RESUMO
Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although the crystal structures of dark- and light-adapted states have been determined, the molecular mechanisms underlying photoactivation remain elusive. Here, we demonstrate that the conserved tongue region of the PHY domain of a 57-kDa photosensory module of Deinococcus radiodurans phytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.
Assuntos
Luz , Fitocromo/química , Escuridão , Deinococcus , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fitocromo/metabolismo , Domínios ProteicosRESUMO
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure-function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
Assuntos
Archaea/metabolismo , Proteínas Arqueais/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Biologia Computacional/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Dobramento de Proteína , Fases de Leitura Aberta , Conformação ProteicaRESUMO
NMR studies of intrinsically disordered proteins and other complex biomolecular systems require spectra with the highest resolution and dimensionality. An efficient approach, extra-large NMR spectroscopy, is presented for experimental data collection, reconstruction, and handling of very large NMR spectra by a combination of the radial and non-uniform sampling, a new processing algorithm, and rigorous statistical validation. We demonstrate the first high-quality reconstruction of a full seven-dimensional HNCOCACONH and two five-dimensional HACACONH and HN(CA)CONH experiments for a representative intrinsically disordered protein α-synuclein. XLSY will significantly enhance the NMR toolbox in challenging biomolecular studies.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Algoritmos , Proteínas Intrinsicamente Desordenadas/química , Conformação Proteica , alfa-Sinucleína/químicaRESUMO
Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics for many sites of interest in globular and intrinsically disordered proteins. We present new three-dimensional pulse sequences for measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions at backbone nitrogen and carbonyl positions. To alleviate increase in the measurement time associated with the additional spectral dimension, we use non-uniform sampling in combination with two distinct methods of spectrum reconstruction: compressed sensing and co-processing with multi-dimensional decomposition. The new methodology was validated using disordered protein CD79A from B-cell receptor and an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that, while providing much better resolution, the 3D NUS experiments give the similar accuracy and precision of the dynamic parameters to ones obtained using traditional 2D experiments. Furthermore, we show that jackknife resampling of the spectra yields robust estimates of peak intensities errors, eliminating the need for recording duplicate data points.
Assuntos
Isótopos de Carbono/análise , Isótopos de Nitrogênio/análise , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas Intrinsicamente Desordenadas/químicaRESUMO
Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker.
Assuntos
Actinina/química , Cálcio/química , Calmodulina/química , Entamoeba histolytica/química , Proteínas de Protozoários/química , Fatores de Virulência/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinina/genética , Actinina/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Clonagem Molecular , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of nonuniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and nonamyloidogenic variants of ß2-microglobulin (ß2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the aggregation-prone ITrans state and population of a less stable, more dynamic species ablate amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Dobramento de Proteína , Escherichia coli/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Plasmídeos/química , Conformação Proteica , Termodinâmica , Microglobulina beta-2/químicaRESUMO
Increasing the dimensionality of NMR experiments strongly enhances the spectral resolution and provides invaluable direct information about atomic interactions. However, the price tag is high: long measurement times and heavy requirements on the computation power and data storage. We introduce sparse fast Fourier transform as a new method of NMR signal collection and processing, which is capable of reconstructing high quality spectra of large size and dimensionality with short measurement times, faster computations than the fast Fourier transform, and minimal storage for processing and handling of sparse spectra. The new algorithm is described and demonstrated for a 4D BEST-HNCOCA spectrum.
Assuntos
Algoritmos , Análise de Fourier , Ressonância Magnética Nuclear Biomolecular/métodos , Humanos , Fatores de Tempo , Ubiquitina/químicaRESUMO
Accelerated multi-dimensional NMR spectroscopy is a prerequisite for high-throughput applications, studying short-lived molecular systems and monitoring chemical reactions in real time. Non-uniform sampling is a common approach to reduce the measurement time. Here, a new method for high-quality spectra reconstruction from non-uniformly sampled data is introduced, which is based on recent developments in the field of signal processing theory and uses the so far unexploited general property of the NMR signal, its low rank. Using experimental and simulated data, we demonstrate that the low-rank reconstruction is a viable alternative to the current state-of-the-art technique compressed sensing. In particular, the low-rank approach is good in preserving of low-intensity broad peaks, and thus increases the effective sensitivity in the reconstructed spectra.
RESUMO
Time-resolved experiments demand high resolution both in spectral dimensions and in time of the studied kinetic process. The latter requirement traditionally prohibits applications of the multidimensional experiments, which, although capable of providing invaluable information about structure and dynamics and almost unlimited spectral resolution, require too lengthy data collection. Our work shows that the problem has a solution in using modern methods of NMR data collection and signal processing. A continuous fast pulsing three-dimensional experiment is acquired using non-uniform sampling during full time of the studied reaction. High sensitivity and time-resolution of a few minutes is achieved by simultaneous processing of the full data set with the multi-dimensional decomposition. The method is verified and illustrated in realistic simulations and by measuring deuterium exchange rates of amide protons in ubiquitin. We applied the method for characterizing kinetics of in vitro phosphorylation of two tyrosine residues in an intrinsically disordered cytosolic domain of the B cell receptor protein CD79b. Signals of many residues including tyrosines in both phosphorylated and unmodified forms of CD79b are found in a heavily crowded region of 2D ¹H-¹5N correlation spectrum and the significantly enhanced spectral resolution provided by the 3D time-resolved approach was essential for the quantitative site-specific analysis.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Antígenos CD79/química , Antígenos CD79/metabolismo , Isótopos de Carbono/química , Humanos , Isótopos de Nitrogênio/química , Fosforilação , Proteínas/metabolismoRESUMO
The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)(2). The helices interact through the extended heptad repeat motif L(535)X(3)G(539)X(2)A(542)X(3)V(546)X(2)L(549) assisted by intermolecular stacking interactions of aromatic rings of (FF(557))(2), whereas the characteristic tandem GG4-like motif A(536)X(3)G(540)X(3)G(544) is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX(3)GX(3)G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.
Assuntos
Membrana Celular/química , Multimerização Proteica , Receptor EphA2/química , Sequência de Aminoácidos , Humanos , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Água/químicaRESUMO
Formation of amyloid-beta (Aß) oligomer pores in the membrane of neurons has been proposed to explain neurotoxicity in Alzheimer's disease (AD). Here, we present the three-dimensional structure of an Aß oligomer formed in a membrane mimicking environment, namely an Aß(1-42) tetramer, which comprises a six stranded ß-sheet core. The two faces of the ß-sheet core are hydrophobic and surrounded by the membrane-mimicking environment while the edges are hydrophilic and solvent-exposed. By increasing the concentration of Aß(1-42) in the sample, Aß(1-42) octamers are also formed, made by two Aß(1-42) tetramers facing each other forming a ß-sandwich structure. Notably, Aß(1-42) tetramers and octamers inserted into lipid bilayers as well-defined pores. To establish oligomer structure-membrane activity relationships, molecular dynamics simulations were carried out. These studies revealed a mechanism of membrane disruption in which water permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core ß-sheets edges of the oligomers.
Assuntos
Peptídeos beta-Amiloides/química , Membrana Celular/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Multimerização Proteica , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Condutividade Elétrica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Síndromes Neurotóxicas/metabolismo , Fragmentos de Peptídeos/metabolismo , Água/metabolismoRESUMO
Practical synthesis of tetrasaccharide sulfates, 6((GlcNAc))-O-Su-SiaLe(X)-OCH(2)CH(2)CH(2)NH(2) and 6((Gal))-O-Su-SiaLe(X)-OCH(2)CH(2)CH(2)NH(2) (Su( )SO(3)H), selectin ligands, and leu- kocyte trafficking agents is presented. Both sulfates were synthesized starting from the same precursor, protected SiaLe(x), by the conventional procedures of carbohydrate chemistry. The sulfated SiaLe(x) derivative was modified at the spacer group to give 6((Gal))-O-Su-SiaLe(x)- OCH(2)CH(2)CH(2)NH-COCH(2)CH(2)C[triple bond]CH, convenient for "click chemistry" mode conjugation with an azido carrier, particularly, for the synthesis of an immunogen.
Assuntos
Oligossacarídeos/química , Oligossacarídeos/síntese química , Sequência de Carboidratos , Dados de Sequência Molecular , Estrutura Molecular , Antígeno Sialil Lewis XRESUMO
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a unique paracaspase protein whose protease activity mediates oncogenic NF-κB signalling in activated B cell-like diffuse large B cell lymphomas (ABC-DLBCLs). ABC-DLBCLs are aggressive lymphomas with high resistance to current chemotherapies. Low survival rate among patients emphasizes the urgent need for alternative treatment options. The characterization of the MALT1 will be an essential tool for developing new target-directed drugs against MALT1 dependent disorders. As the first step in the atomic-level NMR studies of the system, here we report, the (15)N/(13)C/(1)H backbone assignment of the apo form of the MALT1 paracaspase region together with the third immunoglobulin-like (Ig3) domain, 44 kDa, by high resolution NMR. In addition, the non-uniform sampling (NUS) based targeted acquisition procedure is evaluated as a mean of decreasing acquisition and analysis time for larger proteins.
Assuntos
Caspases/química , Proteínas de Neoplasias/química , Humanos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2(120-128)) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2(120-128) region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2.
Assuntos
Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação Puntual/genética , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade , Ubiquitina/metabolismo , Dedos de ZincoRESUMO
The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin-antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SOxH-containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Sistemas Toxina-Antitoxina/fisiologia , Sequência de Aminoácidos , Escherichia coli K12 , Oxirredução , Conformação Proteica , Yersinia/químicaRESUMO
The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeo Hidrolases/química , Vibrio cholerae/enzimologia , Domínio Catalítico , Sequência Conservada , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Vibrio cholerae/químicaRESUMO
Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b in vitro before and after phosphorylation. The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.
Assuntos
Antígenos CD79/química , Processamento de Proteína Pós-Traducional , Antígenos CD79/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tirosina/químicaRESUMO
We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos T/química , Motivos de Aminoácidos , Citosol/metabolismo , Humanos , Espaço Intracelular/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Ligantes , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de SinaisRESUMO
Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.