RESUMO
Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.
Assuntos
Antifúngicos , Rim , Polienos , Esteróis , Animais , Humanos , Camundongos , Anfotericina B/análogos & derivados , Anfotericina B/química , Anfotericina B/toxicidade , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Antifúngicos/toxicidade , Células Cultivadas , Colesterol/química , Colesterol/metabolismo , Farmacorresistência Fúngica , Ergosterol/química , Ergosterol/metabolismo , Rim/efeitos dos fármacos , Cinética , Testes de Sensibilidade Microbiana , Micoses/tratamento farmacológico , Micoses/microbiologia , Polienos/química , Polienos/metabolismo , Polienos/farmacologia , Inoculações Seriadas , Esteróis/química , Esteróis/metabolismo , Fatores de TempoRESUMO
Cholesterol promotes the structural integrity of the fluid cell membrane and interacts dynamically with many membrane proteins to regulate function. Understanding site-resolved cholesterol structural dynamics is thus important. This long-standing challenge has thus far been addressed, in part, by selective isotopic labeling approaches. Here we present a new 3D solid-state NMR (SSNMR) experiment utilizing scalar 13C-13C polarization transfer and recoupling of the 1H-13C interactions in order to determine average dipolar couplings for all 1H-13C vectors in uniformly 13C-enriched cholesterol. The experimentally determined order parameters (OP) agree exceptionally well with molecular dynamics (MD) trajectories and reveal coupling among several conformational degrees of freedom in cholesterol molecules. Quantum chemistry shielding calculations further support this conclusion and specifically demonstrate that ring tilt and rotation are coupled to changes in tail conformation and that these coupled segmental dynamics dictate the orientation of cholesterol. These findings advance our understanding of physiologically relevant dynamics of cholesterol, and the methods that revealed them have broader potential to characterize how structural dynamics of other small molecules impact their biological functions.
Assuntos
Colesterol , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Membrana Celular , Conformação Molecular , Colesterol/químicaRESUMO
Cholesterol (Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol's interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of 13C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.
Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Isótopos de Carbono , Colesterol/análise , Estrutura Molecular , Sensibilidade e Especificidade , Leveduras/metabolismoRESUMO
The plasma membrane of the cell is a complex, tightly regulated, heterogeneous environment shaped by proteins, lipids, and small molecules. Ca2+ ions are important cellular messengers, spatially separated from anionic lipids. After cell injury, disease, or apoptotic events, anionic lipids are externalized to the outer leaflet of the plasma membrane and encounter Ca2+, resulting in dramatic changes in the plasma membrane structure and initiation of signaling cascades. Despite the high chemical and biological significance, the structures of lipid-Ca2+ nanoclusters are still not known. Previously, we demonstrated by solid-state nuclear magnetic resonance (NMR) spectroscopy that upon binding to Ca2+, individual phosphatidylserine lipids populate two distinct yet-to-be-characterized structural environments. Here, we concurrently employ extensive all-atom molecular dynamics (MD) simulations with our accelerated membrane mimetic and detailed NMR measurements to identify lipid-Ca2+ nanocluster conformations. We find that major structural characteristics of these nanoclusters, including interlipid pair distances and chemical shifts, agree with observable NMR parameters. Simulations reveal that lipid-ion nanoclusters are shaped by two characteristic, long-lived lipid structures induced by divalent Ca2+. Using ab initio quantum mechanical calculations of chemical shifts on MD-captured lipid-ion complexes, we show that computationally observed conformations are validated by experimental NMR data. Both NMR measurements of diluted specifically labeled lipids and MD simulations reveal that the basic structural unit that reshapes the membrane is a Ca2+-coordinated phosphatidylserine tetramer. Our combined computational and experimental approach presented here can be applied to other complex systems in which charged membrane-active molecular agents leave structural signatures on lipids.
Assuntos
Cálcio/química , Membrana Celular/química , Lipídeos de Membrana/química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Íons/química , Íons/metabolismo , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Nanoestruturas/química , Fosfatidilserinas/química , Fosfatidilserinas/metabolismoRESUMO
Antilipoperoxidant protein dysfunction is associated with many human diseases, suggesting that bilayer lipid peroxidation may contribute broadly to pathogenesis. Small molecule inhibitors of this membrane-localized chemistry could in theory enable better understanding and/or treatment of such diseases, but currently available compounds have important limitations. Many biological questions thus remain unanswered, and clinical trials have largely been disappointing. Enabled by efficient, building block-based syntheses of three atypical carotenoid natural products produced by microorganisms that thrive in environments of extreme oxidative stress, we found that peridinin is a potent inhibitor of nonenzymatic bilayer lipid peroxidation in liposomes and in primary human endothelial cells. We also found that peridinin blocks monocyte-endothelial cell adhesion, a key step in atherogenesis. A series of frontier solid-state NMR experiments with a site-specifically 13C-labeled isotopolog synthesized using the same MIDA boronate building block-based total synthesis approach revealed that peridinin is completely embedded within and physically spans the hydrophobic core of POPC membranes, maximizing its effective molarity at the site of the targeted lipid peroxidation reactions. Alternatively, the widely used carotenoid astaxanthin is significantly less potent and was found to primarily localize extramembranously. Peridinin thus represents a promising and biophysically well-characterized starting point for the development of small molecule antilipoperoxidants that serve as more effective biological probes and/or therapeutics.
Assuntos
Carotenoides/farmacologia , Bicamadas Lipídicas/antagonistas & inibidores , Peroxidação de Lipídeos/efeitos dos fármacos , Carotenoides/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Estrutura MolecularRESUMO
(2)H quadrupolar line shapes deliver rich information about protein dynamics. A newly designed 3D (2)H-(13)C-(13)C solid-state NMR magic angle spinning (MAS) experiment is presented and demonstrated on the microcrystalline ß1 immunoglobulin binding domain of protein G (GB1). The implementation of (2)H-(13)C adiabatic rotor-echo-short-pulse-irradiation cross-polarization (RESPIRATION CP) ensures the accuracy of the extracted line shapes and provides enhanced sensitivity relative to conventional CP methods. The 3D (2)H-(13)C-(13)C spectrum reveals (2)H line shapes for 140 resolved aliphatic deuterium sites. Motional-averaged (2)H quadrupolar parameters obtained from the line-shape fitting identify side-chain motions. Restricted side-chain dynamics are observed for a number of polar residues including K13, D22, E27, K31, D36, N37, D46, D47, K50, and E56, which we attribute to the effects of salt bridges and hydrogen bonds. In contrast, we observe significantly enhanced side-chain flexibility for Q2, K4, K10, E15, E19, N35, N40, and E42, due to solvent exposure and low packing density. T11, T16, and T17 side chains exhibit motions with larger amplitudes than other Thr residues due to solvent interactions. The side chains of L5, V54, and V29 are highly rigid because they are packed in the core of the protein. High correlations were demonstrated between GB1 side-chain dynamics and its biological function. Large-amplitude side-chain motions are observed for regions contacting and interacting with immunoglobulin G (IgG). In contrast, rigid side chains are primarily found for residues in the structural core of the protein that are absent from protein binding and interactions.
Assuntos
Espectroscopia de Ressonância Magnética , Modelos Moleculares , Receptores de GABA-B/química , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Receptores de GABA-B/metabolismoRESUMO
For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of what are to our knowledge the first derivatives of amphotericin that kill yeast but not human cells.
Assuntos
Anfotericina B/química , Antifúngicos/química , Esteróis/química , Bicamadas Lipídicas , Espectroscopia de Ressonância Magnética , PermeabilidadeRESUMO
The blood coagulation cascade is initiated when the cell-surface complex of factor VIIa (FVIIa, a trypsin-like serine protease) and tissue factor (TF, an integral membrane protein) proteolytically activates factor X (FX). Both FVIIa and FX bind to membranes via their γ-carboxyglutamate-rich domains (GLA domains). GLA domains contain seven to nine bound Ca(2+) ions that are critical for their folding and function, and most biochemical studies of blood clotting have employed supraphysiologic Ca(2+) concentrations to ensure saturation of these domains with bound Ca(2+). Recently, it has become clear that, at plasma concentrations of metal ions, Mg(2+) actually occupies two or three of the divalent metal ion-binding sites in GLA domains, and that these bound Mg(2+) ions are required for full function of these clotting proteins. In this study, we investigated how Mg(2+) influences FVIIa enzymatic activity. We found that the presence of TF was required for Mg(2+) to enhance the rate of FX activation by FVIIa, and we used alanine-scanning mutagenesis to identify TF residues important for mediating this response to Mg(2+). Several TF mutations, including those at residues G164, K166, and Y185, blunted the ability of Mg(2+) to enhance the activity of the TF/FVIIa complex. Our results suggest that these TF residues interact with the GLA domain of FX in a Mg(2+)-dependent manner (although effects of Mg(2+) on the FVIIa GLA domain cannot be ruled out). Notably, these TF residues are located within or immediately adjacent to the putative substrate-binding exosite of TF.
Assuntos
Fator VIIa/química , Magnésio/química , Complexos Multiproteicos/química , Tromboplastina/química , Substituição de Aminoácidos , Cálcio/química , Cálcio/metabolismo , Fator VIIa/genética , Fator VIIa/metabolismo , Fator X/química , Fator X/genética , Fator X/metabolismo , Humanos , Magnésio/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Tromboplastina/genética , Tromboplastina/metabolismoRESUMO
Amphotericin B (AmB) is the archetype for small molecules that form ion channels in living systems and has recently been shown to replace a missing protein ion transporter and thereby restore physiology in yeast. Molecular modeling studies predict that AmB self-assembles in lipid membranes with the polyol region lining a channel interior that funnels to its narrowest region at the C3-hydroxyl group. This model predicts that modification of this functional group would alter conductance of the AmB ion channel. To test this hypothesis, the C3-hydroxyl group was synthetically deleted, and the resulting derivative, C3deoxyAmB (C3deOAmB), was characterized using multidimensional NMR experiments and single ion channel electrophysiology recordings. C3deOAmB possesses the same macrocycle conformation as AmB and retains the capacity to form transmembrane ion channels, yet the conductance of the C3deOAmB channels is 3-fold lower than that of AmB channels. Thus, the C3-hydroxyl group plays an important role in AmB ion channel conductance, and synthetic modifications at this position may provide an opportunity for further tuning of channel functions.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Anfotericina B/química , Antifúngicos/química , Transporte de ÍonsRESUMO
Solid-state NMR (SSNMR) spectroscopy has become an important technique for studying the biophysics and structure biology of proteins. This technique is especially useful for insoluble membrane proteins and amyloid fibrils, which are essential for biological functions and are associated with human diseases. In the past few years, as major contributors to the rapidly advancing discipline of biological SSNMR, we have developed a family of methods for high-resolution structure determination of microcrystalline, fibrous, and membrane proteins. Key developments include order-of-magnitude improvements in sensitivity, resolution, instrument stability, and sample longevity under data collection conditions. These technical advances now enable us to apply new types of 3D and 4D experiments to collect atomic-resolution structural restraints in a site-resolved manner, such as vector angles, chemical shift tensors, and internuclear distances, throughout large proteins. In this Account, we present the technological advances in SSNMR approaches towards protein structure determination. We also describe the application of those methods for large membrane proteins and amyloid fibrils. Particularly, the SSNMR measurements of an integral membrane protein DsbB support the formation of a charge-transfer complex between DsbB and ubiquinone during the disulfide bond transfer pathways. The high-resolution structure of the DsbA-DsbB complex demonstrates that the joint calculation of X-ray and SSNMR restraints for membrane proteins with low-resolution crystal structure is generally applicable. The SSNMR investigations of α-synuclein fibrils from both wild type and familial mutants reveal that the structured regions of α-synuclein fibrils include the early-onset Parkinson's disease mutation sites. These results pave the way to understanding the mechanism of fibrillation in Parkinson's disease.
Assuntos
Amiloide/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Complexos de Coordenação/química , HumanosRESUMO
NMR chemical shift tensors (CSTs) in proteins, as well as their orientations, represent an important new restraint class for protein structure refinement and determination. Here, we present the first determination of both CST magnitudes and orientations for (13)Cα and (15)N (peptide backbone) groups in a protein, the ß1 IgG binding domain of protein G from Streptococcus spp., GB1. Site-specific (13)Cα and (15)N CSTs were measured using synchronously evolved recoupling experiments in which (13)C and (15)N tensors were projected onto the (1)H-(13)C and (1)H-(15)N vectors, respectively, and onto the (15)N-(13)C vector in the case of (13)Cα. The orientations of the (13)Cα CSTs to the (1)H-(13)C and (13)C-(15)N vectors agreed well with the results of ab initio calculations, with an rmsd of approximately 8°. In addition, the measured (15)N tensors exhibited larger reduced anisotropies in α-helical versus ß-sheet regions, with very limited variation (18 ± 4°) in the orientation of the z-axis of the (15)N CST with respect to the (1)H-(15)N vector. Incorporation of the (13)Cα CST restraints into structure calculations, in combination with isotropic chemical shifts, transferred echo double resonance (13)C-(15)N distances and vector angle restraints, improved the backbone rmsd to 0.16 Å (PDB ID code 2LGI) and is consistent with existing X-ray structures (0.51 Å agreement with PDB ID code 2QMT). These results demonstrate that chemical shift tensors have considerable utility in protein structure refinement, with the best structures comparable to 1.0-Å crystal structures, based upon empirical metrics such as Ramachandran geometries and χ(1)/χ(2) distributions, providing solid-state NMR with a powerful tool for de novo structure determination.
Assuntos
Proteínas de Bactérias/química , Anisotropia , Isótopos de Carbono/química , Cristalografia por Raios X , Hidrogênio/química , Modelos Moleculares , Estrutura Molecular , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de ProteínaRESUMO
During therapeutic protein development, two-dimensional (2D) heteronuclear NMR spectra can be a powerful analytical method for measuring protein higher order structure (HOS) in solution since the spectra exhibit much higher resolution than homonuclear 1H spectra. However, 2D NMR capabilities for characterizing protein HOS in crystalline states remain to be assessed, given the low 13C natural abundance and intrinsically broader lines in solid-state NMR (SSNMR). Herein, high-resolution heteronuclear correlation (HETCOR) SSNMR was utilized to directly measure intact crystal drug products of insulin human, insulin lispro and insulin aspart. The fingerprint regions in 2D 1H-13C HETCOR spectra were identified, which distinguished the insulin crystals in their primary structure, HOS heterogeneity and dynamics, as well as the manufacturing processes. The HOS heterogeneity in insulin analogs is consistent with their therapeutic effect of rapid action. Therefore, heteronuclear NMR could be broadly applicable to study protein drug dosage forms from liquid to solid, yielding improved molecular level structure data for assessing drug HOS in biosimilar drug development.
RESUMO
Magic-angle spinning (MAS) solid-state NMR methods are crucial in many areas of biology and materials science. Conventional probe designs have often been specified with 0.1 part per million (ppm) or 100 part per billion (ppb) magnetic field resolution, which is a limitation for many modern scientific applications. Here we describe a novel 5-mm MAS module design that significantly improves the linewidth and line shape for solid samples by an improved understanding of the magnetic susceptibility of probe materials and geometrical symmetry considerations, optimized to minimize the overall perturbation to the applied magnetic field (B0). The improved spinning module requires only first and second order shimming adjustments to achieve a sub-Hz resolution of 13C resonances of adamantane at 150 MHz Larmor frequency (14.1Tesla magnetic field). Minimal use of third and higher order shims improves experimental reproducibility upon sample changes and the exact placement within the magnet. Furthermore, the shimming procedure is faster, and the required gradients smaller, thus minimizing thermal drift of the room temperature (RT) shims. We demonstrate these results with direct polarization (Bloch decay) and cross polarization experiments on adamantane over a range of sample geometries and with multiple superconducting magnet systems. For a direct polarization experiment utilizing the entire active sample volume of a 5-mm rotor (90 µl), we achieved full width at half maximum (FWHM) of 0.76 Hz (5 ppb) and baseline resolved the 13C satellite peaks for adamantane as a consequent of the 7.31 Hz (59 ppb) width at 2% intensity. We expect these approaches to be increasingly pivotal for high-resolution solid-state NMR spectroscopy at and above 1 GHz 1H frequencies.
RESUMO
Improving the spectral sensitivity and resolution of biological solids is one of the long-standing problems in nuclear magnetic resonance (NMR) spectroscopy. In this report, we introduce low-power supercycled variants of two-pulse phase-modulated (TPPM) sequence for heteronuclear decoupling. The utility of the sequence is shown by improvements in the transverse relaxation time of observed nuclei (with 1H decoupling) with its application to different samples (uniformly 13C, 15N, 2H-labeled GB1 back-exchanged with 25% H2O and 75% D2O, uniformly 13C, 15N, 2H-labeled human derived Asyn fibril back-exchanged with 100% H2O and uniformly 13C, 15N -labeled human derived Asyn fibril) at fast MAS using low radiofrequency (RF) fields. To understand the effect of spinning speed, the transverse relaxation time is monitored under different spinning frequencies. In comparison to existing heteronuclear decoupling sequences, the supercycled TPPM (sTPPM) sequence significantly improves the spectral sensitivity and resolution and is robust towards B1 inhomogeneity and decoupler offset.
RESUMO
Sensitivity is the foundation of every NMR experiment, and the signal-to-noise ratio (SNR) should increase with static (B0) magnetic field, by a proportionality that primarily depends on the design of the NMR probe and receiver. In the low B0 field limit, where the coil geometry is much smaller than the wavelength of the NMR frequency, SNR can increase in proportion to B0 to the power 7/4. For modern magic-angle spinning (MAS) probes, this approximation holds for rotor sizes up to 3.2 mm at 14.1 Tesla (T), corresponding to 600 MHz 1H and 151 MHz 13C Larmor frequencies. To obtain the anticipated benefit of larger coils and/or higher B0 fields requires a quantitative understanding of the contributions to SNR, utilizing standard samples and protocols that reproduce SNR measurements with high accuracy and precision. Here, we present such a systematic and comprehensive study of 13C SNR under MAS over the range of 14.1 to 21.1 T. We evaluate a range of probe designs utilizing 1.6, 2.5 and 3.2 mm rotors, including 24 different sets of measurements on 17 probe configurations using five spectrometers. We utilize N-acetyl valine as the primary standard and compare and contrast with other commonly used standard samples (adamantane, glycine, hexamethylbenzene, and 3-methylglutaric acid). These robust approaches and standard operating procedures provide an improved understanding of the contributions from probe efficiency, receiver noise figure, and B0 dependence in a range of custom-designed and commercially available probes. We find that the optimal raw SNR is obtained with balanced 3.2 mm design at 17.6 T, that the best mass-limited SNR is achieved with a balanced 1.6 mm design at 21.1 T, and that the raw SNR at 21.1 T reaches diminishing returns with rotors larger than 2.5 mm.
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
α-Synuclein (AS) is associated with both sporadic and familial forms of Parkinson disease (PD). In sporadic disease, wild-type AS fibrillates and accumulates as Lewy bodies within dopaminergic neurons of the substantia nigra. The accumulation of misfolded AS is associated with the death of these neurons, which underlies many of the clinical features of PD. In addition, a rare missense mutation in AS, A30P, is associated with highly penetrant, autosomal dominant PD, although the pathogenic mechanism is unclear. A30P AS fibrillates more slowly than the wild-type (WT) protein in vitro and has been reported to preferentially adopt a soluble, protofibrillar conformation. This has led to speculation that A30P forms aggregates that are distinct in structure compared with wild-type AS. Here, we perform a detailed comparison of the chemical shifts and secondary structures of these fibrillar species, based upon our recent characterization of full-length WT fibrils. We have assigned A30P AS fibril chemical shifts de novo and used them to determine its secondary structure empirically. Our results illustrate that although A30P forms fibrils more slowly than WT in vitro, the chemical shifts and secondary structure of the resultant fibrils are in high agreement, demonstrating a conserved ß-sheet core.
Assuntos
Proteínas Mutantes/química , Mutação , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica , alfa-Sinucleína/química , Humanos , Cinética , Proteínas Mutantes/genética , Estrutura Secundária de Proteína , alfa-Sinucleína/genéticaRESUMO
Alpha-synuclein is an intrinsically disordered protein whose formation of beta-sheet-rich protein aggregates in the brain is implicated in the development of Parkinson's disease. Due to its believed role in synaptic vesicle trafficking and neurotransmission, many studies have employed simple, synthetic model systems to investigate alpha-synuclein/membrane interactions in an attempt to gain a better understanding of the protein's native and pathogenic functions. Interestingly, these studies seem to suggest that alpha-synuclein interacts differently with rigid vesicle mimics in comparison to malleable vesicle mimics. However, the use of different mimic sizes and surface chemistries across existing studies makes it challenging to directly compare the effects of membrane mechanical properties on protein behavior observed thus far. In this work, we developed a synaptic vesicle mimic library comprising a range of both malleable and rigid synaptic vesicle mimics possessing the same size and biologically representative lipid surface chemistry. Limited proteolysis mass spectrometry experiments revealed distinct fragmentation patterns between rigid and malleable synaptic vesicle mimics. The N-terminal and C-terminal regions of alpha-synuclein were found to become less solvent-accessible upon binding to all synaptic vesicle mimics. Nevertheless, minor variations in digestion pattern were observed in the central region of the protein dependent upon mimic size, rigidity, and lipid composition. Higher binding affinities were observed for alpha-synuclein binding to rigid synaptic vesicle mimics compared to malleable synaptic vesicle mimics. Additionally, the binding affinity of alpha-synuclein toward small lipid vesicles and small lipid-coated gold nanoparticles without cholesterol was found to be lower than that of their respective malleable and rigid counterparts. Interestingly, the binding curves for the rigid synaptic vesicle mimics demonstrated a nontraditional peak and dip shape believed to arise from differences in alpha-synuclein orientation on the particle surface at different protein-to-particle incubation ratios.
Assuntos
Nanopartículas Metálicas , alfa-Sinucleína , alfa-Sinucleína/química , Fosfolipídeos , Ouro/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
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.