RESUMO
The lacrimal film has attracted increasing interest in the last decades as a potential source of biomarkers of physiopathological states, due to its accessibility, moderate complexity, and responsiveness to ocular and systemic diseases. High-performance liquid chromatography-mass spectrometry (LC-MS) has led to effective approaches to tear proteomics, despite the intrinsic limitations in sample amounts. This review focuses on the recent progress in strategy and technology, with an emphasis on the potential for personalized medicine. After an introduction on lacrimal-film composition, examples of applications to biomarker discovery are discussed, comparing approaches based on pooled-sample and single-tear analysis. Then, the most critical steps of the experimental pipeline, that is, tear collection, sample fractionation, and LC-MS implementation, are discussed with reference to proteome-coverage optimization. Advantages and challenges of the alternative procedures are highlighted. Despite the still limited number of studies, tear quantitative proteomics, including single-tear investigation, could offer unique contributions to the identification of low-invasiveness, sustained-accessibility biomarkers, and to the development of personalized approaches to therapy and diagnosis.
Assuntos
Proteômica , Lágrimas , Biomarcadores/análise , Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Lágrimas/químicaRESUMO
Biofluid analysis by optical spectroscopy techniques is attracting considerable interest due to its potential to revolutionize diagnostics and precision medicine, particularly for neurodegenerative diseases. However, the lack of effective biomarkers combined with the unaccomplished identification of convenient biofluids has drastically hampered optical advancements in clinical diagnosis and monitoring of neurodegenerative disorders. Here, we show that vibrational spectroscopy applied to human tears opens a new route, offering a non-invasive, label-free identification of a devastating disease such as amyotrophic lateral sclerosis (ALS). Our proposed approach has been validated using two widespread techniques, namely, Fourier transform infrared (FTIR) and Raman microspectroscopies. In conjunction with multivariate analysis, this vibrational approach made it possible to discriminate between tears from ALS patients and healthy controls (HCs) with high specificity (â¼97% and â¼100% for FTIR and Raman spectroscopy, respectively) and sensitivity (â¼88% and â¼100% for FTIR and Raman spectroscopy, respectively). Additionally, the investigation of tears allowed us to disclose ALS spectroscopic markers related to protein and lipid alterations, as well as to a reduction of the phenylalanine level, in comparison with HCs. Our findings show that vibrational spectroscopy is a new potential ALS diagnostic approach and indicate that tears are a reliable and non-invasive source of ALS biomarkers.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/diagnóstico , Biomarcadores , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Lágrimas , VibraçãoRESUMO
Herein, we compared the ability of linear and cyclic peptides generated in silico to target different protein sites: internal pockets and solvent-exposed sites. We selected human lysozyme (HuL) as a model target protein combined with the computational evolution of linear and cyclic peptides. The sequence evolution of these peptides was based on the PARCE algorithm. The generated peptides were screened based on their aqueous solubility and HuL binding affinity. The latter was evaluated by means of scoring functions and atomistic molecular dynamics (MD) trajectories in water, which allowed prediction of the structural features of the protein-peptide complexes. The computational results demonstrated that cyclic peptides constitute the optimal choice for solvent exposed sites, while both linear and cyclic peptides are capable of targeting the HuL pocket effectively. The most promising binders found in silico were investigated experimentally by surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS) techniques. All tested peptides displayed dissociation constants in the micromolar range, as assessed by SPR; however, both NMR and ESI-MS suggested multiple binding modes, at least for the pocket binding peptides. A detailed NMR analysis confirmed that both linear and cyclic pocket peptides correctly target the binding site they were designed for.
Assuntos
Ligantes , Simulação de Dinâmica Molecular , Muramidase/química , Peptídeos/química , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação , Muramidase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Ressonância de Plasmônio de SuperfícieRESUMO
The interaction of inorganic nanoparticles and many biological fluids often withstands the formation of a Protein Corona enveloping the nanoparticle. This Protein Corona provides the biological identity to the nanoparticle that the immune system will detect. The formation of this Protein Corona depends not only on the composition of the nanoparticle, its size, shape, surface state and exposure time, but also on the type of media, nanoparticle to protein ratio and the presence of ions and other molecular species that interfere in the interaction between proteins and nanoparticles. This has important implications on immune safety, biocompatibility and the use of nanoparticles in medicine.
Assuntos
Materiais Biocompatíveis/metabolismo , Sistema Imunitário , Nanopartículas/metabolismo , Coroa de Proteína/metabolismo , Animais , Materiais Biocompatíveis/química , Humanos , Nanomedicina , Nanopartículas/química , Tamanho da Partícula , Coroa de Proteína/químicaRESUMO
Lacrimal fluid is an attractive source of noninvasive biomarkers, the main limitation being the small sample amounts typically collected. Advanced analytical methods to allow for proteomics profiling from a few microliters are needed to develop innovative biomarkers, with attractive perspectives of applications to precision medicine. This work describes an effective, analytical pipeline for single-tear analysis by ultrahigh-resolution, shotgun proteomics from 23 healthy human volunteers, leading to high-confidence identification of a total of 890 proteins. Highly reproducible quantification was achieved by either peak intensity, peak area, or spectral counting. Hierarchical clustering revealed a stratification of females vs. males that did not emerge from previous studies on pooled samples. Two subjects were monitored weekly over 3 weeks. The samples clustered by withdrawal time of day (morning vs. afternoon) but not by follow-up week, with elevated levels of components of the immune system in the morning samples. This study demonstrates feasibility of single-tear quantitative proteomics, envisaging contributions of this unconventional body fluid to individualized approaches in biomedicine.
Assuntos
Biomarcadores/metabolismo , Proteínas do Olho/metabolismo , Medicina de Precisão , Proteoma/metabolismo , Proteômica/métodos , Lágrimas/metabolismo , Adulto , Feminino , Voluntários Saudáveis , Humanos , Masculino , Proteoma/análise , Adulto JovemRESUMO
α-Synuclein (AS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, a hallmark of Parkinson's disease (PD). AS is particularly exposed to oxidation of its methionine residues, both in vivo and in vitro Oxidative stress has been implicated in PD and oxidized α-synuclein has been shown to assemble into soluble, toxic oligomers, rather than amyloid fibrils. However, the structural effects of methionine oxidation are still poorly understood. In this work, oxidized AS was obtained by prolonged incubations with dopamine (DA) or epigallocatechin-3-gallate (EGCG), two inhibitors of AS aggregation, indicating that EGCG promotes the same final oxidation product as DA. The conformational transitions of the oxidized and non-oxidized protein were monitored by complementary biophysical techniques, including MS, ion mobility (IM), CD, and FTIR spectroscopy assays. Although the two variants displayed very similar structures under conditions that stabilize highly disordered or highly ordered states, differences emerged in the intermediate points of transitions induced by organic solvents, such as trifluoroethanol (TFE) and methanol (MeOH), indicating a lower propensity of the oxidized protein for forming either α- or ß-type secondary structures. Furthermore, oxidized AS displayed restricted secondary-structure transitions in response to dehydration and slightly amplified tertiary-structure transitions induced by ligand binding. This difference in susceptibility to induced folding could explain the loss of fibrillation potential observed for oxidized AS. Finally, site-specific oxidation kinetics point out a minor delay in Met-127 modification, likely due to the effects of AS intrinsic structure.
Assuntos
Catequina/análogos & derivados , Metionina/química , Agregados Proteicos , Dobramento de Proteína , alfa-Sinucleína/química , Catequina/química , Humanos , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Metionina/metabolismo , Oxirredução , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , alfa-Sinucleína/metabolismoRESUMO
Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic-to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here.
Assuntos
Biofísica/métodos , Biologia Computacional/métodos , Espectrometria de Massas/estatística & dados numéricos , Espectrometria de Massas/métodos , Proteínas/análiseRESUMO
The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.
RESUMO
Intrinsically disordered proteins (IDPs) are unable to adopt a unique 3D structure under physiological conditions and thus exist as highly dynamic conformational ensembles. IDPs are ubiquitous and widely spread in the protein realm. In the last decade, compelling experimental evidence has been gathered, pointing to the ability of IDPs and intrinsically disordered regions (IDRs) to undergo liquid-liquid phase separation (LLPS), a phenomenon driving the formation of membrane-less organelles (MLOs). These biological condensates play a critical role in the spatio-temporal organization of the cell, where they exert a multitude of key biological functions, ranging from transcriptional regulation and silencing to control of signal transduction networks. After introducing IDPs and LLPS, we herein survey available data on LLPS by IDPs/IDRs of viral origin and discuss their functional implications. We distinguish LLPS associated with viral replication and trafficking of viral components, from the LLPS-mediated interference of viruses with host cell functions. We discuss emerging evidence on the ability of plant virus proteins to interfere with the regulation of MLOs of the host and propose that bacteriophages can interfere with bacterial LLPS, as well. We conclude by discussing how LLPS could be targeted to treat phase separation-associated diseases, including viral infections.
Assuntos
Interações Hospedeiro-Patógeno , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Extração Líquido-Líquido/métodos , Vírus/crescimento & desenvolvimento , Animais , Desenho de Fármacos , Humanos , Organelas/químicaRESUMO
The abundance of intrinsic disorder in the protein realm and its role in a variety of physiological and pathological cellular events have strengthened the interest of the scientific community in understanding the structural and dynamical properties of intrinsically disordered proteins (IDPs) and regions (IDRs). Attempts at rationalizing the general principles underlying both conformational properties and transitions of IDPs/IDRs must consider the abundance of charged residues (Asp, Glu, Lys, and Arg) that typifies these proteins, rendering them assimilable to polyampholytes or polyelectrolytes. Their conformation strongly depends on both the charge density and distribution along the sequence (i.e., charge decoration) as highlighted by recent experimental and theoretical studies that have introduced novel descriptors. Published experimental data are revisited herein in the frame of this formalism, in a new and possibly unitary perspective. The physicochemical properties most directly affected by charge density and distribution are compaction and solubility, which can be described in a relatively simplified way by tools of polymer physics. Dissecting factors controlling such properties could contribute to better understanding complex biological phenomena, such as fibrillation and phase separation. Furthermore, this knowledge is expected to have enormous practical implications for the design, synthesis, and exploitation of bio-derived materials and the control of natural biological processes.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Polieletrólitos/química , Sequência de Aminoácidos , Agregados Proteicos , Conformação Proteica , Eletricidade EstáticaRESUMO
Intrinsically disordered proteins (IDPs) are systematically under-represented in structural proteomics studies. Their structural characterization implies description of the dynamic conformational ensembles populated by these polymers in solution, posing major challenges to biophysical methods. "Native" MS (native-MS) has emerged as a central tool in this field, conjugating the unique MS analytical power with structurally meaningful descriptors, like solvent-accessible surface area (SASA) and collisional cross section (CCS). This review summarizes recently published papers comparing native-MS and solution methods, with a focus on charge-state-distribution (CSD) analysis for IDP conformational analysis. The results point to substantial agreement, supporting structural interpretation of native-MS spectra of IDPs. The discussion is integrated with data from our group on "synthetic" IDPs, obtained by reduction and alkylation of natively folded proteins, whose fold is stabilized by disulfide bridges. Finally, an MS-based compaction index (CI) is introduced, evaluating SASA with reference to globular and fully disorder proteins. Such a parameter can be calculated for single conformers or the whole conformational ensemble, offering a continuous index for IDP comparison and classification.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Humanos , Proteínas Intrinsicamente Desordenadas/classificação , Conformação Proteica , Dobramento de Proteína , Proteômica/instrumentação , Espectrometria de Massas por Ionização por Electrospray/instrumentaçãoRESUMO
In this review, we detail the most common experimental approaches to assess and characterize protein intrinsic structural disorder, with the notable exception of NMR and EPR spectroscopy, two ideally suited approaches that will be described in depth in two other reviews within this special issue. We discuss the advantages, the limitations, as well as the caveats of the various methods. We also describe less common and more demanding approaches that enable achieving further insights into the conformational properties of IDPs. Finally, we present recent developments that have enabled assessment of structural disorder in living cells, and discuss the currently available methods to model IDPs as conformational ensembles.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Humanos , Hidrodinâmica , Proteínas Intrinsicamente Desordenadas/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Coloração e RotulagemRESUMO
Description of heterogeneous molecular ensembles, such as intrinsically disordered proteins, represents a challenge in structural biology and an urgent question posed by biochemistry to interpret many physiologically important, regulatory mechanisms. Single-molecule techniques can provide a unique contribution to this field. This work applies single molecule force spectroscopy to probe conformational properties of α-synuclein in solution and its conformational changes induced by ligand binding. The goal is to compare data from such an approach with those obtained by native mass spectrometry. These two orthogonal, biophysical methods are found to deliver a complex picture, in which monomeric α-synuclein in solution spontaneously populates compact and partially compacted states, which are differently stabilized by binding to aggregation inhibitors, such as dopamine and epigallocatechin-3-gallate. Analyses by circular dichroism and Fourier-transform infrared spectroscopy show that these transitions do not involve formation of secondary structure. This comparative analysis provides support to structural interpretation of charge-state distributions obtained by native mass spectrometry and helps, in turn, defining the conformational components detected by single molecule force spectroscopy.
Assuntos
Espectrometria de Massas , Conformação Proteica , Imagem Individual de Molécula , alfa-Sinucleína/química , Dicroísmo Circular , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier , alfa-Sinucleína/metabolismoRESUMO
BACKGROUND: Recent theoretical and computational studies have shown that the charge content and, most importantly, the linear distribution of opposite charges are major determinants of conformational properties of intrinsically disordered proteins (IDPs). Charge segregation in a sequence can be measured through κ, which represents a normalized measure of charge asymmetry. A strong inverse correlation between κ and radius of gyration has been previously demonstrated for two independent sets of permutated IDP sequences. METHODS: We used two well-characterized IDPs, namely measles virus NTAIL and Hendra virus PNT4, sharing a very similar fraction of charged residues and net charge per residue, but differing in proline (Pro) content. For each protein, we have rationally designed a low- and a high-κ variant endowed with the highest and the lowest κ values compatible with their natural amino acid composition. Then, the conformational properties of wild-type and κ-variants have been assessed by biochemical and biophysical techniques. RESULTS: We confirmed a direct correlation between κ and protein compaction. The analysis of our original data along with those available from the literature suggests that Pro content may affects the responsiveness to charge clustering. CONCLUSIONS: Charge clustering promotes IDP compaction, but the extent of its effects depends on the sequence context. Proline residues seem to play a role contrasting compaction. GENERAL SIGNIFICANCE: These results contribute to the identification of sequence determinants of IDP conformational properties. They may also serve as an asset for rational design of non-natural IDPs with tunable degree of compactness.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Mutação , Conformação Proteica , Proteínas Virais/química , Sequência de Aminoácidos , Proteínas Intrinsicamente Desordenadas/genética , Dobramento de Proteína , Homologia de Sequência , Proteínas Virais/genéticaRESUMO
BACKGROUND: Protein-nanoparticle (NP) interactions dictate properties of nanoconjugates relevant to bionanotechnology. Non-covalent adsorption generates a protein corona (PC) formed by an inner and an outer layer, the hard and soft corona (HC, SC). Intrinsically disordered proteins (IDPs) exist in solution as conformational ensembles, whose response to the presence of NPs is not known. METHODS: Three IDPs (α-casein, Sic1 and α-synuclein) and lysozyme are compared, describing conformational properties inside HC on silica NPs by circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy. RESULTS: IDPs inside HC are largely unstructured, but display small, protein-specific conformational changes. A minor increase in helical content is observed for α-casein and α-synuclein, reminiscent of membrane effects on α-synuclein. Frozen in their largely disordered conformation, bound proteins do not undergo folding induced by dehydration, as they do in their free forms. While HC thickness approaches the hydrodynamic diameter of the protein in solution for lysozyme, it is much below the respective values for IDPs. NPs boost α-synuclein aggregation kinetics in a dose-dependent manner. CONCLUSIONS: IDPs maintain structural disorder inside HC, experiencing minor, protein-specific, induced folding and stabilization against further conformational transitions, such as formation of intermolecular beta-sheets upon dehydration. The HC is formed by a single layer of protein molecules. SC likely plays a key role stabilizing amyloidogenic α-synuclein conformers. GENERAL SIGNIFICANCE: Protein-NP interactions can mimic those with macromolecular partners, allowing dissection of contributing factors by rational design of NP surfaces. Application of NPs in vivo should be carefully tested for amyloidogenic potential.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Nanopartículas , Conformação Proteica , Coroa de Proteína/química , Animais , Caseínas/química , Bovinos , Embrião de Galinha , Dicroísmo Circular , Proteínas Inibidoras de Quinase Dependente de Ciclina/química , Eletroforese em Gel de Poliacrilamida , Humanos , Muramidase/química , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Dióxido de Silício , Espectroscopia de Infravermelho com Transformada de Fourier , alfa-Sinucleína/químicaRESUMO
Electrospray-ionization mass spectrometry (ESI-MS) is a key tool of structural biology, complementing the information delivered by conventional biochemical and biophysical methods. Yet, the mechanism behind the conformational effects in protein ESI-MS is an object of debate. Two parameters-solvent-accessible surface area (As) and apparent gas-phase basicity (GBapp)-are thought to play a role in controlling the extent of protein ionization during ESI-MS experiments. This review focuses on recent experimental and theoretical investigations concerning the influence of these parameters on ESI-MS results and the structural information that can be derived. The available evidence supports a unified model for the ionization mechanism of folded and unfolded proteins. These data indicate that charge-state distribution (CSD) analysis can provide valuable structural information on normally folded, as well as disordered structures.
Assuntos
Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Conformação Proteica , Dobramento de Proteína , Proteínas/análise , Proteínas/metabolismoRESUMO
Transferrin (Tf) is the major iron-transporting protein in the human body and, for this reason, has been extensively studied in biomedicine. This protein undergoes a complex glycosylation process leading to several glycoforms, some of which are important in the diagnosis of alcohol abuse and of congenital glycosylation defects under the collective name of carbohydrate-deficient transferrin (CDT). Exploiting the Tf ability to bind not only iron but also other ions, specific attention has been devoted to binding activity towards Tb3+, which was reported to greatly enhance its intrinsic fluorescence upon the interaction with Tf. However, the structural properties of the Tb3+-Tf complex have not been described so far. In the present work, the formation of the Tf-Tb3+ complex has been investigated by the employment of several biophysical techniques, such as fluorescence resonance energy transfer (FRET), "native" mass spectrometry (MS), and near-UV circular dichroism (CD). Each method allowed the detection of the Tf-Tb3+ complex, yielding a specific signature. The interaction of Tb3+ with Fe3+-free Tf (apoTf) has been described in terms of stoichiometry, affinity, and structural effects in comparison with Fe3+. These experiments led to the first direct detection of the Tf-Tb3+ complex by MS, indicating a 1:2 stoichiometry and allowing the investigation of structural effects of metal binding. Either Tb3+ or Fe3+ binding affected protein conformation, inducing structural compaction to a similar extent. Nevertheless, near-UV CD and pH-dependence profiles suggested subtle differences in the coordination of the two metals by Tf side chains. Experimental conditions that promote complex formation have been identified, highlighting the importance of alkaline pH and synergistic ions, such as carbonate. On the basis of these studies, sample pretreatment, separation, and detection conditions of a high-performance liquid chromatographic method for CDT analysis are optimized, achieving relevant increase (by a factor of â¼3) of analytical sensitivity. Graphical abstract Schematic representation of HPLC-separated transferrin glycoforms detected by fluorescence emission of the terbium ions bound to the protein.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Complexos de Coordenação/química , Corantes Fluorescentes/química , Térbio/química , Transferrina/análogos & derivados , Quelantes/química , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Transferrina/químicaRESUMO
Lipopolysaccharide (LPS) is an essential glycolipid of the outer membrane (OM) of Gram-negative bacteria with a tripartite structure: lipid A, oligosaccharide core and O antigen. Seven essential LPS-transport proteins (LptABCDEFG) move LPS to the cell surface. Lpt proteins are linked by structural homology, featuring a ß-jellyroll domain that mediates protein-protein interactions and LPS binding. Analysis of LptA-LPS interaction by fluorescence spectroscopy is used here to evaluate the contribution of each LPS moiety in protein-ligand interactions, comparing the wild-type (wt) protein to the I36D mutant. In addition to a crucial role of lipid A, an unexpected contribution emerges for the core region in recognition and binding of Lpt proteins.
Assuntos
Proteínas de Transporte/química , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Lipopolissacarídeos/química , Mutação , Proteínas Recombinantes de Fusão/química , Substituição de Aminoácidos , Naftalenossulfonato de Anilina , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Transporte Biológico , Sequência de Carboidratos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Escherichia coli K12/química , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Isoleucina/química , Isoleucina/metabolismo , Ligantes , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de FluorescênciaRESUMO
The intrinsically disordered and amyloidogenic protein α-synuclein (AS) has been linked to several neurodegenerative states, including Parkinson's disease. Here, nanoelectrospray-ionization mass spectrometry (nano-ESI-MS), ion mobility (IM), and native top-down electron transfer dissociation (ETD) techniques are employed to study AS interaction with small molecules known to modulate its aggregation, such as epigallocatechin-3-gallate (EGCG) and dopamine (DA). The complexes formed by the two ligands under identical conditions reveal peculiar differences. While EGCG engages AS in compact conformations, DA preferentially binds to the protein in partially extended conformations. The two ligands also have different effects on AS structure as assessed by IM, with EGCG leading to protein compaction and DA to its extension. Native top-down ETD on the protein-ligand complexes shows how the different observed modes of binding of the two ligands could be related to their known opposite effects on AS aggregation. The results also show that the protein can bind either ligand in the absence of any covalent modifications, such as oxidation.
Assuntos
Catequina/análogos & derivados , Dopamina/química , alfa-Sinucleína/química , Sítios de Ligação , Catequina/química , Estrutura Molecular , Nanotecnologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
To form extracellular aggregates, amyloidogenic proteins bypass the intracellular quality control, which normally targets unfolded/aggregated polypeptides. Human D76N ß2-microglobulin (ß2m) variant is the prototype of unstable and amyloidogenic protein that forms abundant extracellular fibrillar deposits. Here we focus on the role of the class I major histocompatibility complex (MHCI) in the intracellular stabilization of D76N ß2m. Using biophysical and structural approaches, we show that the MHCI containing D76N ß2m (MHCI76) displays stability, dissociation patterns, and crystal structure comparable with those of the MHCI with wild type ß2m. Conversely, limited proteolysis experiments show a reduced protease susceptibility for D76N ß2m within the MHCI76 as compared with the free variant, suggesting that the MHCI has a chaperone-like activity in preventing D76N ß2m degradation within the cell. Accordingly, D76N ß2m is normally assembled in the MHCI and circulates as free plasma species in a transgenic mouse model.