RESUMO
The combined effects of the cellular environment on proteins led to the definition of a fifth level of protein structural organization termed quinary structure. To explore the implication of potential quinary structure for globular proteins, we studied the dynamics and conformations of Escherichia coli (E. coli) peptidyl-prolyl cis/trans isomerase B (PpiB) in E. coli cells. PpiB plays a major role in maturation and regulation of folded proteins by catalyzing the cis/trans isomerization of the proline imidic peptide bond. We applied electron paramagnetic resonance (EPR) techniques, utilizing both Gadolinium (Gd(III)) and nitroxide spin labels. In addition to using standard spin labeling approaches with genetically engineered cysteines, we incorporated an unnatural amino acid to achieve Gd(III)-nitroxide orthogonal labeling. We probed PpiB's residue-specific dynamics by X-band continuous wave EPR at ambient temperatures and its structure by double electron-electron resonance (DEER) on frozen samples. PpiB was delivered to E. coli cells by electroporation. We report a significant decrease in the dynamics induced by the cellular environment for two chosen labeling positions. These changes could not be reproduced by adding crowding agents and cell extracts. Concomitantly, we report a broadening of the distance distribution in E. coli, determined by Gd(III)-Gd(III) DEER measurements, as compared with solution and human HeLa cells. This suggests an increase in the number of PpiB conformations present in E. coli cells, possibly due to interactions with other cell components, which also contributes to the reduction in mobility and suggests the presence of a quinary structure.
Assuntos
Escherichia coli , Óxidos de Nitrogênio , Proteínas , Humanos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Escherichia coli/genética , Escherichia coli/química , Células HeLa , Marcadores de Spin , Proteínas/químicaRESUMO
UreG is a cytosolic GTPase involved in the maturation network of urease, an Ni-containing bacterial enzyme. Previous investigations in vitro showed that UreG features a flexible tertiary organization, making this protein the first enzyme discovered to be intrinsically disordered. To determine whether this heterogeneous behavior is maintained in the protein natural environment, UreG structural dynamics was investigated directly in intact bacteria by in-cell EPR. This approach, based on site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy, enables the study of proteins in their native environment. The results show that UreG maintains heterogeneous structural landscape in-cell, existing in a conformational ensemble of two major conformers, showing either random coil-like or compact properties. These data support the physiological relevance of the intrinsically disordered nature of UreG and indicates a role of protein flexibility for this specific enzyme, possibly related to the regulation of promiscuous protein interactions for metal ion delivery.
RESUMO
Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the local level, the dynamics of structural transitions in proteins. Here, we consider SDSL-EPR based on the selective grafting of a nitroxide on the protein under study, followed by X-band cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give a reliable interpretation on biological system dynamics, a numerical simulation of the spectra is required. However, regardless of the numerical tool chosen to perform such simulations, the number of parameters is often too high to provide unambiguous results. In this study, we have chosen SimLabel to perform such simulations. SimLabel is a graphical user interface (GUI) of Matlab, using some functions of Easyspin. An exhaustive review of the parameters used in this GUI has enabled to define the adjustable parameters during the simulation fitting and to fix the others prior to the simulation fitting. Among them, some are set once and for all (gy, gz) and others are determined (Az, gx) thanks to a supplementary X-band spectrum recorded on a frozen solution. Finally, we propose guidelines to perform the simulation of X-band cw-EPR spectra of nitroxide labeled proteins at room temperature, with no need of uncommon higher frequency spectrometry and with the minimal number of variable parameters.
Assuntos
Óxidos de Nitrogênio , Proteínas , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Marcadores de Spin , Óxidos de Nitrogênio/química , Proteínas/químicaRESUMO
One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin-labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site-specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time-resolved dynamics of NarJ in cellular context.
Assuntos
Chaperonas Moleculares , Óxidos de Nitrogênio , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Marcadores de Spin , Óxidos de Nitrogênio/química , Chaperonas Moleculares/químicaRESUMO
UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of Helicobacter pylori (Hp) UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution. Here, isothermal titration calorimetry and site-directed spin labeling coupled to electron paramagnetic spectroscopy were successfully combined to investigate HpUreG structural dynamics in solution and the effect of Ni(II) and GTP on protein mobility. The results demonstrate that, although the protein maintains a flexible behavior in the metal and nucleotide bound forms, concomitant addition of Ni(II) and GTP exerts a structural change through the crosstalk of different protein regions.
Assuntos
Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Helicobacter pylori/metabolismo , Níquel/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Infecções por Helicobacter/microbiologia , Helicobacter pylori/química , Humanos , Modelos Moleculares , Proteínas de Ligação a Fosfato/química , Conformação ProteicaRESUMO
Exploring the structure and dynamics of biomolecules in the context of their intracellular environment has become the ultimate challenge for structural biology. As the cellular environment is barely reproducible in vitro, investigation of biomolecules directly inside cells has attracted a growing interest. Among magnetic resonance approaches, site-directed spin labeling (SDSL) coupled to electron paramagnetic resonance (EPR) spectroscopy provides competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in-cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this review, the major advances of in-cell EPR spectroscopy are summarized, as well as the challenges this approach still poses.
Assuntos
Bactérias/ultraestrutura , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Células Eucarióticas/ultraestrutura , Marcadores de Spin , Proteínas de Membrana/ultraestruturaRESUMO
The "Bioénergétique et Ingénierie des Protéines (BIP)" laboratory, CNRS (France), organized its first French workshop on molecular chaperone proteins and protein folding in November 2017. The goal of this workshop was to gather scientists working in France on chaperone proteins and protein folding. This initiative was a great success with excellent talks and fruitful discussions. The highlights were on the description of unexpected functions and post-translational regulation of known molecular chaperones (such as Hsp90, Hsp33, SecB, GroEL) and on state-of-the-art methods to tackle questions related to this theme, including Cryo-electron microscopy, Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR), simulation and modeling. We expect to organize a second workshop in two years that will include more scientists working in France in the chaperone field.
Assuntos
Chaperoninas/metabolismo , Biofísica , FrançaRESUMO
Approaching protein structural dynamics and protein-protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide-based spin labels for in-cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido-proxyl-based spin label (M-TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M-TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.
Assuntos
Óxidos de Nitrogênio/química , Oócitos/metabolismo , Proteínas de Xenopus/química , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Maleimidas/química , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Nitrato Redutase/química , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Marcadores de Spin , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Site-directed spin labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional spin labeling of cysteine residues. Double electron-electron resonance (DEER) EPR measurements were performed for both the nitroxide-nitroxide and Gd(III)-nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Proteínas/química , Marcadores de Spin , Cisteína , Elétrons , Modelos Moleculares , Óxidos de Nitrogênio , TirosinaRESUMO
A growing body of literature on intrinsically disordered proteins (IDPs) led scientists to rethink the structure-function paradigm of protein folding. Enzymes are often considered an exception to the rule of intrinsic disorder (ID), believed to require a unique structure for catalysis. However, recent studies revealed the presence of disorder in several functional native enzymes. In the present work, we address the importance of dynamics for catalysis, by investigating the relationship between folding and activity in Sporosarcina pasteurii UreG (SpUreG), a P-loop GTPase and the first discovered native ID enzyme, involved in the maturation of the nickel-containing urease. The effect of denaturants and osmolytes on protein structure and activity was analyzed using circular dichroism (CD), Site-Directed Spin Labeling (SDSL) coupled to EPR spectroscopy, and enzymatic assays. Our data show that SpUreG needs a "flexibility window" to be catalytically competent, with both too low and too high mobility being detrimental for its activity.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Dobramento de Proteína , Espectroscopia de Ressonância de Spin Eletrônica , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Proteínas de Ligação a Fosfato , Conformação Proteica , Desnaturação Proteica , Marcadores de Spin , Sporosarcina/enzimologia , TemperaturaRESUMO
Proteins are highly variable biological systems, not only in their structures but also in their dynamics. The most extreme example of dynamics is encountered within the family of Intrinsically Disordered Proteins (IDPs), which are proteins lacking a well-defined 3D structure under physiological conditions. Among the biophysical techniques well-suited to study such highly flexible proteins, Site-Directed Spin Labeling combined with EPR spectroscopy (SDSL-EPR) is one of the most powerful, being able to reveal, at the residue level, structural transitions such as folding events. SDSL-EPR is based on selective grafting of a paramagnetic label on the protein under study and is limited neither by the size nor by the complexity of the system. The objective of this mini-review is to describe the basic strategy of SDSL-EPR and to illustrate how it can be successfully applied to characterize the structural behavior of IDPs. Recent developments aimed at enlarging the panoply of SDSL-EPR approaches are presented in particular newly synthesized spin labels that allow the limitations of the classical ones to be overcome. The potentialities of these new spin labels will be demonstrated on different examples of IDPs.
RESUMO
Site Directed Spin Labeling (SDSL) combined with EPR spectroscopy is a very powerful approach to investigate structural transitions in proteins in particular flexible or even disordered ones. Conventional spin labels are based on nitroxide derivatives leading to classical 3-line spectra whose spectral shapes are indicative of the environment of the labels and thus constitute good reporters of structural modifications. However, the similarity of these spectral shapes precludes probing two regions of a protein or two partner proteins simultaneously. To overcome the limitation due to the weak diversity of nitroxide label EPR spectral shapes, we designed a new spin label based on a ß-phosphorylated nitroxide giving 6-line spectra. This paper describes the synthesis of this new spin label, its grafting at four different positions of a model disordered protein able to undergo an induced α-helical folding and its characterization by EPR spectroscopy. For comparative purposes, a classical nitroxide has been grafted at the same positions of the model protein. The ability of the new label to report on structural transitions was evaluated by analyzing the spectral shape modifications induced either by the presence of a secondary structure stabilizer (trifluoroethanol) or by the presence of a partner protein. Taken together the results demonstrate that the new phosphorylated label gives a very distinguishable signature which is able to report from subtle to larger structural transitions, as efficiently as the classical spin label. As a complementary approach, molecular dynamics (MD) calculations were performed to gain further insights into the binding process between the labeled NTAIL and PXD. MD calculations revealed that the new label does not disturb the interaction between the two partner proteins and reinforced the conclusion on its ability to probe different local environments in a protein. Taken together this study represents an important step forward in the extension of the panoply of SDSL-EPR approaches.
Assuntos
Óxidos de Nitrogênio/química , Proteínas/química , Espectroscopia de Ressonância de Spin Eletrônica , Simulação de Dinâmica Molecular , Fosforilação , Estrutura Secundária de Proteína , Proteínas/metabolismo , Marcadores de Spin , Trifluoretanol/químicaRESUMO
CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of the Calvin cycle by forming a supra-molecular complex with at least two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). CP12 shares some similarities with intrinsically disordered proteins (IDPs) depending on its redox state. In this study, site-directed spin labeling (SDSL) combined with EPR spectroscopy was used to probe the dynamic behavior of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH, the first step towards ternary complex formation. The two N-terminal cysteine residues were labeled using the classical approach while the tyrosine located at the C-terminal end of CP12 was modified following an original procedure. The results show that the label grafted at the C-terminal extremity is in the vicinity of the interaction site whereas the N-terminal region remains fully disordered upon binding to GAPDH. In conclusion, GAPDH-CP12 is a fuzzy complex, in which the N-terminal region of CP12 keeps a conformational freedom in the bound form. This fuzziness could be one of the keys to facilitate binding of PRK to CP12-GAPDH and to form the ternary supra-molecular complex.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas de Plantas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Cinética , Modelos Moleculares , Fotossíntese , Proteínas de Plantas/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por SubstratoRESUMO
Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical spin labels are functionalized to be grafted on natural or site-directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based nitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Isoindóis/química , Óxido Nítrico/química , Marcadores de Spin , Tirosina/química , Estrutura Molecular , Óxido Nítrico/síntese químicaRESUMO
Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.
Assuntos
Enzimas/química , Membrana Celular/metabolismo , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Elétrons , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas/métodos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Conformação Molecular , Molibdênio/química , Mutagênese Sítio-Dirigida , Nitrato Redutase/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Espectrometria de Fluorescência/métodos , Marcadores de Spin , Temperatura , Triptofano/químicaAssuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas/química , Marcadores de Spin , Tirosina/química , Clorófitas/metabolismo , Proteínas de Cloroplastos/química , Dicroísmo Circular , Gliceraldeído-3-Fosfato Desidrogenases/química , Bases de Mannich , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The first example of paramagnetic rotaxane containing cucurbit[6]urils has been reported and characterized both by ESR and NMR spectroscopy.
RESUMO
EPR experiments in water-saturated CH(2)Cl(2) solution clearly indicate that oxotempo is not included inside hexameric molecular capsules of resorcin[4]arene. In fact, the tumbling rate of oxotempo only experiences minor changes when resorcinarene is present in the solution. However, NMR spectroscopic data suggest that oxotempo engages in labile hydrogen-bonding interactions with water molecules interacting with the resorcinarene molecular capsules.
RESUMO
A radical-armed guanosine derivative shows drastic magnetic changes by addition (removal) of alkali metal cations corresponding to the reversible assembly (disassembly). In the presence of templating metal ions, the assembly is formed by 8 molecules and 16 open-shell moieties confined in a sphere with a diameter of ca. 30 A.
RESUMO
In quarantine: Nitroxide spin probes are encapsulated by hexameric resorcinarene molecular capsules in dichloromethane solutions (see picture). A substantial reduction in the tumbling rates occurs upon encapsulation of two cationic probes and one neutral probe. As the molecular volume of the probe increases, the tumbling rate of the probe reflects the overall tumbling rate of the entire supramolecular assembly.