mutation of the extra histidines. Our results show that visna p8 ZFs involve three Cys and one His in the canonical spacing in metal ion coordination, and that the two additional histidines appear to interact with nucleic acid bases in p8-DNA complexes.
Assuntos
DNA Viral/metabolismo , Nucleocapsídeo/química , Nucleocapsídeo/metabolismo , Vírus Visna-Maedi/metabolismo , Alquilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cisteína/química , Histidina/química , Técnicas In Vitro , Cinética , Metais/metabolismo , Nucleocapsídeo/genética , Ovinos , Espectrometria de Fluorescência , Espectrofotometria , Vírus Visna-Maedi/genética , Dedos de Zinco/genéticaRESUMO
Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single-stranded polynucleotides with both E. coli single-stranded DNA-binding protein and an E. coli ssb gene product in which Trp-54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp-54 in stabilizing the protein-nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Polinucleotídeos/metabolismo , Proteínas de Ligação a DNA/fisiologia , Luminescência , Análise Espectral/métodosRESUMO
The binding of both wild-type and point-mutated E. coli single-stranded DNA-binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy. Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies. Investigation of a point mutation in the E. coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe-60 is replaced by alanine strongly suggests the participation of Phe-60 in the binding process, possibly by the formation of an extended stacking structure by Trp-54, thymine and Phe-60. This hypothesis is supported by results on the point mutations in which His-55 is replaced by either leucine or tyrosine.
Assuntos
Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli , Fenilalanina , Poli T/metabolismo , Espectrometria de Fluorescência , Relação Estrutura-Atividade , TriptofanoRESUMO
The photoexcited triplet state of Trp-37 in the C-terminal zinc finger of the HIV-1 p7 nucleocapsid protein was used as a probe of p7 interactions with the heavy atom-derivatized RNA homopolymer, poly-5-mercuriuridylic acid (5-HgU). Binding of p7 to 5-HgU (Hg blocked with 2-mercaptoethanol) produces an external heavy atom effect (HAE) on Trp-37 characterized by fluorescence quenching, reduction of the phosphorescence lifetime by three orders of magnitude, and the appearance of the D+E phosphorescence-detected ODMR signal, absent in unperturbed Trp, but induced by a HAE. The details of the HAE are consistent with out-of-plane van der Waals contact of Hg with the indole chromophore of Trp-37. Steric requirements suggest further that the Trp-RNA contact occurs via an aromatic stacking interaction.
Assuntos
Proteínas do Capsídeo , Produtos do Gene gag/metabolismo , Poli U/metabolismo , Proteínas Virais , Sequência de Aminoácidos , Produtos do Gene gag/química , HIV-1/metabolismo , Luminescência , Imageamento por Ressonância Magnética , Dados de Sequência Molecular , Poli U/química , RNA Viral/metabolismo , Triptofano/metabolismo , Dedos de Zinco/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
HIV-1 nucleocapsid, p7, contains two retroviral zinc fingers, which are both necessary for efficient packaging of genomic RNA and infectivity. The nucleocapsid protein is bound tightly to genomic RNA in the mature virion. In this study, the effect of p7 on polymerization of nascent cDNA by viral reverse transcriptase (RT) was examined. An 874-base RNA of HIV-1 was synthesized and used as a template in RT assays with varying concentrations of intact p7, mutants of p7 that have transposed or repeated zinc fingers, and several different peptides that represent various structural regions of p7. Results indicate that at greater than or equal to 50% saturation of p7-binding sites, with p7, there is up to a 90% reduction in total cDNA synthesis, as measured by nucleotide incorporation. However, the cDNA products that are made are almost exclusively full length. Three zinc finger mutants exhibited effects similar to those of wild-type p7. N-terminal and C-terminal halves of p7 inhibited total nucleotide incorporation, but also inhibited synthesis of long cDNA products by RT. In the absence of p7 an array of short transcripts (< 200 bases) was produced by RT. These studies show that full-length p7 is necessary to increase the proportion of long cDNA transcripts produced by RT. The relative position of the two zinc fingers is not critical for this effect.