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1.
Biochim Biophys Acta Bioenerg ; 1862(8): 148433, 2021 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-33932366

RESUMEN

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc1 and aa3-type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O2 reduction activity of the cyt. bc1-aa3 supercomplex. The potential role of the membrane-anchored cyt. cy as a component in supercomplexes was also investigated.


Asunto(s)
Membrana Celular/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Hidroquinonas/metabolismo , Rhodobacter sphaeroides/enzimología , Transporte de Electrón , Complejo III de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/química , Cinética , Oxidación-Reducción
2.
Sci Rep ; 6: 24113, 2016 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-27063297

RESUMEN

Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain.


Asunto(s)
Adenosina Trifosfato/biosíntesis , Liposomas/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Metabolismo Energético , Cinética , Liposomas/química , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismo , Dominios y Motivos de Interacción de Proteínas , Protones , Ubiquinona/química , Ubiquinona/metabolismo
3.
Nat Commun ; 5: 4303, 2014 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-24985889

RESUMEN

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.


Asunto(s)
Vesículas Citoplasmáticas/química , Proteínas SNARE/química , Citocromos c/química , Transporte de Electrón , Complejo II de Transporte de Electrones/química , Escherichia coli , Liposomas , Membranas Artificiales , ATPasas de Translocación de Protón/química , Rhodobacter sphaeroides
4.
J Biol Chem ; 286(18): 16525-32, 2011 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-21454699

RESUMEN

CorA is a family of divalent cation transporters ubiquitously present in bacteria and archaea. Although CorA can transport both Mg(2+) and Co(2+) almost equally well, its main role has been suggested to be that of primary Mg(2+) transporter of prokaryotes and hence the regulator of Mg(2+) homeostasis. The reason is that the affinity of CorA for Co(2+) is relatively low and thus considered non-physiological. Here, we show that Thermotoga maritima CorA (TmCorA) is incapable of regulating the Mg(2+) homeostasis and therefore cannot be the primary Mg(2+) transporter of T. maritima. Further, our in vivo experiments confirm that TmCorA is a highly selective Co(2+) transporter, as it selects Co(2+) over Mg(2+) at >100 times lower concentrations. In addition, we present data that show TmCorA to be extremely thermostable in the presence of Co(2+). Mg(2+) could not stabilize the protein to the same extent, even at high concentrations. We also show that addition of Co(2+), but not Mg(2+), specifically induces structural changes to the protein. Altogether, these data show that TmCorA has the role of being the transporter of Co(2+) but not Mg(2+). The physiological relevance and requirements of Co(2+) in T. maritima is discussed and highlighted. We suggest that CorA may have different roles in different organisms. Such functional diversity is presumably a reflection of minor, but important structural differences within the CorA family that regulate the gating, substrate selection, and transport.


Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Cobalto/metabolismo , Homeostasis/fisiología , Magnesio/metabolismo , Thermotoga maritima/metabolismo , Proteínas de Transporte de Catión/genética , Thermotoga maritima/genética
5.
ACS Nano ; 3(9): 2639-46, 2009 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-19653679

RESUMEN

We have investigated formation of a proteolipid membrane surrounding mesoporous silica particles with a diameter of 550 nm and pore sizes of 3.0 nm. A multisubunit redox-driven proton pump, cytochrome c oxidase, was incorporated into the membrane, and we show that the enzyme is functional, both with respect to catalysis of O(2) reduction to water, and charge separation across the membrane. The orientation of cytochrome c oxidase in the membrane was found to be the same ( approximately 70%) in the lipid vesicles and in the silica-particle-supported lipid membrane, which provides information on the mechanism by which the vesicles adsorb to the surface. Furthermore, cytochrome c oxidase could maintain a proton electrochemical gradient across the supported proteomembrane, that is, the membrane system was proton tight, defining an interior particle compartment that is separated from the surrounding aqueous media. Such a biofunctional cellular interface, supported onto a colloid that has a connected interior cytoskeleton-like pore structure, provides a basis for functional studies of membrane-bound transport proteins, and also for applications within pharmaceutical drug delivery.


Asunto(s)
Membrana Celular/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Dióxido de Silicio/química , Dióxido de Silicio/metabolismo , Adsorción , Biomimética , Cetrimonio , Compuestos de Cetrimonio/química , Electroquímica , Oxígeno/metabolismo , Tamaño de la Partícula , Fotólisis , Porosidad , Rhodobacter sphaeroides/enzimología
6.
Langmuir ; 25(8): 4601-6, 2009 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-19265407

RESUMEN

Silica colloidal particles with functionalized surfaces are used, for example, in studies of membrane proteins or for drug delivery, where novel applications are based on the use of particles covered by lipid membrane bilayers. The mechanism by which such supported lipid bilayers are formed on spherical support is not fully understood. Here, we present results from studies of this process using a new method based on flow cytometry. The approach enabled us to detect particle populations coated and uncoated with lipids in the same sample according to the vesicle:particle surface area ratio. The data suggest that DOPC lipid vesicles efficiently break upon interaction with the silica colloidal particle surface; only a small fraction of the adsorbed vesicles remain unbroken. Furthermore, the data support earlier observations showing that formation of the lipid bilayer at the surface is a cooperative process, where bilayer formation is catalyzed by previously bound membrane fragments.


Asunto(s)
Citometría de Flujo/métodos , Membrana Dobles de Lípidos/química , Dióxido de Silicio/química , Adsorción , Animales , Catálisis , Membrana Celular/metabolismo , Coloides/química , Colorantes Fluorescentes/farmacología , Concentración de Iones de Hidrógeno , Tamaño de la Partícula , Fosfatidilcolinas/química , Dispersión de Radiación , Propiedades de Superficie
7.
Biochemistry ; 47(17): 4948-54, 2008 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-18393441

RESUMEN

The mitochondrial protein frataxin plays a central role in mitochondrial iron homeostasis, and frataxin deficiency is responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1 in 40000 children. Here we present a single-particle reconstruction from cryoelectron microscopic images of iron-loaded 24-subunit oligomeric frataxin particles at 13 and 17 A resolution. Computer-aided classification of particle images showed heterogeneity in particle size, which was hypothesized to result from gradual accumulation of iron within the core structure. Thus, two reconstructions were created from two classes of particles with iron cores of different sizes. The reconstructions show the iron core of frataxin for the first time. Compared to the previous reconstruction of iron-free particles from negatively stained images, the higher resolution of the present reconstruction allowed a more reliable analysis of the overall three-dimensional structure of the 24-meric assembly. This was done after docking the X-ray structure of the frataxin trimer into the EM reconstruction. The structure revealed a close proximity of the suggested ferroxidation sites of different monomers to the site proposed to serve in iron nucleation and mineralization. The model also assigns a new role to the N-terminal helix of frataxin in controlling the channel at the 4-fold axis of the 24-subunit oligomer. The reconstructions show that, together with some common features, frataxin has several unique features which distinguish it from ferritin. These include the overall organization of the oligomers, the way they are stabilized, and the mechanisms of iron core nucleation.


Asunto(s)
Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Hierro/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , Procesamiento de Imagen Asistido por Computador , Proteínas de Unión a Hierro/genética , Modelos Moleculares , Mutación , Estructura Cuaternaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Electricidad Estática , Propiedades de Superficie , Frataxina
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