RESUMEN
The Voltage Dependent Anion Channel (VDAC) is the most abundant protein in the outer membrane of mitochondria. This strategic localization puts it at the heart of a great number of phenomena. Its recent implication in apoptosis is an example of the major importance of this protein and has created a surge of interest in VDAC. There is no atomic-resolution structure allowing a better understanding of the function of VDAC, so alternative techniques to X-ray diffraction have been used to study VDAC. Here we discuss structural models from folding predictions and review data acquired by Atomic Force Microscopy (AFM) imaging that allowed to observe VDAC's structure and supramolecular organization in the mitochondrial outer membrane.
Asunto(s)
Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Pliegue de Proteína , Canales Aniónicos Dependientes del Voltaje/metabolismo , Animales , Humanos , Microscopía de Fuerza Atómica/métodos , Mitocondrias/química , Membranas Mitocondriales/química , Estructura Terciaria de Proteína , Canales Aniónicos Dependientes del Voltaje/químicaRESUMEN
Membrane proteins perform many essential cellular functions. Over the last years, substantial advances have been made in our understanding of the structure and function of isolated membrane proteins. However, like soluble proteins, many membrane proteins assemble into supramolecular complexes that perform specific functions in specialized membrane domains. Since supramolecular complexes of membrane proteins are difficult to study by conventional approaches, little is known about their composition, organization and assembly. The high signal-to-noise ratio of the images that can be obtained with an atomic force microscope (AFM) makes this instrument a powerful tool to image membrane protein complexes within native membranes. Recently, we have reported high-resolution topographs of junctional microdomains in native eye lens membranes containing two-dimensional (2D) arrays of aquaporin-0 (AQP0) surrounded by connexons. While both proteins are involved in cell adhesion, AQP0 is a specific water channel whereas connexons form cell-cell communication channels with broad substrate specificity. Here, we have performed a detailed analysis of the supramolecular organization of AQP0 tetramers and connexon hexamers in junctional microdomains in the native lens membrane. We present first structural models of these junctional microdomains, which we generated by docking atomic models of AQP0 and connexons into the AFM topographs. The AQP0 2D arrays in the native membrane show the same molecular packing of tetramers seen in highly ordered double-layered 2D crystals obtained through reconstitution of purified AQP0. In contrast, the connexons that surround the AQP0 arrays are only loosely packed. Based on our AFM observations, we propose a mechanism that may explain the supramolecular organization of AQP0 and connexons in junctional domains in native lens membranes.
Asunto(s)
Acuaporinas/química , Proteínas del Ojo/química , Glicoproteínas de Membrana/química , Microscopía de Fuerza Atómica , Animales , Acuaporinas/ultraestructura , Cristalización , Proteínas del Ojo/ultraestructura , Cristalino/química , Cristalino/ultraestructura , Lípidos , Glicoproteínas de Membrana/ultraestructura , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Rotación , OvinosRESUMEN
The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane (MOM). Due to its localization, VDAC is involved in a wide range of processes, such as passage of ATP out of mitochondria, and particularly plays a central role in apoptosis. Importantly, the assembly of VDAC provides interaction with a wide range of proteins, some implying oligomerization. However, many questions remain as to the VDAC structure, its supramolecular assembly, packing density, and oligomerization in the MOM is unknown. Here we report the so far highest resolution view of VDAC and its native supramolecular assembly. We have studied yeast MOM by high-resolution atomic force microscopy (AFM) in physiological buffer and found VDAC in two distinct types of membrane domains. We found regions where VDAC was packed at high density (approximately 80%), rendering the membrane a voltage-dependent molecular sieve. In other domains, VDAC has a low surface density (approximately 20%) and the pore assembly ranges from single molecules to groups of up to 20. We assume that these groups are mobile in the lipid bilayer and allow association and dissociation with the large assemblies. VDAC has no preferred oligomeric state and no long-range order was observed in densely packed domains. High-resolution topographs show an eye-shaped VDAC with 3.8 nm x 2.7 nm pore dimensions. Based on the observed VDAC structure and the pair correlation function (PCF) analysis of the domain architectures, we propose a simple model that could explain the phase behavior of VDAC, and illustrates the sensitivity of the molecular organization to conditions in the cell, and the possibility for modulation of its assembly. The implication of VDAC in cytochrome c release from the mitochondria during cell apoptosis has made it a target in cancer research.
Asunto(s)
Mitocondrias/ultraestructura , Canales Aniónicos Dependientes del Voltaje , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Microscopía de Fuerza Atómica , Mitocondrias/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Canales Aniónicos Dependientes del Voltaje/química , Canales Aniónicos Dependientes del Voltaje/metabolismo , Canales Aniónicos Dependientes del Voltaje/ultraestructuraRESUMEN
Biological membranes compartmentalize and define physical borders of cells. They are crowded with membrane proteins that fulfill diverse crucial functions. About one-third of all genes in organisms code for, and the majority of drugs target, membrane proteins. To combine structure and function analysis of membrane proteins, we designed a two-chamber atomic force microscopy (AFM) setup that allows investigation of membranes spanned over nanowells, therefore separating two aqueous chambers. We imaged nonsupported surface layers (S layers) of Corynebacterium glutamicum at sufficient resolution to delineate a 15 A-wide protein pore. We probed the elastic and yield moduli of nonsupported membranes, giving access to the lateral interaction energy between proteins. We combined AFM and fluorescence microscopy to demonstrate the functionality of proteins in the setup by documenting proton pumping by Halobacterium salinarium purple membranes.
Asunto(s)
Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Micromanipulación/métodos , Microscopía de Fuerza Atómica/métodos , Mapeo de Interacción de Proteínas/métodos , Sitios de Unión , Membrana Celular/química , Proteínas de la Membrana/química , Unión Proteica , Estrés Mecánico , Agua/química , Agua/metabolismoRESUMEN
The structural analysis of the individual components of the photosynthetic apparatus of Rhodopseudomonas palustris, or those of related species, is almost complete. To shed light on the assembly and organization of this machinery, we have studied native membranes of Rps.palustris grown under different light conditions using atomic force microscopy (AFM). The organization of the complexes in the membranes is different from any previously observed: with areas of crystalline core-complexes, crystalline peripheral antennae, mixed domains, and apparently pure lipid membranes devoid of protein. Examination of antennae structure shows that chromatic adaptation is associated with modifications in absorption and size of the peripheral light harvesting complexes (LH2) as light intensity is reduced. The core-complex is observed to contain a reaction centre (RC) surrounded by an elliptical assembly of 15 LH1 subunits and a "gap" attributed to the W-subunit. The localization of the W-subunit is not restricted to the periapsis of the core-complex but randomly located with respect to the RC imposed axis.
Asunto(s)
Fotosíntesis , Rhodopseudomonas/química , Cromatóforos Bacterianos/ultraestructura , Membrana Celular/efectos de la radiación , Membrana Celular/ultraestructura , Luz , Complejos de Proteína Captadores de Luz/química , Microscopía de Fuerza Atómica , Rhodopseudomonas/efectos de la radiación , Rhodopseudomonas/ultraestructuraRESUMEN
The ubiquity and importance of photosynthetic organisms in nature has made the molecular mechanisms of photosynthesis a widely studied subject at both structural and functional levels. A current challenge is to understand the supramolecular assembly of the proteins involved in photosynthesis in native membranes. We have used atomic force microscopy to study the architecture of the photosynthetic apparatus and analyze the structure of single molecules in chromatophores of Phaeospirillum molischianum. Core complexes are formed by the reaction center enclosed by an elliptical light harvesting complex 1. LH2 are octameric rings, assembled either with cores or in hexagonally packed LH2 antenna domains. The symmetry mismatch caused by octameric LH2 packing in a hexagonal lattice, that could be avoided in a square lattice, suggests lipophobic effects rather than specific inter-molecular interactions drive protein organization. The core and LH2 complexes are organized to form a supramolecular assembly reminiscent to that found in Rhodospirillum photometricum, and very different from that observed in Rhodobacter sphaeroides, Rb. blasticus, and Blastochloris viridis.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/ultraestructura , Rhodospirillum/enzimología , Cromatóforos Bacterianos/química , Cromatóforos Bacterianos/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Fraccionamiento Celular , Centrifugación por Gradiente de Densidad , Transferencia de Energía , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/ultraestructura , Microscopía de Fuerza Atómica , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/ultraestructura , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodospirillum/química , EspectrofotometríaRESUMEN
Light harvesting complexes 2 (LH2) are the peripheral antenna proteins in the bacterial photosynthetic apparatus and are built of alpha/beta-heterodimers containing three bacteriochlorophylls and two carotenoids each. Previously, we have found in 2D-crystals that the complexes could be inserted within the membrane with a tilt with respect to the membrane plane (Rhodobacter sphaeroides) or without tilt (Rubrivivax gelatinosus). To investigate whether the tilted insertion represents the native state or if it is due to specific 2D-crystal contacts, we have used atomic force microscopy to investigate LH2 from Rhodopseudomonas acidophila reconstituted at different lipid to protein ratios. High-resolution topographs could be acquired of two types of 2D-crystals or of densely packed membranes. Interestingly, in type 2 2D-crystals and in non-crystalline densely packed membranes, cylinders are integrated with their symmetry axis normal to the membrane plane, while in type 1 2D-crystals LH2 cylinders are integrated with a tilt of approximately 4 degrees with respect to the membrane plane. Therefore, we present strong evidence that the tilt of LH2 does not represent the native membrane state and is due to protein-protein contacts in specific 2D-crystals.