RESUMEN
Increased efforts are being made for observing proteins in their native environments. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is a powerful tool for this purpose. Conventionally, PELDOR employs an identical spin pair, which limits the output to a single distance for monomeric samples. Here, we show that the Gd3+-trityl-nitroxide (NO) three-spin system is a versatile tool to study heterooligomeric membrane protein complexes, even within their native membrane. This allowed for an independent determination of four different distances (Gd3+-trityl, Gd3+-NO, trityl-NO, and Gd3+-Gd3+) within the same sample. We demonstrate the feasibility of this approach by observing sequential ligand binding and the dynamics of complex formation in the cobalamin transport system involving four components (cobalamin, BtuB, TonB, and BtuF). Our results reveal that TonB binding alone is sufficient to release cobalamin from BtuB in the native asymmetric bilayers. This approach provides a potential tool for the structural and quantitative analysis of dynamic protein-protein interactions in oligomeric complexes, even within their native surroundings.
Asunto(s)
Membrana Dobles de Lípidos , Vitamina B 12 , Espectroscopía de Resonancia por Spin del Electrón/métodos , Marcadores de SpinRESUMEN
ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO-NO, Mn2+ -NO, and Mn2+ -Mn2+ pulsed electron-electron double-resonance spectroscopy of TmrAB in a time-resolved manner. This type-IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn2+ -ATP, and Mn2+ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates the reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato , Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Nucleótidos/metabolismo , Sitios de Unión , Dominios Proteicos , Conformación ProteicaRESUMEN
The ß-barrel assembly machinery (BAM) consisting of the central ß-barrel BamA and four other lipoproteins mediates the folding of the majority of the outer membrane proteins. BamA is placed in an asymmetric bilayer and its lateral gate is suggested to be the functional hotspot. Here we used in situ pulsed electron-electron double resonance spectroscopy to characterize BamA in the native outer membrane. In the detergent micelles, the data is consistent with mainly an inward-open conformation of BamA. The native membrane considerably enhanced the conformational heterogeneity. The lateral gate and the extracellular loop 3 exist in an equilibrium between different conformations. The outer membrane provides a favorable environment for occupying multiple conformational states independent of the lipoproteins. Our results reveal a highly dynamic behavior of the lateral gate and other key structural elements and provide direct evidence for the conformational modulation of a membrane protein in situ.
Asunto(s)
Proteínas de Escherichia coliRESUMEN
In situ investigation of membrane proteins is a challenging task. Previously we demonstrated that nitroxide labels combined with pulsed ESR spectroscopy is a promising tool for this purpose. However, the nitroxide labels suffer from poor stability, high background labeling, and low sensitivity. Here we show that Finland (FTAM) and OX063 based labels enable labeling of the cobalamin transporter BtuB and BamA, the central component of the ß-barrel assembly machinery (BAM) complex, in E coli. Compared to the methanethiosulfonate spin label (MTSL), trityl labels eliminated the background signals and enabled specific in situ labeling of the proteins with high efficiency. The OX063 labels show a long phase memory time (TM ) of ≈5â µs. All the trityls enabled distance measurements between BtuB and an orthogonally labeled substrate with high selectivity and sensitivity down to a few µm concentration. Our data corroborate the BtuB and BamA conformations in the cellular environment of E. coli.
Asunto(s)
Proteínas de Escherichia coli/análisis , Escherichia coli/química , Proteínas de la Membrana/análisis , Compuestos de Sulfhidrilo/análisis , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/citología , Finlandia , Marcadores de SpinRESUMEN
ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetric inward-facing state, and we show that the C-terminal helices, arranged in a zipper-like fashion, play a crucial role in guiding the conformational changes associated with substrate transport. In conclusion, TmrAB can be regarded as a model system for asymmetric ABC exporters in general, and for TAP in particular.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Thermus thermophilus , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Línea Celular , Resistencia a Múltiples Medicamentos , Humanos , Modelos Moleculares , Conformación Proteica , Thermus thermophilus/metabolismoRESUMEN
ATP-binding cassette (ABC) exporters actively move chemically diverse substrates across biological membranes. Their malfunction leads to human diseases. Many ABC exporters encompass asymmetric nucleotide-binding sites (NBSs), and some of them are inhibited by the transported substrate. The functional relevance of the catalytic asymmetry or the mechanism for trans-inhibition remains elusive. Here, we investigated TmrAB, a functional homologue of the human antigen translocation complex TAP using advanced electron-electron double resonance spectroscopy. In the presence of ATP, the heterodimeric ABC exporter exists in a tunable equilibrium between inward- and outward-facing conformations. The two NBSs exhibit pronounced asymmetry in the open-to-close equilibrium. The closed conformation is more favored at the degenerate NBS, and closure of either of the NBS is sufficient to open the extracellular gate. We define the mechanistic basis for trans-inhibition, which operates by a reverse transition from the outward-facing state through an occluded conformation. These novel findings uncover the central role of reversible conformational equilibrium in the function and regulation of an ABC exporter and establish a mechanistic framework for future investigations on other medically important transporters with imprinted asymmetry. Also, this study demonstrates for the first-time the feasibility to resolve equilibrium populations at multiple domains and their interdependence for global conformational changes in a large membrane protein complex.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Modelos Biológicos , Conformación Proteica , Dominios ProteicosRESUMEN
In BtuB, the Escherichia coli TonB-dependent transporter for vitamin B12, substrate binding to the extracellular surface unfolds a conserved energy coupling motif termed the Ton box into the periplasm. This transmembrane signaling event facilitates an interaction between BtuB and the inner-membrane protein TonB. In this study, continuous-wave and pulse electron paramagnetic resonance in a native outer-membrane preparation demonstrate that signaling also occurs from the periplasmic to the extracellular surface in BtuB. The binding of a TonB fragment to the periplasmic interface alters the configuration of the second extracellular loop and partially dissociates a spin-labeled substrate analog. Moreover, mutants in the periplasmic Ton box that are transport-defective alter the binding site for vitamin B12 in BtuB. This work demonstrates that the Ton box and the extracellular substrate binding site are allosterically coupled in BtuB, and that TonB binding may initiate a partial round of transport.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Transducción de Señal , Regulación Alostérica , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte Biológico , Calcio/metabolismo , Membrana Celular/metabolismo , Escherichia coli/citología , Escherichia coli/metabolismo , Espacio Extracelular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Marcadores de Spin , Vitamina B 12/metabolismoRESUMEN
An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron-electron double resonance (PELDOR) is a well-established technique to follow conformational changes in purified membrane protein complexes. Here we demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells. We exploit the fact that outer membrane proteins usually lack reactive cysteines and that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli. We characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the PELDOR data with high-resolution crystal structures. Our approach avoids detergent extraction, purification, and reconstitution usually required for these systems. With this approach, structure, function, conformational changes, and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Membrana/química , Ligandos , Conformación ProteicaRESUMEN
The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.
Asunto(s)
Ligandos , Proteínas de la Membrana/química , Espectroscopía de Resonancia por Spin del Electrón , Conformación Molecular , Óxidos de Nitrógeno/química , Marcadores de SpinRESUMEN
Double electron-electron resonance is used here to investigate intermediates of the transport cycle of the Escherichia coli vitamin B12 ATP-binding cassette importer BtuCD-F. Previously, we showed the ATP-induced opening of the cytoplasmic gate I in TM5 helices, later confirmed by the AMP-PNP-bound BtuCD-F crystal structure. Here, other key residues are analyzed in TM10 helices (positions 307 and 322) and in the cytoplasmic gate II, i.e. the loop between TM2 and TM3 (positions 82 and 85). Without BtuF, binding of ATP induces detectable changes at positions 307 and 85 in BtuCD in liposomes. Together with BtuF, ATP triggers the closure of the cytoplasmic gate II in liposomes (reported by both positions 82 and 85). This forms a sealed cavity in the translocation channel in agreement with the AMP-PNP·BtuCD-F x-ray structure. When vitamin B12 and AMP-PNP are simultaneously present, the extent of complex formation is reduced, but the short 82-82 interspin distance detected indicates that the substrate does not affect the closed conformation of this gate. The existence of the BtuCD-F complex under these conditions is verified with spectroscopically orthogonal nitroxide and Gd(III)-based labels. The cytoplasmic gate II remains closed also in the vanadate-trapped state, but it reopens in the ADP-bound state of the complex. Therefore, we suggest that the substrate likely trapped in ATP·BtuCD-F can be released after ATP hydrolysis but before the occluded ADP-bound conformation is reached.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Unión Periplasmáticas/química , Vitamina B 12/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hidrólisis , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Vitamina B 12/genética , Vitamina B 12/metabolismoRESUMEN
Root parasitic weeds in Orobanchaceae cause serious damage to worldwide agriculture. Germination of the parasites requires host-derived germination stimulants, such as strigolactones, as indicators of host roots within reach of the parasite's radicles. This unique germination process was focused on to identify metabolic pathways required for germination, and to design a selective control strategy. A metabolomic analysis of germinating seeds of clover broomrape, Orobanche minor, was conducted to identify its distinctive metabolites. Consequently, a galactosyl-sucrose trisaccharide, planteose (α-d-galactopyranosyl-(1â6)-ß-d-fructofuranosyl-(2â1)-α-d-glucopyranoside), was identified as a metabolite that decreased promptly after reception of the germination stimulant. To investigate the importance of planteose metabolism, the effects of several glycosidase inhibitors were examined, and nojirimycin bisulfite (NJ) was found to alter the sugar metabolism and to selectively inhibit the germination of O. minor. Planteose consumption was similar in NJ-treated seeds and non-treated germinating seeds; however, NJ-treated seeds showed lower consumption of sucrose, a possible intermediate of planteose metabolism, resulting in significantly less glucose and fructose. This inhibitory effect was recovered by adding glucose. These results suggest that planteose is a storage carbohydrate required for early stage of germination of O. minor, and NJ inhibits germination by blocking the supply of essential glucose from planteose and sucrose. Additionally, NJ selectively inhibited radicle elongation of germinated seeds of Orobanchaceae plants (Striga hermonthica and Phtheirospermum japonicum). Thus, NJ will be a promising tool to develop specific herbicides to the parasites, especially broomrapes, and to improve our understanding of the molecular mechanisms of this unique germination.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Orobanchaceae/parasitología , Orobanche/metabolismo , Enfermedades de las Plantas/parasitología , Carbohidratos/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas , Germinación , Metabolómica , Orobanche/crecimiento & desarrollo , Raíces de Plantas/parasitología , Malezas , Semillas/crecimiento & desarrollo , Semillas/metabolismoRESUMEN
Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin-labeled in whole cells and outer membranes and interspin distances were measured to a spin-labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions at surface-exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Membrana/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Modelos MolecularesRESUMEN
The role of resource availability in determining the incidence of masting has been widely studied, but how floral transition and initiation are regulated by the resource level is unclear. We tested the hypothesis that floral transition is stimulated by high resource availabiltiy in Fagus crenata based on a new technique, the expression analyses of flowering genes. We isolated F. crenata orthologues of FLOWERING LOCUS T, LEAFY and APETALA1, and confirmed their functions using transgenic Arabidopsis thaliana. We monitored the gene expression levels for 5 years and detected a cycle of on and off years, which was correlated with fluctuations of the shoot-nitrogen concentration. Nitrogen fertilisation resulted in the significantly higher expression of flowering genes than the control, where all of the fertilised trees flowered, whereas the control did not. Our findings identified nitrogen as a key regulator of mast flowering, thereby providing new empirical evidence to support the resource budget model.
Asunto(s)
Fagus/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Nitrógeno/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Carbohidratos/análisis , Fagus/fisiología , Genes de Plantas , Japón , Proteínas de Dominio MADS/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , Estaciones del Año , Factores de Transcripción/genéticaRESUMEN
Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of ß-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd3+-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.
Asunto(s)
Escherichia coli , Proteínas de la Membrana , Espectroscopía de Resonancia por Spin del Electrón/métodos , Marcadores de Spin , Proteínas de la Membrana/metabolismo , Escherichia coli/metabolismo , Conformación MolecularRESUMEN
The ß-barrel assembly machinery (BAM) mediates the folding and insertion of the majority of outer membrane proteins (OMPs) in gram-negative bacteria. BAM is a penta-heterooligomeric complex consisting of the central ß-barrel BamA and four interacting lipoproteins BamB, C, D, and E. The conformational switching of BamA between inward-open (IO) and lateral-open (LO) conformations is required for substrate recognition and folding. However, the mechanism for the lateral gating or how the structural details observed in vitro correspond with the cellular environment remains elusive. In this study, we addressed these questions by characterizing the conformational heterogeneity of BamAB, BamACDE, and BamABCDE complexes in detergent micelles and/or Escherichia coli using pulsed dipolar electron spin resonance spectroscopy (PDS). We show that the binding of BamB does not induce any visible changes in BamA, and the BamAB complex exists in the IO conformation. The BamCDE complex induces an IO to LO transition through a coordinated movement along the BamA barrel. However, the extracellular loop 6 (L6) is unaffected by the presence of lipoproteins and exhibits large segmental dynamics extending to the exit pore. PDS experiments with the BamABCDE complex in intact E. coli confirmed the dynamic behavior of both the lateral gate and the L6 in the native environment. Our results demonstrate that the BamCDE complex plays a key role in the function by regulating lateral gating in BamA.
RESUMEN
ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Movimiento , Cisteína , Citoplasma/metabolismo , Detergentes/química , Dimetilaminas/química , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/citología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Liposomas/metabolismo , Modelos Moleculares , Proteínas de Unión Periplasmáticas/química , Proteínas de Unión Periplasmáticas/metabolismo , Conformación Proteica , Marcadores de Spin , Vitamina B 12/metabolismoRESUMEN
The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels-Alder click chemistry of the genetically encoded amino acid SCO-L-lysine. The previously introduced photoactivatable nitroxide PaNDA prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups. This study describes significant evolution of Diels-Alder-mediated spin labelling on cellular surfaces and opens up new vistas for the the study of membrane proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Escherichia coli/química , Proteínas de Transporte de Membrana/química , Proteínas de la Membrana Bacteriana Externa/genética , Espectroscopía de Resonancia por Spin del Electrón , Código Genético , Proteínas de Transporte de Membrana/genéticaRESUMEN
ATP-binding cassette (ABC) transporters constitute one of the largest protein superfamilies, and they mediate the transport of diverse substrates across the membrane. The molecular mechanism for transducing the energy from ATP binding and hydrolysis into the conformational changes remains elusive. Here, we determined the thermodynamics underlying the ATP-induced global conformational switching for the ABC exporter TmrAB using temperature-resolved pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. We show that a strong entropy-enthalpy compensation mechanism enables the closure of the nucleotide-binding domains (NBDs) over a wide temperature range. This is mechanically coupled with an outward opening of the transmembrane domains (TMDs) accompanied by an entropy gain. The conserved catalytic glutamate plays a key role in the overall energetics. Our results reveal the thermodynamic basis for the chemomechanical energy coupling in an ABC exporter and present a new strategy to explore the energetics of similar membrane protein complexes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato , Catálisis , Cationes Bivalentes/química , Espectroscopía de Resonancia por Spin del Electrón , Hidrólisis , Magnesio/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Temperatura , TermodinámicaRESUMEN
Observation of structure and conformational dynamics of membrane proteins at high resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer-membrane proteins (OMPs) in intact Escherichia coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in situ labeling of engineered cysteines with a methanethiosulfonate spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OMs) under selective conditions. The interspin distances are measured in situ, using pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. The residual background signals, which are problematic for in situ structural biology, contribute specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5-7 d, and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and data analysis typically take 4-5 d. The described protocol provides a general strategy for observing protein ligand-substrate interactions, oligomerization, and conformational dynamics of OMPs in their native OM and intact E. coli.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Espectroscopía de Resonancia por Spin del Electrón/métodos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Cisteína/química , Cisteína/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructura , Mesilatos/química , Mesilatos/metabolismo , Conformación Proteica , Marcadores de SpinRESUMEN
The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.