Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 32
Filtrar
1.
EMBO J ; 40(14): e107294, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031912

RESUMO

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-
2.
Nature ; 559(7714): 423-427, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29995853

RESUMO

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 , Perus
3.
Nat Methods ; 17(5): 505-508, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371966

RESUMO

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 , Ligantes
4.
Angew Chem Int Ed Engl ; 62(36): e202305694, 2023 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-37329506

RESUMO

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 G
5.
Bioinformatics ; 37(24): 4876-4878, 2021 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-34145888

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 Massas
6.
Chemistry ; 27(7): 2537-2542, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33026114

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 , Micelas
7.
Proc Natl Acad Sci U S A ; 115(26): 6691-6696, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891712

RESUMO

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/metabolismo
8.
Angew Chem Int Ed Engl ; 59(36): 15560-15564, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33462887

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 Core 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/análise
9.
Nat Methods ; 13(4): 333-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26901650

RESUMO

Small molecules are known to stabilize membrane proteins and to modulate their function and oligomeric state, but such interactions are often hard to precisely define. Here we develop and apply a high-resolution, Orbitrap mass spectrometry-based method for analyzing intact membrane protein-ligand complexes. Using this platform, we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.


Assuntos
Lipídeos/química , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica
10.
Proc Natl Acad Sci U S A ; 113(29): 8230-5, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27364008

RESUMO

Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Subunidades Proteicas/metabolismo , Acetilação , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/química , Lipídeos/química , Miocárdio/enzimologia , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química
11.
Anal Chem ; 88(14): 7060-7, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27328020

RESUMO

Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.


Assuntos
Gases/efeitos da radiação , Espectrometria de Massas/métodos , Proteínas de Membrana/efeitos da radiação , Complexos Multiproteicos/efeitos da radiação , Acidianus/enzimologia , Avidina/química , Avidina/efeitos da radiação , Chaperonina 60/química , Chaperonina 60/efeitos da radiação , Gases/química , Raios Infravermelhos , Proteínas de Membrana/química , Micelas , Complexos Multiproteicos/química , Fosfatidilgliceróis/química , Subunidades Proteicas/química , Subunidades Proteicas/efeitos da radiação , Thermus thermophilus/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/efeitos da radiação
12.
Nat Methods ; 10(12): 1206-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24122040

RESUMO

We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.


Assuntos
Detergentes/química , Lipídeos/química , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Micelas , Diacilglicerol Quinase/química , Difusão , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Fluorescência Verde/química , Halobacteriaceae/química , Espectroscopia de Ressonância Magnética/métodos , Microscopia Eletrônica de Transmissão/métodos , Proteínas de Transporte de Monossacarídeos/química , Nanopartículas/química , Plasmídeos/metabolismo , Rodopsinas Sensoriais/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Simportadores/química
13.
Angew Chem Int Ed Engl ; 54(15): 4577-81, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25693501

RESUMO

Despite the growing importance of the mass spectrometry of membrane proteins, it is not known how their transfer from solution into vacuum affects their stability and structure. To address this we have carried out a systematic investigation of ten membrane proteins solubilized in different detergents and used mass spectrometry to gain physicochemical insight into the mechanism of their ionization and desolvation. We show that the chemical properties of the detergents mediate the charge state, both during ionization and detergent removal. Using ion mobility mass spectrometry, we monitor the conformations of membrane proteins and show how the surface charge density dictates the stability of folded states. We conclude that the gas-phase stability of membrane proteins is increased when a greater proportion of their surface is lipophilic and is consequently protected by the physical presence of the micelle.


Assuntos
Detergentes/química , Espectrometria de Massas , Proteínas de Membrana/química , Aquaporinas/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Gases/química , Humanos , Espectrometria de Massas/métodos , Micelas , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica
14.
J Am Chem Soc ; 136(49): 17010-2, 2014 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-25402655

RESUMO

The study of intact soluble protein assemblies by means of mass spectrometry is providing invaluable contributions to structural biology and biochemistry. A recent breakthrough has enabled similar study of membrane protein complexes, following their release from detergent micelles in the gas phase. Careful optimization of mass spectrometry conditions, particularly with respect to energy regimes, is essential for maintaining compact folded states as detergent is removed. However, many of the saccharide detergents widely employed in structural biology can cause unfolding of membrane proteins in the gas phase. Here, we investigate the potential of charge reduction by introducing three membrane protein complexes from saccharide detergents and show how reducing their overall charge enables generation of compact states, as evidenced by ion mobility mass spectrometry. We find that charge reduction stabilizes the oligomeric state and enhances the stability of lipid-bound complexes. This finding is significant since maintaining native-like membrane proteins enables ligand binding to be assessed from a range of detergents that retain solubility while protecting the overall fold.


Assuntos
Detergentes/química , Proteínas de Membrana/química , Espectrometria de Massas , Modelos Moleculares , Oxirredução , Estabilidade Proteica
15.
J Am Soc Mass Spectrom ; 35(8): 1891-1901, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39007842

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/química
16.
Angew Chem Weinheim Bergstr Ger ; 135(36): e202305694, 2023 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-38516403

RESUMO

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.

17.
Biochim Biophys Acta Biomembr ; 1864(9): 183958, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35551920

RESUMO

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/metabolismo
18.
Nat Chem ; 14(12): 1375-1382, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36357787

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/metabolismo
19.
RSC Adv ; 12(16): 9671-9680, 2022 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-35424940

RESUMO

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.

20.
Chem Sci ; 11(13): 3538-3546, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-34109026

RESUMO

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.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA