RESUMO
Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.
Assuntos
Espectrometria de Mobilidade Iônica , Proteínas de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/análise , Espectrometria de Mobilidade Iônica/métodos , Micelas , Espectrometria de Massas/métodos , Detergentes/química , Íons/químicaRESUMO
Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.
Assuntos
Detergentes , Micelas , Espectrometria de Massas/métodos , Proteínas de Membrana , Receptores Acoplados a Proteínas GRESUMO
Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.
RESUMO
G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the ß1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor-Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor-Gs complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.
Assuntos
Receptores Adrenérgicos beta 2 , Receptores Acoplados a Proteínas G , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Regulação Alostérica , Isoproterenol/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Ligantes , Proteínas de Ligação ao GTP/metabolismo , Íons , Espectrometria de Massas , Zinco/metabolismoRESUMO
Non-ionic detergents are important tools for the investigation of interactions between membrane proteins and lipid membranes. Recent studies led to the question as to whether the ability to capture protein-lipid interactions depends on the properties of detergents or their concentration in purification buffers. To address this question, we present the synthesis of an asymmetric, hybrid detergent that combines the head groups of detergents with opposing delipidating properties. We discuss detergent properties and protein purification outcomes to reveal whether the properties of detergent micelles or the detergent concentration in purification buffers drive membrane protein delipidation. We anticipate that our findings will enable the development of rationally design detergents for future applications in membrane protein research.
Assuntos
Detergentes , Micelas , Detergentes/metabolismo , Lipídeos , Proteínas de Membrana/metabolismoRESUMO
Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion-ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.
RESUMO
MOTIVATION: Native mass spectrometry is now a well-established method for the investigation of protein complexes, specifically their subunit stoichiometry and ligand binding properties. Recent advances allowing the analysis of complex mixtures lead to an increasing diversity and complexity in the spectra obtained. These spectra can be time-consuming to tackle through manual assignment and challenging for automated approaches. RESULTS: Native Mass Spectrometry Visual Analyser is a web-based tool to augment the manual process of peak assignment. In addition to matching masses to the stoichiometry of its component subunits, it allows raw data processing, assignment and annotation and permits mass spectra to be shared with their respective interpretation. AVAILABILITY AND IMPLEMENTATION: NaViA is open-source and can be accessed online under https://navia.ms. The source code and documentation can be accessed at https://github.com/d-que/navia, under the BSD 2-Clause licence. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Software , Espectrometria de MassasRESUMO
Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
Assuntos
Cloretos/metabolismo , Nucleotídeos/metabolismo , Potássio/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular , Tamanho Celular , Humanos , Fosforilação/fisiologia , Células Sf9 , Transdução de Sinais/fisiologia , Cotransportadores de K e Cl-RESUMO
Dendrons are an important class of macromolecules that can be used for a broad range of applications. Recent studies have indicated that mixtures of oligoglycerol detergent (OGD) regioisomers are superior to individual regioisomers for protein extraction. The origin of this phenomenon remains puzzling. Here we discuss the synthesis and characterization of dendritic oligoglycerol regioisomer mixtures and their implementation into detergents. We provide experimental benchmarks to support quality control after synthesis and investigate the unusual utility of OGD regioisomer mixtures for extracting large protein quantities from biological membranes. We anticipate that our findings will enable the development of mixed detergent platforms in the future.
Assuntos
Misturas Complexas/química , Dendrímeros/química , Detergentes/química , Glicerol/análogos & derivados , Glicerol/química , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Membrana Celular/química , Isomerismo , MicelasRESUMO
Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.
Assuntos
Lipídeos/análise , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Metaboloma , Proteoma/análise , Humanos , LigantesRESUMO
The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/química , Humanos , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Multimerização ProteicaRESUMO
Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Detergentes/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Espectrometria de Massas , Lipídeos de Membrana , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Redobramento de Proteína , SolubilidadeRESUMO
Mass spectrometry enables the in-depth structural elucidation of membrane protein complexes, which is of great interest in structural biology and drug discovery. Recent breakthroughs in this field revealed the need for design rules that allow fine-tuning the properties of detergents in solution and gas phase. Desirable features include protein charge reduction, because it helps to preserve native features of protein complexes during transfer from solution into the vacuum of a mass spectrometer. Addressing this challenge, we here present the first systematic gas-phase study of azobenzene detergents. The utility of gas-phase techniques for monitoring light-driven changes of isomer ratios and molecular properties are investigated in detail. This leads to the first azobenzene detergent that enables the native mass spectrometry analysis of membrane proteins and whose charge-reducing properties can be tuned by irradiation with light. More broadly, the presented work outlines new avenues for the high-throughput characterization of supramolecular systems and opens a new design strategy for detergents in membrane protein research.
RESUMO
The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Herein, we report the development and application of a mass-spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Coreâ 1 and 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the DC-SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites.
Assuntos
Moléculas de Adesão Celular/química , Lectinas Tipo C/química , Polissacarídeos/química , Receptores de Superfície Celular/química , Moléculas de Adesão Celular/análise , Glicosilação , Células HEK293 , Humanos , Espectrometria de Mobilidade Iônica/métodos , Lectinas Tipo C/análise , Espectrometria de Massas/métodos , Polissacarídeos/análise , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Estabilidade Proteica , Receptores de Superfície Celular/análiseRESUMO
G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets1. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Gαsßγ protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Gα subunits (mini-Gαs, mini-Gαi and mini-Gα12)2, we demonstrate that the complex of mini-Gαs with the ß1 adrenergic receptor (ß1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between ß1AR and other Gα subunits (mini-Gαi or mini-Gα12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαißγ complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Simulação de Dinâmica Molecular , Estabilidade Proteica , Ratos , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Neurotensina/química , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Especificidade por Substrato , PerusRESUMO
Strong interactions between lipids and proteins occur primarily through association of charged headgroups and amino acid side chains, rendering the protonation status of both partners important. Here we use native mass spectrometry to explore lipid binding as a function of charge of the outer membrane porin F (OmpF). We find that binding of anionic phosphatidylglycerol (POPG) or zwitterionic phosphatidylcholine (POPC) to OmpF is sensitive to electrospray polarity while the effects of charge are less pronounced for other proteins in outer or mitochondrial membranes: the ferripyoverdine receptor (FpvA) or the voltage-dependent anion channel (VDAC). Only marginal charge-induced differences were observed for inner membrane proteins: the ammonia channel (AmtB) or the mechanosensitive channel. To understand these different sensitivities, we performed an extensive bioinformatics analysis of membrane protein structures and found that OmpF, and to a lesser extent FpvA and VDAC, have atypically high local densities of basic and acidic residues in their lipid headgroup-binding regions. Coarse-grained molecular dynamics simulations, in mixed lipid bilayers, further implicate changes in charge by demonstrating preferential binding of anionic POPG over zwitterionic POPC to protonated OmpF, an effect not observed to the same extent for AmtB. Moreover, electrophysiology and mass-spectrometry-based ligand-binding experiments, at low pH, show that POPG can maintain OmpF channels in open conformations for extended time periods. Since the outer membrane is composed almost entirely of anionic lipopolysaccharide, with similar headgroup properties to POPG, such anionic lipid binding could prevent closure of OmpF channels, thereby increasing access of antibiotics that use porin-mediated pathways.
Assuntos
Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , Porinas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Modelos Químicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Porinas/química , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
With the recent success in determining membrane protein structures, further detailed understanding of the identity and function of the bound lipidome is essential. Using an approach that combines high-energy native mass spectrometry (HE-nMS) and solution-phase lipid profiling, this protocol can be used to determine the identity of the endogenous lipids that directly interact with a protein. Furthermore, this method can identify systems in which such lipid binding has a major role in regulating the oligomeric assembly of membrane proteins. The protocol begins with recording of the native mass spectrum of the protein of interest, under successive delipidation conditions, to determine whether delipidation leads to disruption of the oligomeric state. Subsequently, we propose using a bipronged strategy: first, an HE-nMS platform is used that allows dissociation of the detergent micelle at the front end of the instrument. This allows for isolation of the protein-lipid complex at the quadrupole and successive fragmentation at the collision cell, which leads to identification of the bound lipid masses. Next, simultaneous coupling of this with in-solution LC-MS/MS-based identification of extracted lipids reveals the complete identity of the interacting lipidome that copurifies with the proteins. Assimilation of the results of these two sets of experiments divulges the complete identity of the set of lipids that directly interact with the membrane protein of interest, and can further delineate its role in maintaining the oligomeric state of the protein. The entire procedure takes 2 d to complete.
Assuntos
Espectrometria de Massas/métodos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Multimerização ProteicaRESUMO
G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors belong to the largest family of membrane-embedded cell surface proteins and are involved in a diverse array of physiological processes. Despite progress in the mass spectrometry of membrane protein complexes, G protein-coupled receptors have remained intractable because of their low yield and instability after extraction from cell membranes. We established conditions in the mass spectrometer that preserve noncovalent ligand binding to the human purinergic receptor P2Y1. Results established differing affinities for nucleotides and the drug MRS2500 and link antagonist binding with the absence of receptor phosphorylation. Overall, therefore, our results are consistent with drug binding, preventing the conformational changes that facilitate downstream signaling. More generally, we highlight opportunities for mass spectrometry to probe effects of ligand binding on G protein-coupled receptors.
Assuntos
Ligantes , Espectrometria de Massas , Receptores Acoplados a Proteínas G/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Modelos Moleculares , Conformação Molecular , Fosforilação , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2Y1/química , Receptores Purinérgicos P2Y1/metabolismo , Relação Estrutura-AtividadeRESUMO
Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.
Assuntos
Lipídeos/química , Espectrometria de Massas/métodos , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Antiporters/química , Antiporters/genética , Antiporters/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Metabolismo dos Lipídeos , Simulação de Dinâmica Molecular , Domínios Proteicos , Estabilidade Proteica , Desdobramento de Proteína , Trocadores de Sódio-Hidrogênio/genética , Thermus thermophilus/químicaRESUMO
With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments in crystallography, novel interfaces with other biophysical methods are emerging. Here we consider how mass spectrometry can inform these techniques by providing unambiguous definition of subunit stoichiometry. Moreover recent developments that increase mass spectral resolution enable molecular details to be ascribed to unassigned density within high-resolution maps of membrane and soluble protein complexes. Importantly we also show how developments in mass spectrometry can define optimal solution conditions to guide downstream structure determination, particularly of challenging biomolecules that refuse to crystallise.