RESUMEN
Maintenance of ploidy depends on the mitotic kinase Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC) whose proficient activity is supported by HP1 enriched at inner centromeres. HP1 is known to associate with INCENP of the CPC in a manner that depends on the PVI motif conserved across HP1 interactors. Here, we found that the interaction of INCENP with HP1 requires not only the PVI motif but also its C-terminally juxtaposed domain. Remarkably, these domains conditionally fold the ß-strand (PVI motif) and the α-helix from a disordered sequence upon HP1 binding and render INCENP with high affinity to HP1. This bipartite binding domain termed SSH domain (Structure composed of Strand and Helix) is necessary and sufficient to attain a predominant interaction of HP1 with INCENP. These results identify a unique HP1-binding module in INCENP that ensures enrichment of HP1 at inner centromeres, Aurora B activity, and thereby mitotic fidelity.
Asunto(s)
Aurora Quinasa B , Centrómero , Homólogo de la Proteína Chromobox 5 , Unión Proteica , Humanos , Aurora Quinasa B/metabolismo , Aurora Quinasa B/genética , Sitios de Unión , Centrómero/metabolismo , Homólogo de la Proteína Chromobox 5/genética , Homólogo de la Proteína Chromobox 5/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Células HeLa , MitosisRESUMEN
The pandemic HIV-1, HIV-1 group M, emerged from a single spillover event of its ancestral lentivirus from a chimpanzee. During human-to-human spread worldwide, HIV-1 diversified into multiple subtypes. Here, our interdisciplinary investigation mainly sheds light on the evolutionary scenario of the viral budding system of HIV-1 subtype C (HIV-1C), a most successfully spread subtype. Of the two amino acid motifs for HIV-1 budding, the P(T/S)AP and YPxL motifs, HIV-1C loses the YPxL motif. Our data imply that HIV-1C might lose this motif to evade immune pressure. Additionally, the P(T/S)AP motif is duplicated dependently of the level of HIV-1 spread in the human population, and >20% of HIV-1C harbored the duplicated P(T/S)AP motif. We further show that the duplication of the P(T/S)AP motif is caused by the expansion of the CTG triplet repeat. Altogether, our results suggest that HIV-1 has experienced a two-step evolution of the viral budding process during human-to-human spread worldwide.
Asunto(s)
Seropositividad para VIH , VIH-1 , Humanos , Animales , VIH-1/genética , Pandemias , Lentivirus , División Celular , Pan troglodytesRESUMEN
Nucleosome assembly protein 1 (NAP1) binds to histone H2A-H2B heterodimers, mediating their deposition on and eviction from the nucleosome. Human NAP1 (hNAP1) consists of a dimerization core domain and intrinsically disordered C-terminal acidic domain (CTAD), both of which are essential for H2A-H2B binding. Several structures of NAP1 proteins bound to H2A-H2B exhibit binding polymorphisms of the core domain, but the distinct structural roles of the core and CTAD domains remain elusive. Here, we have examined dynamic structures of the full-length hNAP1 dimer bound to one and two H2A-H2B heterodimers by integrative methods. Nuclear magnetic resonance (NMR) spectroscopy of full-length hNAP1 showed CTAD binding to H2A-H2B. Atomic force microscopy revealed that hNAP1 forms oligomers of tandem repeated dimers; therefore, we generated a stable dimeric hNAP1 mutant exhibiting the same H2A-H2B binding affinity as wild-type hNAP1. Size exclusion chromatography (SEC), multi-angle light scattering (MALS) and small angle X-ray scattering (SAXS), followed by modelling and molecular dynamics simulations, have been used to reveal the stepwise dynamic complex structures of hNAP1 binding to one and two H2A-H2B heterodimers. The first H2A-H2B dimer binds mainly to the core domain of hNAP1, while the second H2A-H2B binds dynamically to both CTADs. Based on our findings, we present a model of the eviction of H2A-H2B from nucleosomes by NAP1.
Asunto(s)
Histonas , Proteína 1 de Ensamblaje de Nucleosomas , Humanos , Histonas/metabolismo , Proteína 1 de Ensamblaje de Nucleosomas/genética , Proteína 1 de Ensamblaje de Nucleosomas/química , Proteína 1 de Ensamblaje de Nucleosomas/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Nucleosomas , Unión ProteicaRESUMEN
The relaxation dispersion (rd) of nuclear magnetic resonance provides thermodynamic and kinetic information regarding molecules for which the conformations are exchanging in equilibrium. Experiments have often been implemented with Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences for heteronuclear S-spin in SI and SI3 spin systems. One of the most common CPMG sequences contains a sequence called a P-element in the middle to average the different relaxation rates of anti-phase and in-phase coherences; however, its drawback is that the artifacts that can be compensated for are only those in one of the two S-spin doublet magnetization components, for example, SIα or SIß in an SI spin system. Thus, when the CPMG sequence is followed by a normal heteronuclear single-quantum correlation (HSQC) sequence, the detected signal will also include the other component with accumulated artifacts. To overcome this issue, we developed a new pulse sequence (AFTAC) that can suppress artifacts in both the magnetization components. Its effectiveness was demonstrated by simulations and actual measurements targeting the methyl groups of dimethylsulfoxide and N, N-dimethyltrichloroacetamide. The results demonstrated that the AFTAC sequence sufficiently suppressed the artifacts, despite being followed by an HSQC sequence that detects both components. AFTAC is particularly suitable for the rd measurements of small molecules, which are usually not deuterated and are not subject to transverse relaxation optimized spectroscopy (TROSY) sequences. AFTAC does not require 1H continuous wave irradiation for I-spin decoupling, which is necessary for certain CPMG methods that maintain S-spin in-phase coherence during the CPMG period (Tcpmg). Therefore, AFTAC places less burden on the probe, even with a long Tcpmg. Furthermore, the AFTAC method achieves a similar artifact suppression quality not only in SI but also in SI2 and SI3 spin systems.
Asunto(s)
Artefactos , Branquias , Animales , Resonancia Magnética Nuclear Biomolecular/métodos , Espectroscopía de Resonancia Magnética , Conformación MolecularRESUMEN
INTRODUCTION: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) receptors play a central role in neurotransmission and neuronal function. A positron emission tomography (PET) tracer for AMPA receptors, [11C]K-2, was recently developed by us to visualize AMPA receptors in the living human brain. [11C]K-2 is a derivative of 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difuluoro-phenoxyacetamide (PEPA), and is labeled with the radioactive isotope 11C, which has a short half-life. PET drugs are usually labeled with 18F because of its long half-life. Therefore, we screened and identified potential 18F-labeled PET drugs for AMPA receptors (AMPA-PET drugs), which could provide an image equivalent to that of [11C]K-2. METHODS: Derivatives of K-2 labeled with 18F were synthesized and administered to rats and PET imaging was performed. The transferability of each compound to the brain and its correlation with the PET image of [11C]K-2 were evaluated from the obtained PET images. Furthermore, the specific binding ability of promising compounds to the AMPA receptor was evaluated by the PET imaging of rats, which we specifically knocked down the expression of AMPA by the lentivirus-mediated introduction of short hairpin RNA (shRNA) targeted to subunits of the AMPA receptor (GluA1-A3). The specific binding ability was also evaluated through electrophysiological experiments with acute brain slices. RESULTS: Some of the synthesized 18F-labeled candidate compounds showed a distribution similar to that of K-2, with reasonable transferability to the brain. In addition, from the evaluation of the specific binding ability to the AMPA receptor, a promising structure of an 18F-labeled AMPA PET drug was identified. This study also revealed that the alkylation of the sulfonamide group of PEPA enhances brain transferability.
Asunto(s)
Flúor , Receptores AMPA , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Flúor/metabolismo , Radioisótopos de Flúor/metabolismo , Tomografía de Emisión de Positrones/métodos , Radiofármacos/metabolismo , Ratas , Receptores AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five ß-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel ß-barrel structure. However, the ß-strands were found to display a unique topology, one pair of these ß-strands formed a parallel ß-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern-triggered immunity in N. benthamiana.
Asunto(s)
Colletotrichum/metabolismo , Proteínas Fúngicas/fisiología , Inmunidad de la Planta/fisiología , Agrobacterium/patogenicidad , Secuencia de Aminoácidos , Colletotrichum/patogenicidad , Proteínas Fúngicas/química , Interacciones Huésped-Patógeno , Conformación Proteica , Especies Reactivas de Oxígeno/metabolismo , Homología de Secuencia de Aminoácido , Nicotiana/metabolismo , Nicotiana/microbiología , VirulenciaRESUMEN
The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)2, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.
Asunto(s)
ADN/metabolismo , Histonas/química , Histonas/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/genética , Humanos , Simulación de Dinámica Molecular , Nucleosomas/metabolismo , Conformación ProteicaRESUMEN
A preceding experiment suggested that a compound, which inhibits binding of the REST/NRSF segment to the cleft of a receptor protein mSin3B, can be a potential drug candidate to ameliorate many neuropathies. We have recently developed an enhanced conformational sampling method, genetic-algorithm-guided multi-dimensional virtual-system-coupled canonical molecular dynamics, and in the present study, applied it to three systems consisting of mSin3B and one of three compounds, sertraline, YN3, and acitretin. Other preceding experiments showed that only sertraline inhibits the binding of REST/NRSF to mSin3B. The current simulation study produced the spatial distribution of the compounds around mSin3B, and showed that sertraline and YN3 bound to the cleft of mSin3B with a high propensity, although acitretin did not. Further analyses of the simulation data indicated that only the sertraline-mSin3B complex produced a hydrophobic core similar to that observed in the molecular interface of the REST/NRSF-mSin3B complex: An aromatic ring of sertraline sunk deeply in the mSin3B's cleft forming a hydrophobic core contacting to hydrophobic amino-acid residues located at the bottom of the cleft. The present study proposes a step to design a compound that inhibits competitively the binding of a ligand to its receptor.
Asunto(s)
Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
BACKGROUND: Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state. RESULTS: We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis. CONCLUSIONS: The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of KV channels.
Asunto(s)
Venenos de Cnidarios/toxicidad , Canal de Potasio ERG1/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Venenos de Cnidarios/química , Venenos de Cnidarios/genética , Análisis Mutacional de ADN , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulación del Acoplamiento Molecular , Proteínas Mutantes/metabolismo , Mutación/genética , Proteínas Recombinantes/toxicidad , Soluciones , Xenopus laevisRESUMEN
TFIIH is a crucial transcription and DNA repair factor consisting of the seven-subunit core. The core subunit p62 contains a pleckstrin homology domain (PH-D), which is essential for locating TFIIH at transcription initiation and DNA damage sites, and two BSD (BTF2-like transcription factors, synapse-associated proteins and DOS2-like proteins) domains. A recent cryo-electron microscopy (cryo-EM) structure of human TFIIH visualized most parts of core, except for the PH-D. Here, by nuclear magnetic resonance spectroscopy we have established the solution structure of human p62 PH-D connected to the BSD1 domain by a highly flexible linker, suggesting the flexibility of PH-D in TFIIH. Based on this dynamic character, the PH-D was modeled in the cryo-EM structure to obtain the whole human TFIIH core structure, which indicates that the PH-D moves around the surface of core with a specific but limited spatial distribution; these dynamic structures were refined by molecular dynamics (MD) simulations. Furthermore, we built models, also refined by MD simulations, of TFIIH in complex with five p62-binding partners, including transcription factors TFIIEα, p53 and DP1, and nucleotide excision repair factors XPC and UVSSA. The models explain why the PH-D is crucially targeted by these factors, which use their intrinsically disordered acidic regions for TFIIH recruitment.
Asunto(s)
Factor de Transcripción TFIIH/química , Microscopía por Crioelectrón , Humanos , Simulación de Dinámica Molecular , Dominios Homólogos a Pleckstrina , Dominios ProteicosRESUMEN
Dysregulation of repressor-element 1 silencing transcription factor REST/NRSF is related to several neuropathies, including medulloblastoma, glioblastoma, Huntington's disease, and neuropathic pain. Inhibitors of the interaction between the N-terminal repressor domain of REST/NRSF and the PAH1 domain of its corepressor mSin3 may ameliorate such neuropathies. In-silico screening based on the complex structure of REST/NRSF and mSin3 PAH1 yielded 52 active compounds, including approved neuropathic drugs. We investigated their binding affinity to PAH1 by NMR, and their inhibitory activity toward medulloblastoma cell growth. Interestingly, three antidepressant and antipsychotic medicines, sertraline, chlorprothixene, and chlorpromazine, were found to strongly bind to PAH1. Multivariate analysis based on NMR chemical shift changes in PAH1 residues induced by ligand binding was used to identify compound characteristics associated with cell growth inhibition. Active compounds showed a new chemo-type for inhibitors of the REST/NRSF-mSin3 interaction, raising the possibility of new therapies for neuropathies caused by dysregulation of REST/NRSF.
Asunto(s)
Meduloblastoma/tratamiento farmacológico , Dominios Proteicos/efectos de los fármacos , Proteínas Represoras/química , Animales , Sitios de Unión/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Clorpromazina/química , Clorpromazina/farmacología , Clorprotixeno/química , Clorprotixeno/farmacología , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Sertralina/química , Sertralina/farmacologíaRESUMEN
The neuron-restrictive silencing factor NRSF/REST binds to neuron-restrictive silencing elements in neuronal genes and recruits corepressors such as mSin3 to inhibit epigenetically neuronal gene expression. Because dysregulation of NRSF/REST is related to neuropathic pain, here, we have designed compounds to target neuropathic pain based on the mSin3-binding helix structure of NRSF/REST and examined their ability to bind to mSin3 by NMR. One compound, mS-11, binds strongly to mSin3 with a binding mode similar to that of NRSF/REST. In a mouse model of neuropathic pain, mS-11 was found to ameliorate abnormal pain behavior and to reverse lost peripheral morphine analgesia. Furthermore, even in the less well epigenetically defined case of fibromyalgia, mS-11 ameliorated symptoms in a mouse model, suggesting that fibromyalgia is related to the dysfunction of NRSF/REST. Taken together, these findings show that the chemically optimized mimetic mS-11 can inhibit mSin3-NRSF/REST binding and successfully reverse lost peripheral and central morphine analgesia in mouse models of pain.
Asunto(s)
Proteínas Portadoras/metabolismo , Dolor Crónico/tratamiento farmacológico , Compuestos Heterocíclicos con 2 Anillos/metabolismo , Proteínas Represoras/metabolismo , Analgésicos Opioides/uso terapéutico , Animales , Sitios de Unión , Proteínas Portadoras/química , Dolor Crónico/patología , Frío , Modelos Animales de Enfermedad , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/uso terapéutico , Ratones , Simulación del Acoplamiento Molecular , Morfina/uso terapéutico , Neuralgia/tratamiento farmacológico , Neuralgia/patología , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Represoras/químicaRESUMEN
During chromatin-regulated processes, the histone H2A-H2B heterodimer functions dynamically in and out of the nucleosome. Although detailed crystal structures of nucleosomes have been established, that of the isolated full-length H2A-H2B heterodimer has remained elusive. Here, we have determined the solution structure of human H2A-H2B by NMR coupled with CS-Rosetta. H2A and H2B each contain a histone fold, comprising four α-helices and two ß-strands (α1-ß1-α2-ß2-α3-αC), together with the long disordered N- and C-terminal H2A tails and the long N-terminal H2B tail. The N-terminal αN helix, C-terminal ß3 strand, and 310 helix of H2A observed in the H2A-H2B nucleosome structure are disordered in isolated H2A-H2B. In addition, the H2A α1 and H2B αC helices are not well fixed in the heterodimer, and the H2A and H2B tails are not completely random coils. Comparison of hydrogen-deuterium exchange, fast hydrogen exchange, and {(1)H}-(15)N hetero-nuclear NOE data with the CS-Rosetta structure indicates that there is some conformation in the H2A 310 helical and H2B Lys11 regions, while the repression domain of H2B (residues 27-34) exhibits an extended string-like structure. This first structure of the isolated H2A-H2B heterodimer provides insight into its dynamic functions in chromatin.
Asunto(s)
Histonas/química , Multimerización de Proteína , Humanos , Espectroscopía de Resonancia Magnética , Conformación Proteica , Pliegue de ProteínaRESUMEN
The (1)H-(19) F heteronuclear NMR experiments were achieved using the conventional spectrometer equipped with a single high band amplifier and a (1)H/(19)F/(13) C double-tuned probe. Although double high band amplifiers are generally required to perform such experiments, a simple modification of pathway in the conventional spectrometer was capable of acquiring various (1)H-(19)F heteronuclear spectra. The efficiency of the present technique was demonstrated in an application for (19)F{(1)H} and (1)H{(19)F} saturation transfer difference experiments.
Asunto(s)
Amplificadores Electrónicos , Flúor/análisis , Flúor/química , Resonancia Magnética Nuclear Biomolecular/instrumentación , Espectroscopía de Protones por Resonancia Magnética/instrumentación , Procesamiento de Señales Asistido por Computador/instrumentación , Algoritmos , Diseño de Equipo , Análisis de Falla de Equipo , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
FBP11/HYPA is a mammalian homologue of yeast splicing factor Prp40. The first WW domain of FBP11/HYPA (FBP11 WW1) is essential for preventing severe neurological diseases such as Huntington disease and Rett syndrome and strongly resembles the WW domain of FCA, the essential regulator for flowering time control. We have solved the structure of FBP11 WW1 and a Pro-Pro-Leu-Pro ligand complex, and demonstrated the binding mechanism with mutational analysis using surface plasmon resonance. The overall structure of FBP11 WW1 in the complex form is quite similar to the structures of WW domains from Group I and IV in complexes. In addition, conformation of FBP11 WW1 does not change much upon ligand binding. The binding orientation of the ligand against FBP11 WW1 is the same as that of the Group IV WW domain-ligand complex, but opposite to that of the Group I complex. The ligand interacts with two grooves formed by surface aromatic residues. The Pro and Leu residues in the ligand interact with the grooves and the Loop I region of FBP11 WW1, respectively, which are necessary interactions for binding the ligand. Interestingly, the two aromatic grooves recognize the Pro residues in entirely different manners, which allows FBP11 WW1 to recognize shorter sequences than the SH3 domain. Combined with homology models of other WW domains, the present report shows the detailed mechanism of ligand binding by Group II/III WW domains, and provides information useful in designing drugs to treat neurodegenerative diseases.
Asunto(s)
Proteínas Portadoras/química , Leucina/química , Péptidos/química , Prolina/química , Triptófano/química , Secuencias de Aminoácidos/fisiología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Humanos , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Leucina/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Péptidos/metabolismo , Prolina/metabolismo , Dominios Proteicos Ricos en Prolina , Estructura Terciaria de Proteína , Síndrome de Rett/metabolismo , Síndrome de Rett/patología , Ribonucleoproteína Nuclear Pequeña U1/química , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Triptófano/metabolismoRESUMEN
The chemical shifts of nuclei that have chemical shielding anisotropy, such as the 15N amide in a protein, show significant changes in their chemical shifts when the sample is altered from an isotropic state to an aligned state. Such orientation-dependent chemical shift changes provide information on the magnitudes and orientation of the chemical shielding tensors relative to the molecule's alignment frame. Because of the extremely high sensitivity of the chemical shifts to the sample conditions, the changes in chemical shifts induced by adding aligned bicelles do not arise only from the protein alignment but should also include the accumulated effects of environmental changes including protein-bicelle interactions. With the aim of determining accurate 15N chemical shielding tensor values for solution proteins, here we have used magic angle sample spinning (MAS) to observe discriminately the orientation-dependent changes in the 15N chemical shift. The application of MAS to an aligned bicelle solution removes the torque that aligns the bicelles against the magnetic field. Thus, the application of MAS to a protein in a bicelle solution eliminates only the molecular alignment effect, while keeping all other sample conditions the same. The observed chemical shift differences between experiments with and without MAS therefore provide accurate values of the orientation-dependent 15N chemical shifts. From the values for ubiquitin in a 7.5% (w/v) bicelle medium, we determined the 15N chemical shielding anisotropy (CSA) tensor. For this evaluation, we considered uncertainties in measuring the 1H-15N dipolar couplings and the 15N chemical shifts and also structural noise present in the reference X-ray structure, assuming a random distribution of each NH bond vector in a cone with 5 degrees deviation from the original orientation. Taking into account these types of noise, we determined the average 15N CSA tensor for the residues in ubiquitin as Delta sigma=-162.0+/-4.3 ppm, eta=0.18+/-0.02, and beta=18.6+/-0.5 degrees, assuming a 1H-15N bond length of 1.02 A. These tensor values are consistent with those obtained from solid-state NMR experiments.