RESUMEN
Microtubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). Here we outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed points through four conserved hydrophobic regions. By NMR and live cell analyses we reveal that two neighboring residues in the first hydrophobic binding motif are crucial for the microtubule interaction. The microtubule-binding mechanism of CC1 is reminiscent to that of the prominent neuropathology-related protein Tau, indicating evolutionary convergence of MAP functions across animal and plant cells.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tolerancia a la Sal/fisiología , Proteínas tau/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Celulosa/biosíntesis , Glucosiltransferasas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Asociadas a Microtúbulos/genética , Tolerancia a la Sal/genética , Plantones/crecimiento & desarrolloRESUMEN
Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, ß-sheet-rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/química , Biopelículas/crecimiento & desarrollo , Bacillus subtilis/química , Proteínas Bacterianas/metabolismo , Calorimetría , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Metaloendopeptidasas/química , Microscopía Electrónica , Modelos Moleculares , Peso Molecular , Conformación Proteica , Homología Estructural de Proteína , UltracentrifugaciónRESUMEN
ß-barrel proteins mediate nutrient uptake in bacteria and serve vital functions in cell signaling and adhesion. For the 14-strand outer membrane protein G of Escherichia coli, opening and closing is pH-dependent. Different roles of the extracellular loops in this process were proposed, and X-ray and solution NMR studies were divergent. Here, we report the structure of outer membrane protein G investigated in bilayers of E. coli lipid extracts by magic-angle-spinning NMR. In total, 1847 inter-residue 1H-1H and 13C-13C distance restraints, 256 torsion angles, but no hydrogen bond restraints are used to calculate the structure. The length of ß-strands is found to vary beyond the membrane boundary, with strands 6-8 being the longest and the extracellular loops 3 and 4 well ordered. The site of barrel closure at strands 1 and 14 is more disordered than most remaining strands, with the flexibility decreasing toward loops 3 and 4. Loop 4 presents a well-defined helix.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Membrana Dobles de Lípidos/química , Porinas/química , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.
Asunto(s)
Leucina/metabolismo , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/química , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Bacterias/enzimología , Sitios de Unión , Espectroscopía de Resonancia Magnética , Unión ProteicaRESUMEN
Phytochromes are red/far-red photochromic photoreceptors acting as master regulators of development in higher plants, thereby controlling transcription of about 20 % of their genes. Light-induced isomerization of the bilin chromophore leads to large rearrangements in protein structure, whereby the role of protonation dynamics and charge distribution is of particular interest. To help unravel the inherent mechanisms, we present two-dimensional dynamic nuclear polarization (DNP) enhanced solid-state magic-angle spinning (MAS) NMR spectra of the functional sensory module of the cyanobacterial phytochrome Cph1. To this end, the pyrrole ring nitrogen signals were assigned unequivocally, enabling us to locate the positive charge of the phycocyanobilin (PCB) chromophore. To help analyze proton exchange pathways, the proximity of PCB ring nitrogen atoms and functionally relevant H2 O molecules was also determined. Our study demonstrates the value of DNP in biological solid-state MAS NMR spectroscopy.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Fotorreceptores de Plantas/química , Fitocromo/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K. The combined effects of enhancement, depolarization, and proton longitudinal relaxation are surprisingly sample-specific. At 200 K, DNP on SH3 domain standard samples yields a 15-fold increase in signal-to-noise over a sample without radicals. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours, respectively. Decreasing enhancements with increasing 2H-content at the CH2 sites of the TEMPO rings in CD3-TOTAPOL highlight the importance of protons in a sphere of 4-6 Å around the nitroxyl group, presumably for polarization pickup from electron spins.
RESUMEN
Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Señales de Clasificación de Proteína , Ribosomas/química , Secuencia de Aminoácidos , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes , Ribosomas/genéticaRESUMEN
Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long-range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.
Asunto(s)
Amiloide/química , Óxidos N-Cíclicos/química , Espectroscopía de Resonancia Magnética/métodos , Propanoles/química , Animales , Sitios de Unión , Humanos , Estructura Molecular , Propiedades de SuperficieRESUMEN
Curli are functional bacterial amyloids produced by an intricate biogenesis machinery. Insights into their folding and regulation can advance our understanding of amyloidogenesis. However, gaining detailed structural information of amyloids, and their tendency for structural polymorphisms, remains challenging. Herein we compare high-quality solid-state NMR spectra from biofilm-derived and recombinantly produced curli and provide evidence that they adopt a similar, well-defined ß-solenoid arrangement. Curli subunits consist of five sequence repeats, resulting in severe spectral overlap. Using segmental isotope labeling, we obtained the unambiguous sequence-specific resonance assignments and secondary structure of one repeat, and demonstrate that all repeats are most likely structurally equivalent.
Asunto(s)
Amiloide/química , Biopelículas , Fimbrias Bacterianas/química , Fimbrias Bacterianas/metabolismo , Secuencia de Aminoácidos , Espectroscopía de Resonancia MagnéticaRESUMEN
Membrane proteins are largely underrepresented among available atomic-resolution structures. The use of detergents in protein purification procedures hinders the formation of well-ordered crystals for X-ray crystallography and leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy is an emerging method for membrane-protein structural biology that can overcome these technical problems. Here we present the solid-state NMR structure of the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA). The sample was derived from crystallization trials that yielded only poorly diffracting microcrystals. We solved the structure using a single, uniformly (13)C- and (15)N-labeled sample. In addition, solid-state NMR allowed us to acquire information on the flexibility and mobility of parts of the structure, which, in combination with evolutionary conservation information, presents new insights into the autotransport mechanism of YadA.
Asunto(s)
Adhesinas Bacterianas/química , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , Cristalización , Modelos MolecularesRESUMEN
We report the complete solid-state MAS NMR resonance assignment of a medium-sized, trimeric membrane protein, YadA-M. The protein YadA (Yersinia adhesin A) is an important virulence factor of enteropathogenic Yersinia species (such as Yersinia enterocolitica and Yersinia pseudotuberculosis). YadA is localized on the bacterial cell surface and is involved in adhesion to host cells and tissues. It is anchored in the outer membrane by a transmembrane anchor domain (YadA-M). This domain hosts the so-called autotransporter function of YadA: it transports its own N-terminal domain through the outer membrane. The assignment is based on a dataset that consisted of several MAS NMR correlation spectra, recorded on a single, uniformly (13)C, (15)N- labelled microcrystalline preparation. Except for the single C-terminal residue and the mobile strep tag, we were able to completely assign YadA-M. From this, secondary structure elements were predicted, which, combined with several long-range interstrand restraints, yielded the architecture of the ß-sheet.
RESUMEN
An efficient approach to determine the structures of symmetric protein aggregates from liquid and solid-state NMR data is presented. Any symmetry can be used (cyclic or dihedral point symmetries, helical symmetries, crystallographic symmetries). Because the starting point is the random structure of the monomer, the knowledge of the 3D structure of the monomer is not required.
Asunto(s)
Espectroscopía de Resonancia Magnética , Modelos Moleculares , Proteínas/química , Cristalografía por Rayos X , Conformación Proteica , Multimerización de ProteínaRESUMEN
In nuclear magnetic resonance spectroscopy, experimental limits due to the radiofrequency transmitter and/or coil means that conventional radiofrequency pulses ("hard pulses") are sometimes not sufficiently powerful to excite magnetization uniformly over a desired range of frequencies. Effects due to nonuniform excitation are most frequently encountered at high magnetic fields for nuclei with a large range of chemical shifts. Using optimal control theory, we have designed broadband excitation pulses that are suitable for solid-state samples under magic-angle-spinning conditions. These pulses are easy to implement, robust to spinning frequency variations, and radiofrequency inhomogeneities, and only four times as long as a corresponding hard pulse. The utility of these pulses for uniformly exciting (13) C nuclei is demonstrated on a 900 MHz (21.1 T) spectrometer.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Isótopos de Carbono , Simulación por Computador , Campos Magnéticos , Ondas de RadioRESUMEN
With the technique of dynamic nuclear polarization (DNP) signal intensity in solid-state MAS-NMR experiments can be enhanced by 2-3 orders of magnitude. DNP relies on the transfer of electron spin polarization from unpaired electrons to nuclear spins. For this reason, stable organic biradicals such as TOTAPOL are commonly added to samples used in DNP experiments. We investigated the effects of biradical concentration on the relaxation, enhancement, and intensity of NMR signals, employing a series of samples with various TOTAPOL concentrations and uniformly (13)C, (15)N labeled proline. A considerable decrease of the NMR relaxation times (T(1), T(2)(∗), and T(1)(ρ)) is observed with increasing amounts of biradical due to paramagnetic relaxation enhancement (PRE). For nuclei in close proximity to the radical, decreasing T(1)(ρ) reduces cross-polarization efficiency and decreases in T(2)(∗) broaden the signal. Additionally, paramagnetic shifts of (1)H signals can cause further line broadening by impairing decoupling. On average, the combination of these paramagnetic effects (PE; relaxation enhancement, paramagnetic shifts) quenches NMR-signals from nuclei closer than 10Å to the biradical centers. On the other hand, shorter T(1) times allow the repetition rate of the experiment to be increased, which can partially compensate for intensity loss. Therefore, it is desirable to optimize the radical concentration to prevent additional line broadening and to maximize the signal-to-noise observed per unit time for the signals of interest.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Algoritmos , Isótopos de Carbono , Espectroscopía de Resonancia por Spin del Electrón , Marcaje Isotópico , Neurotoxinas/química , Isótopos de Nitrógeno , Prolina/química , Protones , Receptores Colinérgicos/metabolismoRESUMEN
Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Resonancia Magnética Nuclear Biomolecular/instrumentación , Resonancia Magnética Nuclear Biomolecular/métodos , Animales , Humanos , Ligandos , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol "glass" leads to well-resolved spectra, yet a considerable enhancement (ε = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.
Asunto(s)
Membrana Celular/metabolismo , Proteínas Neurotóxicas de Elápidos/metabolismo , Elapidae/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Receptores Colinérgicos/metabolismo , Animales , Órgano Eléctrico/citología , Modelos Moleculares , Unión Proteica , TorpedoRESUMEN
Solid-state magic-angle-spinning (MAS) NMR of proteins has undergone many rapid methodological developments in recent years, enabling detailed studies of protein structure, function and dynamics. Software development, however, has not kept pace with these advances and data analysis is mostly performed using tools developed for solution NMR which do not directly address solid-state specific issues. Here we present additions to the CcpNmr Analysis software package which enable easier identification of spinning side bands, straightforward analysis of double quantum spectra, automatic consideration of non-uniform labelling schemes, as well as extension of other existing features to the needs of solid-state MAS data. To underpin this, we have updated and extended the CCPN data model and experiment descriptions to include transfer types and nomenclature appropriate for solid-state NMR experiments, as well as a set of experiment prototypes covering the experiments commonly employed by solid-sate MAS protein NMR spectroscopists. This work not only improves solid-state MAS NMR data analysis but provides a platform for anyone who uses the CCPN data model for programming, data transfer, or data archival involving solid-state MAS NMR data.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Programas Informáticos , Análisis de Elementos Finitos , Modelos Químicos , Estadística como AsuntoRESUMEN
X-ray crystallography using synchrotron radiation and the technique of dynamic nuclear polarization (DNP) in nuclear magnetic resonance (NMR) require samples to be kept at temperatures below 100 K. Protein dynamics are poorly understood below the freezing point of water and down to liquid nitrogen temperatures. Therefore, we investigate the α-spectrin SH3 domain by magic angle spinning (MAS) solid state NMR (ssNMR) at various temperatures while cooling slowly. Cooling down to 95 K, the NMR-signals of SH3 first broaden and at lower temperatures they separate into several peaks. The coalescence temperature differs depending on the individual residue. The broadening is shown to be inhomogeneous by hole-burning experiments. The coalescence behavior of 26 resolved signals (of 62) was compared to water proximity and crystal structure Debye-Waller factors (B-factors). Close proximity to the solvent and large B-factors (i.e. mobility) lead, generally, to a higher coalescence temperature. We interpret a high coalescence temperature as indicative of a large number of magnetically inequivalent populations at cryogenic temperature.
Asunto(s)
Frío , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Conformación Proteica , Espectrina/química , Dominios Homologos srcRESUMEN
Save the last WALTZ for me: the use of simultaneous proton and deuterium cross-polarization for (13)C CPMAS NMR spectroscopy in highly deuterated proteins is discussed. The aim of the new method introduced herein, triple-resonance cross-polarization, is to increase the sensitivity of the carbon-detected methods in such systems.
RESUMEN
We present a Floquet theory approach for the analysis of homonuclear recoupling assisted by radio frequency (RF) irradiation of surrounding heteronuclear spins. This description covers a broad range of systems from fully protonated to deuterated proteins, focusing in detail on recoupling via protons and deuterons separately as well as simultaneously by the double nucleus enhanced recoupling (DONER) scheme. The theoretical description, supported by numerical simulations and compared to experimental results from a partially deuterated model compound, indicates that in perdeuterated systems setting the RF amplitude equal to the magic angle spinning (MAS) frequency is not necessarily optimal for recoupling via (1)H and/or (2)H nuclei and modified recoupling conditions are identified.