RESUMO
BACKGROUND: Caustic ingestion is relatively common in developing countries and can result in life-threatening sequelae. There is limited understanding of the epidemiology and incidence in Australia. AIMS: This statewide 10-year audit aims to document the rate of caustic injury in a defined Australian pouplation. METHODS: A retrospective review was conducted over 10 years (2007-2016), including all admissions to hospitals in Victoria. This includes a population of 5.9 million people and 22 hospitals. RESULTS: Three hundred and eighty-four cases of caustic ingestion were admitted to hospital between January 2007 and December 2016. The overall incidence was 7 cases/million/year. This cohort included 217 (56.5%) females, 193 (50.2%) overseas born patients and 196 (51%) people with a history of mental illness. The countries of birth with the highest incidence of caustic ingestion were Ethiopia (11 patients; 227 cases/million/year; relative risk (RR) 31.7; P < 0.0001), Sudan (11 patients; 161 cases/million/year; RR 22.6; P < 0.0001) and India (38 patients; 27 cases/million/year; RR 3.9; P < 0.0001). All had a significantly higher incidence than the Australian-born population of only 6.5 cases/million/year (RR 0.4; P < 0.0001). Of those born in India, Sudan and Ethiopia, rates of females (72%) were considerably higher than males. The overall mortality rate in this cohort was 2.3%. CONCLUSIONS: Caustic ingestion remains a significant cause of morbidity and health expenditure in Victoria, particularly among vulnerable groups such as recent female migrants from areas in Africa and India. The high frequency of events seen in migrant populations highlights the significant need for awareness of risks in these groups for the development of possible prevention strategies that are required.
Assuntos
Queimaduras Químicas , Cáusticos , Migrantes , Queimaduras Químicas/etiologia , Ingestão de Alimentos , Feminino , Humanos , Masculino , Vitória/epidemiologiaRESUMO
Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Salmonella typhimurium , Sistemas de Secreção Tipo III/química , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Citoplasma/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Salmonella typhimurium/química , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Sistemas de Secreção Tipo III/metabolismoRESUMO
The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein-protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid-protein interactions.
Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Cardiolipinas/metabolismo , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Translocador 1 do Nucleotídeo Adenina/química , Animais , Sítios de Ligação , Cardiolipinas/química , Bovinos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Camundongos , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica , Proteína Desacopladora 2/química , Proteína Desacopladora 2/metabolismoRESUMO
Amyloids are proteinaceous aggregates known for their role in debilitating degenerative diseases involving protein dysfunction. Many forms of functional amyloid are also produced in nature and often these systems require careful control of their assembly to avoid the potentially toxic effects. The best-characterised functional amyloid system is the bacterial curli system. Three natural inhibitors of bacterial curli amyloid have been identified and recently characterised structurally. Here, we compare common structural features of CsgC, CsgE and CsgH and discuss the potential implications for general inhibition of amyloid.
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Amiloide/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Amiloide/química , Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Conformação Proteica , Eletricidade EstáticaRESUMO
The twin-arginine translocase (Tat) carries out the remarkable process of translocating fully folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Tat is required for bacterial pathogenesis and for photosynthesis in plants. TatA, the protein-translocating element of the Tat system, is a small transmembrane protein that assembles into ring-like oligomers of variable size. We have determined a structural model of the Escherichia coli TatA complex in detergent solution by NMR. TatA assembly is mediated entirely by the transmembrane helix. The amphipathic helix extends outwards from the ring of transmembrane helices, permitting assembly of complexes with variable subunit numbers. Transmembrane residue Gln8 points inward, resulting in a short hydrophobic pore in the center of the complex. Simulations of the TatA complex in lipid bilayers indicate that the short transmembrane domain distorts the membrane. This finding suggests that TatA facilitates protein transport by sensitizing the membrane to transient rupture.
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Proteínas de Escherichia coli/química , Escherichia coli/química , Bicamadas Lipídicas/química , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Multimerização Proteica , Transporte Biológico Ativo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Plantas/química , Plantas/genética , Plantas/metabolismo , Estrutura Quaternária de Proteína , Tilacoides/química , Tilacoides/genética , Tilacoides/metabolismoRESUMO
Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.
Assuntos
Biologia Computacional/métodos , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Simulação de Dinâmica Molecular , Domínio Catalítico , Membrana Celular , Heme/química , Heme/metabolismo , Humanos , Maleabilidade , Ligação Proteica , Conformação Proteica , Varfarina/química , Varfarina/metabolismoRESUMO
The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common ß-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.
Assuntos
Pseudomonas aeruginosa/metabolismo , Alginatos/química , Sequência de Bases , Membrana Celular/metabolismo , Cristalografia por Raios X , Primers do DNA , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/química , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
The glucagon receptor family are typical class B1 G protein-coupled receptors (GPCRs) with important roles in metabolism, including the control of pancreas, brain, and liver function. As proteins with seven transmembrane domains, GPCRs are intimately in contact with lipid bilayers and therefore can be putatively regulated by interactions with their lipidic components, including cholesterol, sphingolipids, and other lipid species. Additionally, these receptors, as well as the agonists they bind to, can undergo lipid modifications, which can influence their binding capacity and/or elicit modified or biased signalling profiles. While the effect of lipids, and in particular cholesterol, has been widely studied for other GPCR classes, information about their role in regulating the glucagon receptor family is only beginning to emerge. Here we summarise our current knowledge on the effects of cholesterol modulation of glucagon receptor family signalling and trafficking profiles, as well as existing evidence for specific lipid-receptor binding and indirect effects of lipids via lipid modification of cognate agonists. Finally, we discuss the different methodologies that can be employed to study lipid-receptor interactions and summarise the importance of this area of investigation to increase our understanding of the biology of this family of metabolically relevant receptors.
Assuntos
Colesterol , Receptores de Glucagon , Transdução de Sinais , Humanos , Receptores de Glucagon/metabolismo , Animais , Colesterol/metabolismo , Transdução de Sinais/fisiologia , Metabolismo dos Lipídeos/fisiologiaRESUMO
Molecular dynamics simulations have been used to characterize the effects of transfer from aqueous solution to a vacuum to inform our understanding of mass spectrometry of membrane-protein-detergent complexes. We compared two membrane protein architectures (an α-helical bundle versus a ß-barrel) and two different detergent types (phosphocholines versus an alkyl sugar) with respect to protein stability and detergent packing. The ß-barrel membrane protein remained stable as a protein-detergent complex in vacuum. Zwitterionic detergents formed conformationally destabilizing interactions with an α-helical membrane protein after detergent micelle inversion driven by dehydration in vacuum. In contrast, a nonionic alkyl sugar detergent resisted micelle inversion, maintaining the solution-phase conformation of the protein. This helps to explain the relative stability of membrane proteins in the presence of alkyl sugar detergents such as dodecyl maltoside.
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Glucosídeos/farmacologia , Proteínas de Membrana/química , Proteínas Virais/química , Sequência de Aminoácidos , Desidratação , Glucosídeos/química , Proteínas de Membrana/metabolismo , Micelas , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Terciária de Proteína , Vácuo , Proteínas Virais/metabolismoRESUMO
The PRELID-TRIAP1 family of proteins is responsible for lipid transfer in mitochondria. Multiple structures have been resolved of apo and lipid substrate bound forms, allowing us to begin to piece together the molecular level details of the full lipid transfer cycle. Here, we used molecular dynamics simulations to demonstrate that the lipid binding is mediated by an extended, water-mediated hydrogen bonding network. A key mutation, R53E, was found to disrupt this network, causing lipid to be released from the complex. The X-ray crystal structure of R53E was captured in a fully closed and apo state. Lipid transfer assays and molecular simulations allow us to interpret the observed conformation in the context of the biological role. Together, our work provides further understanding of the mechanistic control of lipid transport by PRELID-TRIAP1 in mitochondria.
Assuntos
Hidrogênio , Simulação de Dinâmica Molecular , Ligação de Hidrogênio , LipídeosRESUMO
Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a â¼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.
Assuntos
Adenovírus Humanos , COVID-19 , Humanos , Capsídeo/química , Capsídeo/metabolismo , Proteínas Virais/análise , Adenoviridae/genética , Adenovírus Humanos/genética , Vetores GenéticosRESUMO
Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage.
Assuntos
Peptídeo Hidrolases , Streptococcus pyogenes , Imunidade InataRESUMO
The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells, and cross biological barriers. The glideosome-associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the solution properties together with membrane and F-actin binding interfaces suggests that GAC adopts several conformations from closed to open and extended. A multi-conformational model for assembly and regulation of GAC within the glideosome is proposed.
Assuntos
Toxoplasma , Toxoplasma/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Miosinas/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
[This corrects the article DOI: 10.1016/j.csbj.2021.11.014.].
RESUMO
Two proteins of the Escherichia coli membrane protein complex, CsgG and CsgF, are studied as proteinaceous nanopores for DNA sequencing. It is highly desirable to control the DNA as it moves through the pores, this requires characterisation of DNA translocation and subsequent optimization of the pores. In order to inform protein engineering to improve the pores, we have conducted a series of molecular dynamics simulations to characterise the mechanical strength and conformational dynamics of CsgG and the CsgG-CsgF complex and how these impact ssDNA, water and ion movement. We find that the barrel of CsgG is more susceptible to damage from external electric fields compared to the protein vestibule. Furthermore, the presence of CsgF within the CsgG-CsgF complex enables the complex to withstand higher electric fields. We find that the eyelet loops of CsgG play a key role in both slowing the translocation rate of DNA and modulating the conductance of the pore. CsgF also impacts the DNA translocation rate, but to a lesser degree than CsgG.
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Although less studied than interspecific interactions, interactions among members of the same species can influence space use and temporal activity. Using techniques commonly applied to the analysis of interspecific interactions-multispecies occupancy modeling and the analysis of temporal activity patterns-we studied intraspecific interactions within a high-density population of Persian leopards (Panthera pardus saxicolor) in Tandoureh National Park, northeastern Iran. Using camera-trap data, we investigated spatiotemporal interactions between male leopards, lone female leopards, and families (cubs/females with cubs). While we hypothesized that male and female leopards would display different temporal activity patterns, we did not predict spatial avoidance between these groups. We also predicted that leopard families would exhibit spatiotemporal avoidance from male leopards due to the risk of infanticide. Contrary to our expectations, we did not find any evidence for spatial or temporal avoidance between leopard families and adult male leopards. Male and lone female leopards exhibited positive pairwise co-occurrence, consistent with reports of high overlap between male and female leopard home ranges. While a high level of overlap in temporal activity patterns was found between males/lone females and males/families, there was evidence for variation in the proportion of time each leopard group was active in particular periods of the diel cycle. Male leopards showed cathemeral activity, while lone females and families were more active during daylight hours. The application of these techniques to interactions within a species has improved understanding of the ecology and behavior of this endangered solitary carnivore.
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Advances in molecular dynamics simulations have led to large increases across spatial and complexity scales, providing valuable molecular level insight into processes occurring on the subcellular level. An increasing repertoire of methods to assemble and analyse complex membrane simulations, alongside advances in structural biology methods for membrane proteins, have contributed to our increased understanding of the roles of specific lipid interactions for multiple membrane protein systems. Large scale simulations of crowded protein solutions have provided a model describing the biophysical basis for experimentally observed diffusion properties. In this review we discuss recent approaches that pave the way towards linking molecular level detail to the cellular level.
Assuntos
Estruturas Celulares/química , Estruturas Celulares/ultraestrutura , Simulação de Dinâmica Molecular , Membrana Celular/química , Membrana Celular/ultraestrutura , Espaço Intracelular , Modelos Moleculares , Proteínas/químicaRESUMO
BACKGROUND: Improved survival of children with congenital heart disease (CHD) into adult life has led to further study of their quality of life (QoL) and its determinants. The QoL including the symptoms of anxiety and depression of adults with CHD was analyzed to determine the relationship, if any, between prior cardiac surgery and QoL. METHODS: Adults with CHD who were recruited from a single community-based cardiology practice completed self-reported questionnaires on their QoL, which included symptoms of anxiety and depression. Standard linear regression analysis was used to determine whether prior cardiac surgery predicted lower QoL scores. RESULTS: One hundred forty-nine adult patients with CHD were sent QoL questionnaires. Completed questionnaires were received from 135 patients: 71 (53%) males and 64 (47%) females, with a mean age of 26.3 years (standard deviation: 7.8, min: 17, max: 49). Respondents were assigned to two groups: those who had (n = 89, 66%) or had not (n = 46, 34%) previously undergone one or more cardiac surgical interventions. Results from standard linear regression analyses revealed no predictive relationship between history of previous cardiac surgery, whether one or more operations, and QoL. CONCLUSIONS: Among adult patients with CHD who completed QoL questionnaires, we observed no association between a patient's history of prior cardiac surgery and self-reported QoL measures. This welcome and important finding may be a reflection of the good functional capacity of both groups (postsurgical and nonsurgical) irrespective of the original CHD diagnosis and need for surgical intervention.
Assuntos
Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Cardiopatias Congênitas/psicologia , Cardiopatias Congênitas/cirurgia , Qualidade de Vida , Adolescente , Adulto , Ansiedade/complicações , Criança , Depressão/complicações , Feminino , Cardiopatias Congênitas/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Autorrelato , Inquéritos e Questionários , Resultado do TratamentoRESUMO
The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Recent applications of mass spectrometry (MS) to study membrane protein complexes are yielding valuable insights into the binding of lipids and their structural and functional roles. To date, most native MS experiments with membrane proteins are based on detergent solubilization. Many insights into the structure and function of membrane proteins have been obtained using detergents; however, these can promote local lipid rearrangement and can cause fluctuations in the oligomeric state of protein complexes. To overcome these problems, we developed a method that does not use detergents or other chemicals. Here we report a detailed protocol that enables direct ejection of protein complexes from membranes for analysis by native MS. Briefly, lipid vesicles are prepared directly from membranes of different sources and subjected to sonication pulses. The resulting destabilized vesicles are concentrated, introduced into a mass spectrometer and ionized. The mass of the observed protein complexes is determined and this information, in conjunction with 'omics'-based strategies, is used to determine subunit stoichiometry as well as cofactor and lipid binding. Within this protocol, we expand the applications of the method to include peripheral membrane proteins of the S-layer and amyloid protein export machineries overexpressed in membranes from which the most abundant components have been removed. The described experimental procedure takes approximately 3 d from preparation to MS. The time required for data analysis depends on the complexity of the protein assemblies embedded in the membrane under investigation.