RESUMEN
The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its p-HBI chromophore, corresponding to the cis and trans isomers, with each one either protonated (neutral) or deprotonated (anionic) at the benzylidene ring. The relative populations and interconversion kinetics of these chromophore species depend on sample pH and buffer composition that alter in a complex way the strength of H-bonds that contribute in stabilizing the chromophore within the protein scaffold. We show in particular the important role of histidine-149 in stabilizing the neutral trans chromophore at intermediate pH values, leading to ground-state cis-trans isomerization with a peculiar pH dependence. We discuss the potential implications of our findings on the pH dependence of the photoswitching contrast, a critical parameter in nanoscopy applications.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Resonancia Magnética Nuclear Biomolecular , Concentración de Iones de Hidrógeno , Conformación ProteicaRESUMEN
Specific isotopic labeling of methyl groups in a perdeuterated protein background enables the detection of long range NOEs in proteins or high molecular weight complexes. We introduce here an approach, combining an optimized isotopic labeling scheme with a specifically tailored NMR pulse sequence, to distinguish between intramolecular and intermolecular NOE connectivities. In hetero-oligomeric complexes, this strategy enables sign encoding of intra-subunit and inter-subunit NOEs. For homo-oligomeric assemblies, our strategy allows the specific detection of intra-chain NOEs in high resolution 3D NOESY spectra. The general principles, possibilities and limitations of this approach are presented. Applications of this approach for the detection of intermolecular NOEs in a hetero-hexamer, and the assignment of methyl 1H and 13C resonances in a homo-tetrameric protein complex are shown.
Asunto(s)
Complejos Multiproteicos/química , Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Chaperonas Moleculares/química , Conformación ProteicaRESUMEN
The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a nonfluorescent state. Our results add a dynamic view to the static structures obtained by x-ray crystallography. Including a custom-tailored NMR toolbox in fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins and their correlation with the observed photochemical and photophysical properties.
Asunto(s)
Luz , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Espectroscopía de Resonancia Magnética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Oxidación-Reducción , Estructura Secundaria de Proteína , Temperatura , ViscosidadRESUMEN
We present NMRlib, a suite of jython-based tools designed for Bruker spectrometers (TopSpin versions 3.2-4.0) that allow easy setup, management, and exchange of NMR experiments. A NMR experiment can be set up and executed in a few clicks by navigating through the NMRlib GUI tree structure, without any further parameter adjustment. NMRlib is magnetic-field independent, and thus particularly helpful for laboratories operating multiple NMR spectrometers. NMRlib is easily personalized by adding, deleting, or reorganizing experiments. Additional tools are provided for data processing, visualization, and analysis. In particular, NMRlib contains all the polarization-enhanced fast-pulsing NMR experiments (SOFAST, BEST, HADAMAC, ) developed in our laboratory over the last decade. We also discuss some specific features that have been implemented to make these experiments most efficient and user friendly.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/análisisRESUMEN
Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.
Asunto(s)
Proteínas y Péptidos de Choque por Frío/química , Proteínas y Péptidos de Choque por Frío/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Pliegue del ARN , ARN/química , Aminoácidos Aromáticos/química , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , ARN/metabolismoRESUMEN
Aromatic amino-acid side chains are essential components for the structure and function of proteins. We present herein a set of NMR experiments for time-efficient resonance assignment of histidine and tyrosine side chains in uniformly 13C/15N-labeled proteins. The use of band-selective 13C pulses allows to deal with linear chains of coupled spins, thus avoiding signal loss that occurs in branched spin systems during coherence transfer. Furthermore, our pulse schemes make use of longitudinal 1H relaxation enhancement, Ernst-angle excitation, and simultaneous detection of 1H and 13C steady-state polarization to achieve significant signal enhancements.
Asunto(s)
Histidina , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Tirosina , Algoritmos , Isótopos de Carbono , Isótopos de Nitrógeno , Conformación ProteicaRESUMEN
The Achaete-scute homolog 1 (Ascl1) protein regulates a large subset of genes that leads neuronal progenitor cells to distinctive differentiation pathways during human brain development. Although it is well known that Ascl1 binds DNA as a homo- or heterodimer via its basic helix-loop-helix (bHLH) motif, little is known about the conformational sampling properties of the DNA-free full-length protein, and in particular about the bHLH domain-flanking N- and C-terminal segments, which are predicted to be highly disordered in solution. The structural heterogeneity, low solubility, and high aggregation propensity of Ascl1 in aqueous buffer solutions make high-resolution studies of this protein a challenging task. Here, we have adopted a fragment-based strategy that allowed us to obtain high-quality NMR data providing, to our knowledge, the first comprehensive high-resolution information on the structural propensities and conformational dynamics of Ascl1. The emerging picture is that of an overall extended and highly dynamic polypeptide chain comprising three helical segments and lacking persistent long-range interactions. We also show that the C-terminal helix of the bHLH domain is involved in intermolecular interactions, even in the absence of DNA. Our results contribute to a better understanding of the mechanisms of action that govern the regulation of proneural transcription factors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Espectroscopía de Resonancia Magnética , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , ADN/metabolismo , Humanos , Dominios ProteicosRESUMEN
NMR spectroscopy is a powerful tool for studying molecular dynamics at atomic resolution simultaneously for a large number of nuclear sites. In this communication, we combine two powerful NMR techniques, relaxation-dispersion NMR and real-time NMR, in order to obtain unprecedented information on the conformational exchange dynamics present in short-lived excited protein states, such as those transiently accumulated during protein folding. We demonstrate the feasibility of the approach for the amyloidogenic protein ß2-microglobulin that folds via an intermediate state which is believed to be responsible for the onset of the aggregation process leading to amyloid formation.
Asunto(s)
Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Microglobulina beta-2/química , Humanos , Conformación Proteica , Pliegue de Proteína , Factores de TiempoRESUMEN
Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (â¼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and ß-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.
Asunto(s)
Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Sitios de Unión , Escherichia coli , Cinética , Espectroscopía de Resonancia Magnética , Fosforilación , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Electricidad Estática , Proteínas no Estructurales Virales/aislamiento & purificación , Difracción de Rayos X , Dominios Homologos srcRESUMEN
Thanks to recent improvements in NMR instrumentation, pulse sequence design, and sample preparation, a panoply of new NMR tools has become available for atomic resolution characterization of intrinsically disordered proteins (IDPs) that are optimized for the particular chemical and spectroscopic properties of these molecules. A wide range of NMR observables can now be measured on increasingly complex IDPs that report on their structural and dynamic properties in isolation, as part of a larger complex, or even inside an entire living cell. Herein we present basic NMR concepts, as well as optimised tools available for the study of IDPs in solution. In particular, the following sections are discussed hereafter: a short introduction to NMR spectroscopy and instrumentation (Sect. 3.1), the effect of order and disorder on NMR observables (Sect. 3.2), particular challenges and bottlenecks for NMR studies of IDPs (Sect. 3.3), 2D HN and CON NMR experiments: the fingerprint of an IDP (Sect. 3.4), tools for overcoming major bottlenecks of IDP NMR studies (Sect. 3.5), 13C detected experiments (Sect. 3.6), from 2D to 3D: from simple snapshots to site-resolved characterization of IDPs (Sect. 3.7), sequential NMR assignment: 3D experiments (Sect. 3.8), high-dimensional NMR experiments (nD, with n>3) (Sect. 3.9) and conclusions and perspectives (Sect. 3.10).
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación ProteicaRESUMEN
HET(ex)-SOFAST NMR (Schanda et al. in J Biomol NMR 33:199-211, 2006) has been proposed some years ago as a fast and sensitive method for semi-quantitative measurement of site-specific amide-water hydrogen exchange effects along the backbone of proteins. Here we extend this concept to BEST readout sequences that provide a better resolution at the expense of some loss in sensitivity. We discuss the theoretical background and implementation of the experiment, and demonstrate its performance for an intrinsically disordered protein, 2 well folded globular proteins, and a transiently populated folding intermediate state. We also provide a critical evaluation of the level of accuracy that can be obtained when extracting quantitative exchange rates from HET(ex) NMR measurements.
Asunto(s)
Hidrógeno/química , Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Espectroscopía de Protones por Resonancia Magnética , Agua/química , Amidas/química , Proteínas y Péptidos de Choque por Frío/química , Proteínas de Escherichia coli/química , Humanos , Isótopos de Nitrógeno , Ubiquitina/química , alfa-Sinucleína/química , Microglobulina beta-2/químicaRESUMEN
Three experiments, BEST-TROSY HNCA+, HNCO+ and HNCACB+ are presented for sequential backbone resonance assignment of (13)C, (15)N labelled proteins. The novelty of these experiments with respect to conventional pulse sequences is the detection of additional orthogonal coherence transfer pathways that results in enhanced sensitivity for sequential correlations without significantly compromising the intensity of intra-residue correlation peaks. In addition, a 2-step phase cycle separates peaks originating from the orthogonal coherence transfer pathways in 2 sub-spectra, thus providing similar information as obtained from performing a pair of sequential and intra-residue correlation experiments.
Asunto(s)
Isótopos de Carbono/química , Isótopos de Nitrógeno/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/químicaRESUMEN
Intrinsically disordered proteins (IDPs) are a class of highly flexible proteins whose characterization by NMR spectroscopy is complicated by severe spectral overlaps. The development of experiments designed to facilitate the sequence-specific assignment procedure is thus very important to improve the tools for the characterization of IDPs and thus to be able to focus on IDPs of increasing size and complexity. Here, we present and describe the implementation of a set of novel ¹H-detected 5D experiments, (HACA)CON(CACO)NCO(CA)HA, BT-(H)NCO(CAN)CONNH and BT-HN(COCAN)CONNH, optimized for the study of highly flexible IDPs that exploit the best resolved correlations, those involving the carbonyl and nitrogen nuclei of neighboring amino acids, to achieve sequence-specific resonance assignment. Together with the analogous recently proposed pulse schemes based on ¹³C detection, they form a complete set of experiments for sequence-specific assignment of highly flexible IDPs. Depending on the particular sample conditions (concentration, lifetime, pH, temperature, etc.), these experiments present certain advantages and disadvantages that will be discussed. Needless to say, that the availability of a variety of complementary experiments will be important for accurate determination of resonance frequencies in complex IDPs.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono/química , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single-particle-tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side-chain protonation of the conserved residues E212 and H62, altering the hydrogen-bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green-to-Red photoconversion properties differ between the A and B states. The chromophore in the A-state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B-state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.
Asunto(s)
Colorantes , Microscopía , Proteínas Luminiscentes/químicaRESUMEN
Src homology 3 (SH3) domains are widely known for their ability to interact with other proteins using the canonical PxxP binding motif. Besides those well-characterized interaction modes, there is an increasing number of SH3 domain-containing complexes that lack this motif. Here we characterize the interaction of SH3 domains, in particular the Bin1-SH3 domain, with the intrinsically disordered part of nonstructural protein 5A of the hepatitis C virus using noncanonical binding sites in addition to its PxxP motif. These binding regions partially overlap with regions that have previously been identified as having an increased propensity to form α-helices. Remarkably, upon interaction with the Bin1-SH3 domain, the α-helical propensity decreases and a fuzzy complex is formed.
Asunto(s)
Proteínas no Estructurales Virales/química , Dominios Homologos src/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Hepacivirus/metabolismo , Modelos Moleculares , Proteínas no Estructurales Virales/metabolismoRESUMEN
The characterization of the conformational properties of intrinsically disordered proteins (IDPs), and their interaction modes with physiological partners has recently become a major research topic for understanding biological function on the molecular level. Although multidimensional NMR spectroscopy is the technique of choice for the study of IDPs at atomic resolution, the intrinsically low resolution, and the large peak intensity variations often observed in NMR spectra of IDPs call for resolution- and sensitivity-optimized pulse schemes. We present here a set of amide proton-detected 3D BEST-TROSY correlation experiments that yield the required sensitivity and spectral resolution for time-efficient sequential resonance assignment of large IDPs. In addition, we introduce two proline-edited 2D experiments that allow unambiguous identification of residues adjacent to proline that is one of the most abundant amino acids in IDPs. The performance of these experiments, and the advantages of BEST-TROSY pulse schemes are discussed and illustrated for two IDPs of similar length (~270 residues) but with different conformational sampling properties.
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Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/análisis , Proteínas/química , Conformación ProteicaRESUMEN
The characterization of both the structure and the conformational dynamics of biological macromolecules, namely proteins and nucleic acids, is required for understanding the molecular mechanisms underlying physiological function and disease. Molecular dynamics involves the transient departure from the ground-state structures to populate short-lived excited state conformations that can play important functional roles. Real-time multi-dimensional NMR spectroscopy represents a unique tool for investigating dynamic molecular processes occurring on time scales of seconds or longer, providing atomic-resolution information about short-lived states. In this minireview, we discuss recent progress made in the field of fast real-time multidimensional NMR. The potential of these new methods is illustrated for several biomolecular systems that have recently been studied by fast real-time multidimensional NMR.
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Enfermedad , Espectroscopía de Resonancia Magnética/métodos , Ácidos Nucleicos/química , Pliegue de Proteína , Proteínas/química , Humanos , Lactalbúmina/química , LigandosRESUMEN
RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2-K209 residue in the RIPK2-XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.
Asunto(s)
Bioensayo , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/genética , Microscopía por Crioelectrón , Fosforilación , UbiquitinaciónRESUMEN
Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Uranio , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Compuestos Férricos/metabolismo , Membrana Celular/metabolismo , Cationes/química , Cationes/metabolismo , Uranio/química , Proteínas de Unión al Calcio/metabolismoRESUMEN
Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g., states that become transiently populated during protein folding. Here we present a strategy for obtaining sequential NMR assignments as well as atom-resolved information on structural and dynamic features within a folding intermediate of the amyloidogenic protein ß2-microglobulin that has a half-lifetime of only 20 min.