RESUMO
Magic-angle spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) spectroscopy is a powerful and versatile technique for probing structure and dynamics in large, insoluble biological systems at atomic resolution. With many recent advances in instrumentation and polarization methods, technology development in SSNMR remains an active area of research and presents opportunities to further improve data collection, processing, and analysis of samples with low sensitivity and complex tertiary and quaternary structures. SSNMR spectra are often collected as multidimensional data, requiring stable experimental conditions to minimize signal fluctuations (t1 noise). In this work, we examine the factors adversely affecting signal stability as well as strategies used to mitigate them, considering laboratory environmental requirements, configuration of amplifiers, and pulse sequence parameter selection. We show that Thermopad® temperature variable attenuators (TVAs) can partially compensate for the changes in amplifier output power as a function of temperature and thereby ameliorate one significant source of instability for some spectrometers and pulse sequences. We also consider the selection of tangent ramped cross polarization (CP) waveform shapes, to balance the requirements of sensitivity and instrumental stability. These findings collectively enable improved stability and overall performance for CP-based multidimensional spectra of microcrystalline, membrane, and fibrous proteins performed at multiple magnetic field strengths.
Assuntos
Espectroscopia de Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Temperatura , AlgoritmosRESUMO
The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn.
Assuntos
Doença por Corpos de Lewy , Doença de Parkinson , Humanos , alfa-Sinucleína/química , Microscopia Crioeletrônica , Corpos de Lewy/patologia , Doença por Corpos de Lewy/patologia , Doença de Parkinson/patologiaRESUMO
Sleep, a state of reduced consciousness, affects brain oxygen metabolism and lowers cerebral metabolic rate of oxygen (CMRO2). Previously, we quantified CMRO2 during sleep via Fick's Principle, with a single-band MRI sequence measuring both hemoglobin O2 saturation (SvO2) and superior sagittal sinus (SSS) blood flow, which was upscaled to obtain total cerebral blood flow (tCBF). The procedure involves a brief initial calibration scan to determine the upscaling factor (fc), assumed state-invariant. Here, we used a dual-band sequence to simultaneously provide SvO2 in SSS and tCBF in the neck every 16 seconds, allowing quantification of fc dynamically. Ten healthy subjects were scanned by MRI with simultaneous EEG for 80 minutes, yielding 300 temporal image frames per subject. Four volunteers achieved slow-wave sleep (SWS), as evidenced by increased δ-wave activity (per American Academy of Sleep Medicine criteria). SWS was maintained for 13.5 ± 7.0 minutes, with CMRO2 28.6 ± 5.5% lower than pre-sleep wakefulness. Importantly, there was negligible bias between tCBF obtained by upscaling SSS-blood flow, and tCBF measured directly in the inflowing arteries of the neck (intra-class correlation 0.95 ± 0.04, averaged across all subjects), showing that the single-band approach is a valid substitute for quantifying tCBF, simplifying image data collection and analysis without sacrificing accuracy.
Assuntos
Seio Sagital Superior , Vigília , Humanos , Vigília/fisiologia , Seio Sagital Superior/diagnóstico por imagem , Oxigênio/metabolismo , Encéfalo/irrigação sanguínea , Sono , Consumo de Oxigênio/fisiologia , Circulação Cerebrovascular/fisiologia , Imageamento por Ressonância Magnética/métodosRESUMO
Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.
RESUMO
Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.
Assuntos
Doença por Corpos de Lewy , Atrofia de Múltiplos Sistemas , Doença de Parkinson , Humanos , alfa-Sinucleína/química , Doença por Corpos de Lewy/patologia , Ressonância Magnética Nuclear Biomolecular , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Atrofia de Múltiplos Sistemas/metabolismo , Atrofia de Múltiplos Sistemas/patologiaRESUMO
The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.
RESUMO
The assembly of proteins into amyloid fibrils has become linked not only with the progression of myriad human diseases, but also important biological functions. Understanding and controlling the formation, structure, and stability of amyloid fibrils is therefore a major scientific goal. Here we utilize electron microscopy-based approaches combined with quantitative statistical analysis to show how recently developed kind of amyloid modulators-multivalent polymer-peptide conjugates (mPPCs)-can be applied to control the structure and stability of amyloid fibrils. In doing so, we demonstrate that mPPCs are able to convert 40-residue amyloid beta fibrils into ordered nanostructures through a combination of fragmentation and bundling. Fragmentation is shown to be consistent with a model where the rate constant of fibril breakage is independent of the fibril length, suggesting a local and specific interaction between fibrils and mPPCs. Subsequent bundling, which was previously not observed, leads to the formation of sheet-like nanostructures which are surprisingly much more uniform than the starting fibrils. These nanostructures have dimensions independent of the molecular weight of the mPPC and retain the molecular-level ordering of the starting amyloid fibrils. Collectively, we reveal quantitative and nanoscopic understanding of how mPPCs can be applied to control amyloid structure and stability, and demonstrate approaches to elucidate nanoscale amyloid phase behavior in the presence of functional macromolecules and other modulators.
RESUMO
Fibrils of the protein α-synuclein (α-syn) are implicated in the pathogenesis of Parkinson's disease and related neurodegenerative disorders. We have reported a high-resolution structure (PDB 2N0A) of an α-syn fibril form prepared by in vitro incubation of monomeric protein in 50 mM sodium phosphate buffer pH 7.4 with 0.1 mM EDTA and 0.01% sodium azide. In parallel with this structure determination, ongoing studies of small molecule ligands binding to α-syn fibrils, prepared in 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer, have been in progress, and it is therefore of interest to determine the structural similarity of these forms. Here we report the 13C and 15N resonance assignments for α-syn fibrils prepared with Tris-HCl buffer (pH 7.7 at 37 °C) and 100 mM NaCl. These fibrillization conditions yield a form with fibril core chemical shifts highly similar to those we reported (BMRB 16939) in the course of determining the high-resolution 2N0A structure, with the exception of some small perturbations from T44 to V55, including two sets of peaks observed for residues T44-V48. Additional differences occur in the patterns of observed residues in the primarily unstructured N-terminus. These results demonstrate a common fold of the fibril core for α-syn fibrils prepared in phosphate or Tris-HCl buffer at moderate ionic strength.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Concentração Osmolar , alfa-Sinucleína/química , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
Solid-state NMR spectroscopy (SSNMR) is an established and invaluable tool for the study of amyloid fibril structure with atomic-level detail. Optimization of the homogeneity and concentration of fibrils enhances the resolution and sensitivity of SSNMR spectra. Here, we present a fibrillization and fibril processing protocol, starting from purified monomeric α-synuclein, that enables the collection of high-resolution SSNMR spectra suitable for site-specific structural analysis. This protocol does not rely on any special features of α-synuclein and should be generalizable to any other amyloid protein.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Espectroscopia de Ressonância Magnética/métodos , Amiloide/isolamento & purificação , Proteínas Amiloidogênicas/isolamento & purificação , Humanos , Conformação Proteica , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson's disease (PD). We present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register ß-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.