RESUMEN
Biofilm-protected pathogenic Staphylococcus aureus causes chronic infections that are difficult to treat. An essential building block of these biofilms are functional amyloid fibrils that assemble from phenol-soluble modulins (PSMs). PSMα1 cross-seeds other PSMs into cross-ß amyloid folds and is therefore a key element in initiating biofilm formation. However, the paucity of high-resolution structures hinders efforts to prevent amyloid assembly and biofilm formation. Here, we present a 3.5 Å resolution density map of the major PSMα1 fibril form revealing a left-handed cross-ß fibril composed of two C2-symmetric U-shaped protofilaments whose subunits are unusually tilted out-of-plane. Monomeric α-helical PSMα1 is extremely cytotoxic to cells, despite the moderate toxicity of the cross-ß fibril. We suggest mechanistic insights into the PSM functional amyloid formation and conformation transformation on the path from monomer-to-fibril formation. Details of PSMα1 assembly and fibril polymorphism suggest how S. aureus utilizes functional amyloids to form biofilms and establish a framework for developing therapeutics against infection and antimicrobial resistance.
Asunto(s)
Amiloide , Biopelículas , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/fisiología , Biopelículas/crecimiento & desarrollo , Amiloide/metabolismo , Amiloide/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Conformación Proteica , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Modelos MolecularesRESUMEN
Functional bacterial amyloids play a crucial role in the formation of biofilms, which mediate chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC amyloid fibrillar protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone-like protein FapA on this process. Using solution nuclear magnetic resonance (NMR), we recently showed that both FapC and FapA are intrinsically disordered proteins (IDPs). Here, the secondary structure propensities (SSPs) are compared to alphafold (DeepMind, protein structure prediction tool/algorithm: https://alphafold.ebi.ac.uk/) models. We further demonstrate that the FapA chaperone interacts with FapC and significantly slows down the formation of FapC fibrils. Our NMR titrations reveal ~ 18% of the resonances show FapA-induced chemical shift perturbations (CSPs), which has not been previously observed, the largest being for A82, N201, C237, C240, A241, and G245. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the repeat-1 (R1)/loop-2 (L2) and L2/R3 transition areas and at the C-terminus of FapC. Remarkably, ~ 90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC, the largest being for S63, A69, A80, and I92. A temperature-dependent effect of FapA was observed on FapC by thioflavin T (ThT) and NMR experiments. This study provides a detailed understanding of the interaction between the FapA and FapC, shedding light on the regulation and slowing down of amyloid formation, and has important implications for the development of therapeutic strategies targeting biofilms and associated infections.
Asunto(s)
Amiloide , Proteínas Bacterianas , Biopelículas , Chaperonas Moleculares , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Amiloide/metabolismo , Amiloide/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Pseudomonas/metabolismo , Estructura Secundaria de Proteína , Resonancia Magnética Nuclear BiomolecularRESUMEN
We present a multidimensional magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native Pseudomonas fluorescens colony biofilms at natural abundance without isotope-labelling. By using a high-resolution INEPT-based 2D 1H-13C ssNMR spectrum and thorough peak deconvolution at the 1D ssNMR spectra, approximately 80/134 (in 1D/2D) distinct biofilm chemical sites were identified. We compared CP and INEPT 13C ssNMR spectra to differentiate signals originating from the mobile and rigid fractions of the biofilm, and qualitatively determined dynamical changes by comparing CP buildup behaviors. Protein and polysaccharide signals were differentiated and identified by utilizing FapC protein signals as a template, a biofilm forming functional amyloid from Pseudomonas. We identified several biofilm polysaccharide species such as glucose, mannan, galactose, heptose, rhamnan, fucose and N-acylated mannuronic acid by using 1H and 13C chemical shifts obtained from the 2D spectrum. To our knowledge, this study marks the first high-resolution multidimensional ssNMR characterization of a native bacterial biofilm. Our experimental pipeline can be readily applied to other in vitro biofilm model systems and natural biofilms and holds the promise of making a substantial impact on biofilm research, fostering new ideas and breakthroughs to aid in the development of strategic approaches to combat infections caused by biofilm-forming bacteria.
Asunto(s)
Amiloide , Pseudomonas , Espectroscopía de Resonancia Magnética , Biopelículas , PolisacáridosRESUMEN
We present a high-resolution 1D and 2D magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native Pseudomonas fluorescens colony biofilms at natural abundance without isotope-labelling. By using a high-resolution INEPT-based 2D 1 H- 13 C ssNMR spectrum and thorough peak deconvolution approach at the 1D ssNMR spectra, approximately 80/134 (in 1D/2D) distinct biofilm chemical sites were identified. We compared CP and INEPT 13 C ssNMR spectra to different signals originating from the mobile and rigid fractions of the biofilm, and qualitative determined dynamical changes by comparing CP buildup behaviors. Protein and polysaccharide signals were differentiated and identified by utilizing FapC signals as a template, a biofilm forming functional amyloid from Pseudomonas . We also attempted to identify biofilm polysaccharide species by using 1 H/ 13 C chemical shifts obtained from the 2D spectrum. This study marks the first demonstration of high-resolution 2D ssNMR spectroscopy for characterizing native bacterial biofilms and expands the scope of ssNMR in studying biofilms. Our experimental pipeline can be readily applied to other in vitro biofilm model systems and natural biofilms and holds the promise of making a substantial impact on biofilm research, fostering new ideas and breakthroughs to aid in the development of strategic approaches to combat infections caused by biofilm-forming bacteria.
RESUMEN
FapA is an accessory protein within the biofilm forming functional bacterial amyloid related fap-operon in Pseudomonas, and maybe a chaperone for FapC controlling its fibrillization. To allow further structural analysis, here we present a complete sequential assignment of 1Hamide, 13Cα, 13Cß, and 15N NMR resonances for the functional form of the monomeric soluble FapA protein, comprising amino acids between 29 and 152. From these observed chemical shifts, the secondary structure propensities (SSPs) were determined. FapA predominantly adopts a random coil conformation, however, we also identified small propensities for α-helical and ß-strand conformations. Notably, these observed SSPs are smaller compared to the ones we recently observed for the monomeric soluble FapC protein. These NMR results provide valuable insights into the activity of FapA in functional amyloid formation and regulation, that will also aid developing strategies targeting amyloid formation within biofilms and addressing chronic infections.
Asunto(s)
Proteínas Amiloidogénicas , Pseudomonas , Pseudomonas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Amiloide/química , BiopelículasRESUMEN
Functional bacterial amyloids provide structural scaffolding to bacterial biofilms. In contrast to the pathological amyloids, they have a role in vivo and are tightly regulated. Their presence is essential to the integrity of the bacterial communities surviving in biofilms and may cause serious health complications. Targeting amyloids in biofilms could be a novel approach to prevent chronic infections. However, structural information is very scarce on them in both soluble monomeric and insoluble fibrillar forms, hindering our molecular understanding and strategies to fight biofilm related diseases. Here, we present solution-state NMR assignment of 250 amino acid long biofilm-forming functional-amyloid FapC from Pseudomonas aeruginosa. We studied full-length (FL) and shorter minimalistic-truncated (L2R3C) FapC constructs without the signal-sequence that is required for secretion. 91% and 100% backbone NH resonance assignments for FL and L2R3C constructs, respectively, indicate that soluble monomeric FapC is predominantly disordered, with sizeable secondary structural propensities mostly as PP2 helices, but also as α-helices and ß-sheets highlighting hotspots for fibrillation initiation interface. A shorter construct showing almost identical NMR chemical shifts highlights the promise of utilizing it for more demanding solid-state NMR studies that require methods to alleviate signal redundancy due to almost identical repeat units. This study provides key NMR resonance assignments for future structural studies of soluble, pre-fibrillar and fibrillar forms of FapC.
Asunto(s)
Amiloide , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Resonancia Magnética Nuclear Biomolecular , Amiloide/química , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/metabolismo , BiopelículasRESUMEN
A novel deuterium-excited and proton-detected quadruple-resonance three-dimensional (3D) 2HαcαNH MAS nuclear magnetic resonance (NMR) method is presented to obtain site-specific 2Hα deuterium quadrupolar couplings from protein backbone, as an extension to the 2D version of the experiment reported earlier. Proton-detection results in high sensitivity compared to the heteronuclei detection methods. Utilizing four independent radiofrequency (RF) channels (quadruple-resonance), we managed to excite the 2Hα, then transfer deuterium polarization to its attached Cα, followed by polarization transfers to the neighboring backbone nitrogen and then to the amide proton for detection. This experiment results in an easy to interpret HSQC-like 2D 1H-15N fingerprint NMR spectrum, which contains site-specific deuterium quadrupolar patterns in the indirect third dimension. Provided that four-channel NMR probe technology is available, the setup of the 2HαcαNH experiment is relatively straightforward, by using low power deuterium excitation and polarization transfer schemes we have been developing. To our knowledge, this is the first demonstration of a quadruple-resonance MAS NMR experiment to link 2Hα quadrupolar couplings to proton-detection, extending our previous triple-resonance demonstrations. Distortion-free excitation and polarization transfer of â¼160-170 kHz 2Hα quadrupolar coupling were presented by using a deuterium RF strength of â¼20 kHz. From these 2Hα patterns, an average backbone order parameter of S = 0.92 was determined on a deuterated SH3 sample, with an average η = 0.22. These indicate that SH3 backbone represents sizable dynamics in the microsecond timescale where the 2Hα lineshape is sensitive. Moreover, site-specific 2Hα T1 relaxation times were obtained for a proof of concept. This 3D 2HαcαNH NMR experiment has the potential to determine structure and dynamics of perdeuterated proteins by utilizing deuterium as a novel reporter.
Asunto(s)
Proteínas , Protones , Deuterio/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Espectroscopía de Resonancia Magnética/métodosRESUMEN
Protein aggregation and amyloid formation have historically been linked with various diseases such as Alzheimer's and Parkinson's disease, but recently functional amyloids have gained a great deal of interest in not causing a disease and having a distinct function in vivo. Functional bacterial amyloids form the structural scaffold in bacterial biofilms and provide a survival strategy for the bacteria along with antibiotic resistance. The formation of functional amyloids happens extracellularly which differs from most disease related amyloids. Studies of functional amyloids have revealed several distinctions compared to disease related amyloids including primary structures designed to optimize amyloid formation while still retaining a controlled assembly of the individual subunits into classical cross-ß-sheet structures, along with a unique cross-α-sheet amyloid fold. Studies have revealed that functional amyloids interact with components found in the extracellular matrix space such as lipids from membranes and polymers from the biofilm. Intriguingly, a level of complexity is added as functional amyloids also interact with several disease related amyloids and a causative link has even been established between functional amyloids and neurodegenerative diseases. It is hence becoming increasingly clear that functional amyloids are not inert protein structures found in bacterial biofilms but interact with many different components including human proteins related to pathology. Gaining a clear understanding of the factors governing the interactions will lead to improved strategies to combat biofilm associated infections and the correlated antibiotic resistance. In the current review we summarize the current state of the art knowledge on this exciting and fast growing research field of biofilm forming bacterial functional amyloids, their structural features and interaction partners.
RESUMEN
Determination of protein structure and dynamics is key to understand the mechanism of protein action. Perdeuterated proteins have been used to obtain high resolution/sensitivty NMR experiments via proton-detection. These methods utilizes 1H, 13C and 15N nuclei for chemical shift dispersion or relaxation probes, despite the existing abundant deuterons. However, a high-sensitivity NMR method to utilize deuterons and e.g. determine site-specific deuterium quadrupolar pattern information has been lacking due to technical difficulties associated with deuterium's large quadrupolar couplings. Here, we present a novel deuterium-excited and proton-detected three-dimensional 2H-13C-1H MAS NMR experiment to utilize deuterons and to obtain site-specific methyl 2H quadrupolar patterns on detuterated proteins for the first time. A high-resolution fingerprint 1H-15N HSQC-spectrum is correlated with the anisotropic deuterium quadrupolar tensor in the third dimension. Results from a model perdeuterated protein has been shown.
Asunto(s)
Proteínas , Protones , Deuterio/química , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/químicaRESUMEN
Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.
Asunto(s)
Atrofia de Múltiples Sistemas/genética , Enfermedades Neurodegenerativas/genética , Sinucleinopatías/patología , alfa-Sinucleína/genética , Animales , Línea Celular , Humanos , Cuerpos de Inclusión/patología , Ratones , Ratones Transgénicos , Atrofia de Múltiples Sistemas/patología , Proteínas del Tejido Nervioso/genética , Oligodendroglía/metabolismo , Conformación Proteica , Deficiencias en la Proteostasis/genética , Sustancia Negra/patología , alfa-Sinucleína/toxicidadRESUMEN
We describe a 2H based MAS nuclear magnetic resonance (NMR) method to obtain site-specific molecular dynamics of biomolecules. The method utilizes the use of deuterium nucleus as a spin label that is proven to be very useful in dynamics studies of solid biological and functional materials. The aim is to understand overall characteristics of protein backbone and side-chain motions for CD3, CD2 and CD groups, in terms of timescale, type and activation energy of the underlying processes. Variable temperature two-dimensional (2D) 2H-13C correlation MAS NMR spectra were recorded for the uniformly 2H,13C,15N labelled Alanine and microcrystalline SH3 at a broad temperature range, from 320 K down to 100 K. First, the deuterium quadrupolar-coupling constant from specific D-C sites is obtained with the 2D experiment by utilizing carbon chemical shifts. Second, the static quadrupolar patterns are obtained at 100 K. Third, variable temperature approach enabled the observation of quadrupolar pattern over different motional regimes; slow, intermediate and fast. And finally, the apparent activation energies for C-D sites are determined and compared, by evaluating the temperature induced signal intensities. This information led to the determination of the dynamic processes for different D-C sites at a broad range of temperature and motional timescales. This is a first representation of 2D 2H-13C MAS NMR approach applied to fully isotope labelled deuterated protein covering 220 K temperature range.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas , Deuterio , Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear BiomolecularRESUMEN
Protein fibrillation is traditionally associated with misfolding, loss of functional phenotype, and gain of toxicity in neurodegenerative diseases. However, many organisms exploit fibrils in the form of functional amyloids (FA), as seen in bacteria, such as E. coli, Salmonella, Bacillus, and Pseudomonas. Here, we provide structural information and mechanistic data for fibrillation of the smallest amyloidogenic truncation unit along with the full-length version (FL) of the major amyloid protein FapC from Pseudomonas, predicted to consist of three ß-hairpin-forming imperfect repeats separated by disordered regions. Using a series of truncation mutants, we establish that the putative loops (linkers) increase the rate of aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates in a pathway dominated by secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. SAXS on FapC FL, R3C, and remaining truncation constructs resolves two major coexisting species in the fibrillation process, namely pre-fibrillar loosely aggregated monomers, and cylindrical, elliptical cross-section fibrils. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα-Cß chemical shifts, indicative of a predominant ß-sheet topology with some α-helix or loop chemical shifts. Our work emphasizes the complex nature of FapC fibrillation. In addition, we are able to deduce the importance of non-repeat regions (i.e., predicted loops), which enhance the amyloid protein aggregation and their influence on the polymorphism of the fibril architecture.
Asunto(s)
Amiloide/química , Proteínas Amiloidogénicas/metabolismo , Proteínas Bacterianas/metabolismo , Agregado de Proteínas , Pseudomonas/metabolismo , Secuencia de Aminoácidos , Proteínas Amiloidogénicas/genética , Proteínas Bacterianas/genética , Mutación , Pseudomonas/genéticaRESUMEN
In this article we give an overview over the use of DNP-enhanced solid-state NMR spectroscopy for the investigation of unfolded, disordered and misfolded proteins. We first provide an overview over studies in which DNP spectroscopy has successfully been applied for the structural investigation of well-folded amyloid fibrils formed by short peptides as well as full-length proteins. Sample cooling to cryogenic temperatures often leads to severe line broadening of resonance signals and thus a loss in resolution. However, inhomogeneous line broadening at low temperatures provides valuable information about residual dynamics and flexibility in proteins, and, in combination with appropriate selective isotope labeling techniques, inhomogeneous linewidths in disordered proteins or protein regions may be exploited for evaluation of conformational ensembles. In the last paragraph we highlight some recent studies where DNP-enhanced MAS-NMR-spectroscopy was applied to the study of disordered proteins/protein regions and inhomogeneous sample preparations.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Desplegamiento Proteico , Proteínas/química , Humanos , Estabilidad Proteica , Proteínas/metabolismo , TemperaturaRESUMEN
The new compound Sr(BH4)2(NH3BH3)2 has been synthesized and characterized with in situ powder X-ray diffraction and fast (28 or 60 kHz) magic angle spinning 1H, 11B and 15N NMR and structurally optimized with density functional theory calculations. This investigation reveals complex structural rearrangements for this compound as a function of temperature. A room temperature orthorhombic polymorph, α-Sr(BH4)2(NH3BH3)2, with the space group symmetry Pbca, has been determined with a layered structure of alternating ammonia borane and Sr(BH4)2, partially stabilized by dihydrogen bonding. Surprisingly the crystal symmetry is lowered upon heating, as evidenced both by in situ synchrotron powder X-ray diffraction and 11B MAS NMR data, resulting in an intermediate polymorph, ß'-Sr(BH4)2(NH3BH3)2, present from â¼65 to 115 °C. ß-Sr(BH4)2(NH3BH3)2, a sub structure of the ß'-polymorph showing higher symmetry with the space group symmetry Aba2, forms upon further heating. Ab initio molecular dynamics simulations show that the ammonia borane molecule can dynamically alternate between a bidentate and a tridentate coordination to Sr at finite temperature. The dynamic properties of the ammonia borane molecule in the solid state are suggested to cause the observed structural complexity. Based on simultaneous thermogravimetric analysis, differential scanning calorimetry and mass spectrometry, the decomposition of the compound was investigated showing a stabilization of ammonia borane in the structure relative to other metal borohydride ammonia boranes and neat ammonia borane.
RESUMEN
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
Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These "competence pili" are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three α-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second α-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.
Asunto(s)
Proteínas Fimbrias/química , Fimbrias Bacterianas/ultraestructura , Modelos Moleculares , Streptococcus pneumoniae/fisiología , Factores de Virulencia/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Dimerización , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Eliminación de Gen , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Operón , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Conformación Proteica en Hélice alfa , Proteínas Recombinantes de Fusión , Solubilidad , Streptococcus pneumoniae/ultraestructura , Transactivadores/genética , Transactivadores/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
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
Dynamic nuclear polarization (DNP) is a versatile option to improve the sensitivity of NMR and MRI. This versatility has elicited interest for overcoming potential limitations of these techniques, including the achievement of solid-state polarization enhancement at ambient conditions, and the maximization of (13) C signal lifetimes for performing inâ vivo MRI scans. This study explores whether diamond's (13) C behavior in nano- and micro-particles could be used to achieve these ends. The characteristics of diamond's DNP enhancement were analyzed for different magnetic fields, grain sizes, and sample environments ranging from cryogenic to ambient temperatures, in both solution and solid-state experiments. It was found that (13) Câ NMR signals could be boosted by orders of magnitude in either low- or room-temperature solid-state DNP experiments by utilizing naturally occurring paramagnetic P1 substitutional nitrogen defects. We attribute this behavior to the unusually long electronic/nuclear spin-lattice relaxation times characteristic of diamond, coupled with a time-independent cross-effect-like polarization transfer mechanism facilitated by a matching of the nitrogen-related hyperfine coupling and the (13) C Zeeman splitting. The efficiency of this solid-state polarization process, however, is harder to exploit in dissolution DNP-enhanced MRI contexts. The prospects for utilizing polarized diamond approaching nanoscale dimensions for both solid and solution applications are briefly discussed.
RESUMEN
Dynamic Nuclear Polarization (DNP) has long been an aim for increasing sensitivity of nuclear magnetic resonance (NMR) spectroscopy, delivering spectra in shorter experiment times or of smaller sample amounts. In recent years, it has been applied in magic angle spinning (MAS) solid-state NMR to a large range of samples, including biological macromolecules and functional materials. New research directions in structural biology can be envisaged by DNP, facilitating investigations on very large complexes or very heterogeneous samples. Here we present a summary of state of the art DNP MAS NMR spectroscopy and its applications to structural biology, discussing the technical challenges and factors affecting DNP performance.
Asunto(s)
Algoritmos , Biopolímeros/análisis , Biopolímeros/química , Espectroscopía de Resonancia Magnética/métodos , Procesamiento de Señales Asistido por Computador , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Smart materials are created in nature at interfaces between biomolecules and solid materials. The ability to probe the structure of functional peptides that engineer biogenic materials at this heterogeneous setting can be facilitated tremendously by use of DNP-enhanced solid-state NMR spectroscopy. This sensitive NMR technique allows simple and quick measurements, often without the need for isotope enrichment. Here, it is used to characterize a pentalysine peptide, derived from a diatom's silaffin protein. The peptide accelerates the formation of bioinspired silica and gets embedded inside the material as it is formed. Two-dimensional DNP MAS NMR of the silica-bound peptide and solution NMR of the free peptide are used to derive its secondary structure in the two states and to pinpoint some subtle conformational changes that the peptide undergoes in order to adapt to the silica environment. In addition, interactions between abundant lysine residues and silica surface are identified, and proximity of other side chains to silica and to neighboring peptide molecules is discussed.