All organisms have to monitor the folding state of cellular proteins precisely. The heat-shock protein DegP is a protein quality control factor in the bacterial envelope that is involved in eliminating misfolded proteins and in the biogenesis of outer-membrane proteins. Here we describe the molecular mechanisms underlying the regulated protease and chaperone function of DegP from Escherichia coli. We show that binding of misfolded proteins transforms hexameric DegP into large, catalytically active 12-meric and 24-meric multimers. A structural analysis of these particles revealed that DegP represents a protein packaging device whose central compartment is adaptable to the size and concentration of substrate. Moreover, the inner cavity serves antagonistic functions. Whereas the encapsulation of folded protomers of outer-membrane proteins is protective and might allow safe transit through the periplasm, misfolded proteins are eliminated in the molecular reaction chamber. Oligomer reassembly and concomitant activation on substrate binding may also be critical in regulating other HtrA proteases implicated in protein-folding diseases.

Type IV secretion systems (T4SSs) are important virulence factors used by Gram-negative bacterial pathogens to inject effectors into host cells or to spread plasmids harboring antibiotic resistance genes. We report the 15 angstrom resolution cryo-electron microscopy structure of the core complex of a T4SS. The core complex is composed of three proteins, each present in 14 copies and forming a approximately 1.1-megadalton two-chambered, double membrane-spanning channel. The structure is double-walled, with each component apparently spanning a large part of the channel. The complex is open on the cytoplasmic side and constricted on the extracellular side. Overall, the T4SS core complex structure is different in both architecture and composition from the other known double membrane-spanning secretion system that has been structurally characterized.

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I 1 III 2 and I 1 III 2 IV 1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I 1 III 2 and I 1 III 2 IV 1 closely fits the x-ray structure of monomeric complex IV and shows its location in the assembly. By comparing different views of supercomplex I 1 III 2 IV 1 , the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.All living organisms use a series of integral membrane protein complexes for energy conversion and ATP synthesis. In eukaryotes, electrons are transported by the respiratory chain, starting from NADH via complex I (NADH:ubiquinone oxidoreductase) or from succinate via complex II (succinate:ubiquinone oxidoreductase), the membrane integral electron carrier ubiquinol, complex III (ubiquinol:cytochrome c oxidoreductase), the peripheral electron carrier cytochrome c, and complex IV (cytochrome c oxidase) to the terminal acceptor molecular oxygen (1). The electron transport chain generates a proton gradient across the inner mitochondrial membrane, which is used by complex V (F O F 1 -ATP synthase) to synthesize ATP. In the last decade, structures of the individual respiratory chain complexes from various organisms have been determined. Atomic models exist for bovine heart mitochondrial complex III (2) and IV (3). A high resolution structure of complex I is not yet available, but electron microscopy indicates that it is L-shaped in all organisms investigated, and a 2.2-nm resolution map from cryoelectron microscopy exists for the bovine heart complex I (4).Two alternative models for the arrangement of the respiratory chain complexes in the membrane have been proposed. According to the currently favored random collision model (5), all components of the respiratory chain diffuse individually in the membrane, and electron transfer depends on the random, transient encounter of the individual protein complexes and the smaller electron carriers. In the solid state model (6) proposed 50 years ago, the substrate is channeled directly from one enzyme to the next. Recently isolated stoichiometric assemblies, so-called supercomplexes, support this model. Respiratory supercomplexes of different compositions have been described in bact...

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein.membrane protein ͉ Escherichia coli ͉ blue native PAGE ͉ single particle electron microscopy

The respiratory chain complexes can arrange into multienzyme assemblies, so-called supercomplexes. We present the first 3D map of a respiratory chain supercomplex. It was determined by random conical tilt electron microscopy analysis of a bovine supercomplex consisting of complex I, dimeric complex III, and complex IV (I1III2IV1). Within this 3D map the positions and orientations of all the individual complexes in the supercomplex were determined unambiguously. Furthermore, the ubiquinone and cytochrome c binding sites of each complex in the supercomplex could be located. The mobile electron carrier binding site of each complex was found to be in proximity to the binding site of the succeeding complex in the respiratory chain. This provides structural evidence for direct substrate channeling in the supercomplex assembly with short diffusion distances for the mobile electron carriers.

The liquid state model that envisions respiratory chain complexes diffusing freely in the membrane is increasingly challenged by reports of supramolecular organization of the complexes in the mitochondrial inner membrane. Supercomplexes of complex III with complex I and/or IV can be isolated after solubilisation with mild detergents like digitonin. Electron microscopic studies have shown that these have a distinct architecture and are not random aggregates. A 3D reconstruction of a I1III2IV1 supercomplex shows that the ubiquinone and cytochrome c binding sites of the individual complexes are facing each other, suggesting a role in substrate channelling. Formation of supercomplexes plays a role in the assembly and stability of the complexes, suggesting that the supercomplexes are the functional state of the respiratory chain. Furthermore, a supramolecular organisation of ATP synthases has been observed in mitochondria, where ATP synthase is organised in dimer rows. Dimers can be isolated by mild detergent extraction and recent electron microscopic studies have shown that the membrane domains of the two partners in the dimer are at an angle to each other, indicating that in vivo the dimers would cause the membrane to bend. The suggested role in crista formation is supported by the observation of rows of ATP synthase dimers in the most curved parts of the cristae. Together these observations show that the mitochondrial inner membrane is highly organised and that the molecular events leading to ATP synthesis are carefully coordinated.

By the elucidation of high-resolution structures the view of the bioenergetic processes has become more precise. But in the face of these fundamental advances, many problems are still unresolved. We have examined a variety of aspects of energy-transducing membranes from large protein complexes down to the level of protons and functional relevant picosecond protein dynamics. Based on the central role of the ATP synthase for supplying the biological fuel ATP, one main emphasis was put on this protein complex from both chloroplast and mitochondria. In particular the stoichiometry of protons required for the synthesis of one ATP molecule and the supramolecular organisation of ATP synthases were examined. Since formation of supercomplexes also concerns other complexes of the respiratory chain, our work was directed to unravel this kind of organisation, e.g. of the OXPHOS supercomplex I(1)III(2)IV(1), in terms of structure and function. Not only the large protein complexes or supercomplexes work as key players for biological energy conversion, but also small components as quinones which facilitate the transfer of electrons and protons. Therefore, their location in the membrane profile was determined by neutron diffraction. Physico-chemical features of the path of protons from the generators of the electrochemical gradient to the ATP synthase, as well as of their interaction with the membrane surface, could be elucidated by time-resolved absorption spectroscopy in combination with optical pH indicators. Diseases such as Alzheimer's dementia (AD) are triggered by perturbation of membranes and bioenergetics as demonstrated by our neutron scattering studies.

The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimer's and Parkinson's Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.