RESUMEN
Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach, crosslinking of segmentally isotope-labeled RNA and tandem mass spectrometry (CLIR-MS/MS), to localize protein-RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on polypyrimidine tract binding protein 1 and U1 small nuclear RNP. Our method provides distance restraints to support integrative atomic-scale structural modeling and to gain mechanistic insights into RNP-regulated processes.
Asunto(s)
Ribonucleoproteínas Nucleares Heterogéneas/química , Modelos Moleculares , Conformación de Ácido Nucleico , Proteína de Unión al Tracto de Polipirimidina/química , ARN/química , Ribonucleoproteína Nuclear Pequeña U1/química , Sitios de Unión , Isótopos de Carbono , Cromatografía Líquida de Alta Presión , Ribonucleoproteínas Nucleares Heterogéneas/genética , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Proteína de Unión al Tracto de Polipirimidina/genética , Unión Proteica , Ribonucleoproteína Nuclear Pequeña U1/genética , Programas Informáticos , Espectrometría de Masas en Tándem , Rayos UltravioletaRESUMEN
The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121-230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrP(C)) that is formed by the C-terminal part of helix 3, with residues 218-230, and a loop of residues 166-172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized "protein X," and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121-230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized "conformational markers" could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166-172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166-172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).
Asunto(s)
Variación Genética , Priones/química , Secuencia de Aminoácidos , Animales , Bovinos , Cricetinae , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Priones/genética , Isoformas de Proteínas/química , Estructura Secundaria de Proteína , OvinosRESUMEN
A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly 15N, 13C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable 15N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse 15N relaxation rates of unfolded polypeptides in high resolution constant-time [1H, 15N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the 15N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence.
Asunto(s)
Isótopos de Carbono , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Péptidos/química , Humanos , Movimiento (Física) , Priones/química , Prolina/química , Estructura Secundaria de ProteínaRESUMEN
The NMR structures of the recombinant human prion protein, hPrP(23-230), and two C-terminal fragments, hPrP(90-230) and hPrP(121-230), include a globular domain extending from residues 125-228, for which a detailed structure was obtained, and an N-terminal flexibly disordered "tail." The globular domain contains three alpha-helices comprising the residues 144-154, 173-194, and 200-228 and a short anti-parallel beta-sheet comprising the residues 128-131 and 161-164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167-171, the residues 187-194 at the end of helix 2, and the residues 219-228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187-193 in helix 2 and 219-226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23-230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167-171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.
Asunto(s)
Priones/química , Animales , Clonación Molecular , Cricetinae , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Genes Bacterianos , Complejos Multienzimáticos/genética , Familia de Multigenes , Oxidorreductasas/genética , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Transactivadores , Factores de Transcripción/metabolismo , Aerobiosis , Secuencia de Aminoácidos , Anaerobiosis , Proteínas Bacterianas/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Hierro-Azufre/genética , Datos de Secuencia Molecular , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupos CH-NH2 , Control Biológico de Vectores , Enfermedades de las Plantas/microbiología , Plantas Tóxicas , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genética , Homología de Secuencia de Aminoácido , Nicotiana/microbiología , Factores de Transcripción/genéticaRESUMEN
An efficient method is presented for the production of intact mammalian prion proteins and partial sequences thereof. As an illustration we describe the production of polypeptides comprising residues 23-231, 81-231, 90-231 and 121-231 of the human prion protein (hPrP). Polypeptides were expressed as histidine tail fusion proteins into inclusion bodies in the cytoplasm of Escherichia coli and refolded and oxidized while N-terminally immobilized on a nickel-NTA agarose resin. This 'high-affinity column refolding' facilitates the preparation of prion proteins by preventing protein aggregation and intermolecular disulfide formation. After elution from the resin the histidine tail can be removed using thrombin without cleaving the prion protein polypeptide chain. The same protocol as used here for hPrP has been successfully applied with bovine and murine prion proteins. The protein preparations are stable for weeks at room temperature in concentrated solution and are thus suitable for detailed structural studies. Preliminary biophysical characterization of hPrP(23-231) suggests that the C-terminal half of the polypeptide chain forms a well-structured globular domain, and that the N-terminal half does not form extensive regular secondary structures.