RESUMEN
Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental pKa values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (â¼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with pKas above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical pKa values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.
Asunto(s)
Fibroínas , Arañas , Animales , Fibroínas/química , Seda/química , Dimerización , Espectroscopía de Resonancia Magnética , Arañas/metabolismoRESUMEN
INTRODUCTION: Sensitive methods are necessary to identify the residual structure in an unfolded protein, which may be similar to the functionally native structure. Signal intensity in NMR experiments is useful for analyzing the line width for a dynamic structure; however, another contribution is contained. METHODS: Here, the signal-intensity difference along the sequence was used for probability to calculate the standard deviation. RESULTS: The relative values of the standard deviations were 0.57, 0.57, and 0.66 for alpha-synuclein wild-type, A53T, and A30P, respectively. This revealed that the flexible region was mainly in the Cterminal region of alpha-synuclein at higher temperatures as observed by the amide-proton exchange studies. CONCLUSION: In particular, the flexible structure was induced by the A30P mutation.
Asunto(s)
alfa-Sinucleína , alfa-Sinucleína/química , Espectroscopía de Resonancia Magnética , MutaciónRESUMEN
HIV-1 capsid is comprised of over a hundred p24 protein molecules, arranged as either pentamers or hexamers. Three p24 mutants with amino acid substitutions in capsid N-terminal domain protein were examined: G60W (α3-4 loop), M68T (helix 4), and P90T (α4-5 loop), which exhibited no viability for biological activity. One common structural feature of the three p24 N-domain mutants, examined by NMR, was the long-range effect of more ß-structures at the ß2-strand in the N-terminal region compared with the wild-type. In addition, the presence of fewer helical structures was observed in M68T and P90T, beyond the broad area from helix 1 to the C-terminal part of helix 4. This suggests that both N-terminal beta structures and helices play important roles in the formation of p24 hexamers and pentamers. Next, compared with P90T, we examined cis-conformation or trans-conformation of wild-type adopted by isomerization at G89-P90. Since P90T mutant adopts only a trans-conformation, comparison of chemical shifts and signal intensities between each spectra revealed that the major peaks (about 85%) in the spectrum of wild-type correspond to trans-conformation. Furthermore, it was indicated that the region in cis-conformation (minor; 15%) was more stabilized than that observed in trans-conformation, based on the analyses of heteronuclear Overhauser effect as well as the order-parameter. Therefore, it was concluded that the cis-conformation is more favorable than the trans-conformation for the interaction between the p24 N-terminal domain and cyclophilin-A. This is because HIV-1 with a P90T protein, which adopts only a trans-conformation, is associated with non-viability of biological activity.
Asunto(s)
Sustitución de Aminoácidos , Proteína p24 del Núcleo del VIH/química , VIH-1/química , Modelos Moleculares , Mutación Missense , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Conformación Proteica en Hélice alfa , Dominios ProteicosRESUMEN
Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy-in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.
Asunto(s)
Aptámeros de Nucleótidos/química , G-Cuádruplex , Luminol/metabolismo , Mioglobina/metabolismo , Peroxidasa/metabolismo , Aptámeros de Nucleótidos/metabolismo , Simulación por Computador , Hemo/metabolismo , Luminiscencia , Luminol/química , Resonancia Magnética Nuclear Biomolecular , Técnica SELEX de Producción de Aptámeros , Especificidad por SustratoRESUMEN
Lanthanide ions (Ln3+) show similar physicochemical properties in aqueous solutions, wherein they exist as + 3 cations and exhibit ionic radii differences of less than 0.26 Å. A flexible linear peptide lanthanide binding tag (LBT), which recognizes a series of 15 Ln3+, shows an interesting characteristic in binding specificity, i.e., binding affinity biphasically changes with an increase in the atomic number, and shows a greater than 60-fold affinity difference between the highest and lowest values. Herein, by combining experimental and computational investigations, we gain deep insight into the reaction mechanism underlying the specificity of LBT3, an LBT mutant, toward Ln3+. Our results clearly show that LBT3-Ln3+ binding can be divided into three, and the large affinity difference is based on the ability of Ln3+ in a complex to be directly coordinated with a water molecule. When the LBT3 recognizes a Ln3+ with a larger ionic radius (La3+ to Sm3+), a water molecule can interact with Ln3+ directly. This extra water molecule infiltrates the complex and induces dissociation of the Asn5 sidechain (one of the coordinates) from Ln3+, resulting in a destabilizing complex and low affinity. Conversely, with recognition of smaller Ln3+ (Sm3+ to Yb3+), the LBT3 completely surrounds the ions and constructs a stable high affinity complex. Moreover, when the LBT3 recognizes the smallest Ln3+, namely Lu3+, although it completely surrounds Lu3+, an entropically unfavorable phenomenon specifically occurs, resulting in lower affinity than that of Yb3+. Our findings will be useful for the design of molecules that enable the distinction of sub-angstrom size differences.
Asunto(s)
Cationes/química , Elementos de la Serie de los Lantanoides/química , Simulación de Dinámica Molecular , Péptidos/química , Sitios de Unión , Calorimetría/métodos , Cationes/metabolismo , Cristalografía por Rayos X , Elementos de la Serie de los Lantanoides/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Estructura Molecular , Péptidos/metabolismo , Termodinámica , Agua/químicaRESUMEN
Spider dragline silk is well recognized due to its excellent mechanical properties. Dragline silk protein mainly consists of two proteins, namely, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). The MaSp N-terminal domain (NTD) conformation displays a strong dependence on ion and pH gradients, which is crucial for the self-assembly behavior of spider silk. In the spider major ampullate gland, where the pH is neutral and concentration of NaCl is high, the NTD forms a monomer. In contrast, within the spinning duct, where pH becomes more acidic (to pH ~ 5) and the concentration of salt is low, NTD forms a dimer in antiparallel orientation. In this study, we report near-complete backbone and side chain chemical shift assignment of the monomeric form of NTD of MaSp2 from Nephila clavipes at pH 7 in the presence of 300 mM NaCl. Our NMR data demonstrate that secondary structure of monomeric form of NTD MaSp2 consists of five helix regions.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Fibroínas/química , Espectroscopía de Protones por Resonancia Magnética , Arañas/metabolismo , Secuencia de Aminoácidos , Estructuras Animales , Animales , Concentración de Iones de Hidrógeno , Isótopos de Nitrógeno , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
The residual solution structures of two alpha-synuclein mutants, A30P and A53T, observed in family members of patients with Parkinson's disease were compared with that of wild-type by NMR. The A53T substitution had been shown to accelerate fibril formation of alpha-synuclein, whereas the A30P mutation has the negative and positive effects on the formation of the fibril and spherical oligomer, respectively. The remaining structure was analyzed via amide-proton exchange and signal intensity measurements using NMR. Amide-proton exchange was used for both the calculation of kex values and ratio of kex at different temperatures. Effects of the A30P (N-terminal region) mutation were observed at the C-terminal region as a more flexible structure, suggesting that long-range interactions exist between the N- and C-terminal regions in alpha-synuclein. In addition, the N-terminal region adopted a more rigid structure in the A53T and A30P mutants than in the wild-type. It was concluded that the structural change caused by the mutations is related to the formation of a beta-hairpin at the initiation site of the N-terminal core structure. Furthermore, the signal intensity was used to estimate the rigidity of the structure. Higher signal intensities were observed for A30P at the 112, 113, and 116 C-terminal residues, suggesting that this region adopts more flexible structure. The ratio of the intensities at different temperatures indicated more flexible or rigid structures in the N-terminal region of A30P than in that of wild-type. Thus, using different approaches and temperatures is a good method to analyze residual structure in intrinsically disordered proteins.
Asunto(s)
Amidas/química , Proteínas Intrínsecamente Desordenadas/genética , Protones , alfa-Sinucleína/química , Humanos , Proteínas Intrínsecamente Desordenadas/química , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Proteínas Mutantes/química , Mutación , Enfermedad de Parkinson/genética , Estructura Secundaria de Proteína , Temperatura , alfa-Sinucleína/genéticaRESUMEN
The effect of ions on the structure and dynamics of a spider silk protein is elucidated. Chaotropic ions prevent intra- and inter-molecular interactions on the repetitive domain, which are required to maintain the solubility, while kosmotropic ions promote hydrogen bond interactions in the glycine-rich region, which are a prerequisite for ß-sheet formation.
Asunto(s)
Conformación Proteica en Lámina beta , Seda/química , Animales , Cloruros/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Sodio/química , Solubilidad , ArañasRESUMEN
The objective of this study is to control and elucidate the mechanism of molecular degradation in a polysaccharide hydrogel. Glycidyl methacrylate (GMA) immobilized dextran (Dex-GMA) was oxidized by periodate to introduce aldehyde groups (oxidized Dex-GMA). The hydrogel was formed by the addition of dithiothreitol to the oxidized Dex-GMA solution through thiol Michael addition with the preservation of the aldehyde group for degradation points. It was experimentally determined that the degradation of this hydrogel can be controlled by the addition of amino groups and the speed of degradation can be controlled independently of mechanical properties because crosslinking and degradation points are different. In addition, the molecular mechanism of the crosslinking between the thiol and aldehyde groups was found to control the degradation of dextran derivatives. It is expected that these results will be beneficial in the design of polymer materials in which the speed of degradation can be precisely controlled. In addition, the cytotoxicity of oxidized Dex-GMA was approximately 3000 times lower than that of glutaraldehyde. The low cytotoxicity of the aldehyde in oxidized Dex-GMA was the likely reason for the harmless functionalized polysaccharide material. Possible future clinical applications include cell scaffolds in regenerative medicine and carriers for drug delivery systems.
RESUMEN
The ß-sheet is the key structure underlying the excellent mechanical properties of spider silk. However, the comprehensive mechanism underlying ß-sheet formation from soluble silk proteins during the transition into insoluble stable fibers has not been elucidated. Notably, the assembly of repetitive domains that dominate the length of the protein chains and structural features within the spun fibers has not been clarified. Here we determine the conformation and dynamics of the soluble precursor of the repetitive domain of spider silk using solution-state NMR, far-UV circular dichroism and vibrational circular dichroism. The soluble repetitive domain contains two major populations: ~65% random coil and ~24% polyproline type II helix (PPII helix). The PPII helix conformation in the glycine-rich region is proposed as a soluble prefibrillar region that subsequently undergoes intramolecular interactions. These findings unravel the mechanism underlying the initial step of ß-sheet formation, which is an extremely rapid process during spider silk assembly.
Asunto(s)
Conformación Proteica en Lámina beta/fisiología , Seda/química , Arañas/metabolismo , Animales , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Estrés Mecánico , Resistencia a la Tracción/fisiologíaRESUMEN
The increasing demand for rare earth (RE) elements in advanced materials for permanent magnets, rechargeable batteries, catalysts and lamp phosphors necessitates environmentally friendly approaches for their recovery and separation. Here, we propose a mineralization concept for direct extraction of RE ions with Lamp (lanthanide ion mineralization peptide). In aqueous solution containing various metal ions, Lamp promotes the generation of RE hydroxide species with which it binds to form hydrophobic complexes that accumulate spontaneously as insoluble precipitates, even under physiological conditions (pH â¼6.0). This concept for stabilization of an insoluble lanthanide hydroxide complex with an artificial peptide also works in combination with stable scaffolds like synthetic macromolecules and proteins. Our strategy opens the possibility for selective separation of target metal elements from seawater and industrial wastewater under mild conditions without additional energy input.
RESUMEN
To test the existence of the salt bridge and stability of the HIV-1 p17 matrix protein, an E12A (mutated at helix 1) was established to abolish possible electrostatic interactions. The chemical shift perturbation from the comparison between wild type and E12A suggested the existence of an electrostatic interaction in wild type between E12 and H89 (located in helix 4). Unexpectedly, the studies using urea denaturation indicated that the E12A substitution slightly stabilized the protein. The dynamic structure of E12A was examined under physiological conditions by both amide proton exchange and relaxation studies. The quick exchange method of amide protons revealed that the residues with faster exchange were located at the mutated region, around A12, compared to those of the wild-type protein. In addition, some residues at the region of helix 4, including H89, exhibited faster exchange in the mutant. In contrast, the average values of the kinetic rate constants for amide proton exchange for residues located in all loop regions were slightly lower in E12A than in wild type. Furthermore, the analyses of the order parameter revealed that less flexible structures existed at each loop region in E12A. Interestingly, the structures of the regions including the alpha1-2 loop and helix 5 of E12A exhibited more significant conformational exchanges with the NMR time-scale than those of wild type. Under lower pH conditions, for further destabilization, the helix 1 and alpha2-3 loop in E12A became more fluctuating than at physiological pH. Because the E12A mutant lacks the activities for trimer formation on the basis of the analytical ultra-centrifuge studies on the sedimentation distribution of p17 (Fledderman et al. Biochemistry 49, 9551-9562, 2010), it is possible that the changes in the dynamic structures induced by the absence of the E12-H89 interaction in the p17 matrix protein contributes to a loss of virus assembly.
Asunto(s)
Glutamina/química , Antígenos VIH/química , Histidina/química , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Conformación Proteica , Termodinámica , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Amidas , Codón , Glutamina/genética , Antígenos VIH/genética , Histidina/genética , Humanos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Protones , Relación Estructura-Actividad , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Human mitochondrial dysfunction can lead to severe and often deadly diseases, for which there are no known cures. Although the targeted delivery of therapeutic gene to mitochondria is a promising approach to alleviate these disorders, gene carrier systems for the selective delivery of functional DNA into the mitochondria of living mammalian cells are currently unavailable. Here we rationally developed dual-domain peptides containing DNA-condensing/cell-penetrating/endosome-disruptive and mitochondria-targeting sequences. Secondary structures of the dual-domain peptides were analyzed, and variations in the physicochemical properties (stability, size, and ζ potential) of peptide/DNA complexes were studied as a function of peptide-to-DNA ratio and serum addition. An optimized formulation, identified through qualitative and quantitative studies, fulfills the fundamental prerequisites for mitochondria-specific DNA delivery, successfully transfecting a high proportion (82 ± 2%) of mitochondria in a human cell line with concomitant biocompatibility. Nuclear magnetic resonance studies confirmed the effectiveness of our bipartite peptide design with segregated functions: a helical domain necessary for mitochondrial import and an unstructured region for interaction with DNA involving lysine residues. Further analyses revealed that the lysine-specific interaction assisted the self-organization of the peptide and the DNA cargo, leading to a structural arrangement within the formed complex that is crucial for its biological efficiency. Thus the reported gene vector represents a new and reliable tool to uncover the complexity of mitochondrial transfection.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética , Mitocondrias/genética , Péptidos/genética , Endosomas/química , Endosomas/genética , Vectores Genéticos , Humanos , Mitocondrias/patología , Péptidos/química , TransfecciónRESUMEN
The stereochemical theory claims that primitive coded translation initially occurred in the RNA world by RNA-directed amino acid coupling. In this study, we show that the HIV Tat aptamer RNA is capable of recognizing two consecutive arginine residues within the Tat peptide, thus demonstrating how RNA might be able to position two amino acids for sequence-specific coupling. We also show that this RNA can act as a template to accelerate the coupling of a single arginine residue to the N-terminal arginine residue of a peptide primer. The results might have implications for our understanding of the origin of translation.
Asunto(s)
Arginina/metabolismo , ARN Viral/metabolismo , Secuencia de Aminoácidos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Arginina/química , Sitios de Unión , VIH/genética , VIH/metabolismo , Humanos , Conformación de Ácido Nucleico , Péptidos/química , Péptidos/metabolismo , ARN Viral/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
A modular structure of ribonucleopeptide (RNP) affords a framework to construct macromolecular receptors and fluorescent sensors. We have isolated ATP-binding RNP with the minimum of nucleotides for ATP binding, in which the RNA consensus sequence is different from those reported for RNA aptamers against the ATP analogues. The three-dimensional structure of the substrate-binding complex of RNP was studied to understand the ATP-binding mechanism of RNP. A combination of NMR measurements, enzymatic and chemical mapping, and nucleotide mutation studies of the RNP-adenosine complex show that RNP interacts with the adenine ring of adenosine by forming a U:A:U triple with two invariant U nucleotides. The observed recognition mode for the adenine ring is different from those of RNA aptamers for ATP derivatives reported previously. The RNP-adenosine complex is folded into a particular structure by formation of the U:A:U triple and a Hoogsteen type A:U base pair. This recognition mechanism was successfully utilized to convert the substrate-binding specificity of RNP from ATP- to GTP-binding with a C(+):G:C triple recognition mode.
Asunto(s)
Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Aptámeros de Nucleótidos/química , Espectroscopía de Resonancia Magnética , Conformación ProteicaRESUMEN
The sheath of Leptothrix cholodnii is constructed from a structural glycoconjugate, a straight-chained amphoteric heteropolysaccharide modified with glycine and cysteine. Though the structure of the glycan core is already determined, its modifications with amino acids and other molecules are not fully resolved. In this study, we aimed to determine the chemical structure of the glycoconjugate as a whole. Enantiomeric determination of cysteine in the sheath was performed and as a result, L-cysteine was detected. NMR spectroscopy was endeavored to determine overall structure of the glycoconjugate. Prior to NMR analysis, solubilization of the glycoconjugate was attempted by adding denaturing reagents or by derivatization. As far as tested, sulfonation by performic acid oxidation was suitable for solubilization, but further improvement was achieved by N-acetylation. The approximate molecular weight of the derivative was estimated to be 4.5 x 10(4) by size-exclusion chromatography. The NMR studies for the sulfonated glycoconjugate and its N-acetylated derivative revealed that the sheath glycoconjugate is a glycosaminoglycan consisting of a pentasaccharide repeating unit which is substoichiometrically esterified with 3-hydroxypropionic acid and stoichiometrically amidated with acetic acid and glycyl-L-cysteine.
Asunto(s)
Glicoconjugados/química , Leptothrix/química , Acetilación , Aminoácidos/análisis , Cisteína/química , Glicoconjugados/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Solubilidad , Estereoisomerismo , Sulfatos/metabolismoRESUMEN
Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) is known to play a role in intrinsic cellular immunity against human immunodeficiency virus type 1 (HIV-1). The antiretroviral activity of APOBEC3G is associated with hypermutation of viral DNA through cytidine deamination. APOBEC3G contains two cytidine deaminase domains that are characterized by a highly conserved zinc-coordinating motif. It is known that only the C-terminal domain of APOBEC3G (c-APOBEC3G) is involved in the catalytic activity. Here, we present the solution structure and the interaction with single-stranded DNA of c-APOBEC3G. Furthermore, we have succeeded for the first time in monitoring the deamination reaction of c-APOBEC3G in real-time using NMR signals. The monitoring has demonstrated that the deamination reaction occurs in a strict 3'-->5'
Asunto(s)
Citidina Desaminasa/química , ADN de Cadena Simple/química , Desaminasa APOBEC-3G , VIH-1/genética , Humanos , Cinética , Resonancia Magnética Nuclear BiomolecularRESUMEN
A ribonucleopeptide aptamer against ATP was obtained by the in vitro selection method. This ribonucleopeptide aptamer comprises a randomized and selected RNA linked to the Rev-responsive element (RRE) in complex with a peptide derived from an HIV Rev protein. The ribonucleopeptide aptamer selectively binds ATP in the presence of the Rev-derived peptide, exclusively. Here, we present the structural analysis of the ribonucleopeptide aptamer with NMR. The secondary structure of the RNA part of the aptamer, the selected RNA region linked to RRE, in the presence of the Rev-derived peptide was determined in an Ado-bound form. G:A and G:G base pairs, together with canonical base pairs, are formed in a duplex of RRE. The selected RNA region plays a crucial role in target binding. It has been found that the two U residues located in the selected RNA region trap Ado through the formation of the U:A:U base triple. This was directly confirmed by the HNN-COSY experiment through the detection of spin-spin couplings across the hydrogen bonds for Watson-Crick and Hoogsteen A:U base pairs in the U:A:U base triple.
Asunto(s)
Adenosina Trifosfato/metabolismo , Aptámeros de Nucleótidos/química , ARN/química , Aptámeros de Nucleótidos/metabolismo , Conformación de Ácido Nucleico , Péptidos/química , Elementos de Respuesta , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
RNA aptamers against bovine prion protein (bPrP) were obtained, most of the obtained aptamers being found to contain the r(GGAGGAGGAGGA) (R12) sequence. Then, it was revealed that R12 binds to both bPrP and its beta-isoform with high affinity. Here, we present the structure of R12. This is the first report on the structure of an RNA aptamer against prion protein. R12 forms an intramolecular parallel quadruplex. The quadruplex contains G:G:G:G tetrad and G(:A):G:G(:A):G hexad planes. Two quadruplexes form a dimer through intermolecular hexad-hexad stacking. Two lysine clusters of bPrP have been identified as binding sites for R12. The electrostatic interaction between the uniquely arranged phosphate groups of R12 and the lysine clusters is suggested to be responsible for the affinity of R12 to bPrP. The stacking interaction between the G:G:G:G tetrad planes and tryptophan residues may also contribute to the affinity. One R12 dimer molecule is supposed to simultaneously bind the two lysine clusters of one bPrP molecule, resulting in even higher affinity. The atomic coordinates of R12 would be useful for the development of R12 as a therapeutic agent against prion diseases and Alzheimer's disease.
Asunto(s)
Aptámeros de Nucleótidos/química , G-Cuádruplex , Priones/química , Animales , Sitios de Unión , Bovinos , Dimerización , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Priones/antagonistas & inhibidores , Priones/metabolismoRESUMEN
Nuclear magnetic resonance (NMR) studies have shown that RNA/DNA oligomers with GGA repeat sequences contain unique G-quadruplex structures in the presence of K(+) or Na(+) ions. In this study, we used microchip electrophoresis to study the structure of an RNA aptamer against bovine prion protein that possessed four GGA-triplet repeats (wt2). We analyzed the structural changes and characterized dimer formation of the aptamer. Mutational, circular dichroism, and one-dimensional NMR studies of wt2 revealed that K(+) ions induce wt2 to assume a thermostable dimer in an intramolecular G-quadruplex with parallel orientation.