Holger Seelert

Structural biology. Proton-powered turbine of a plant motor.

ATP synthases are enzymes that can work in two directions to catalyse either the synthesis or breakdown of ATP, and they constitute the smallest rotary motors in biology. The flow of protons propels the rotation of a membrane-spanning complex of identical protein subunits, the number of which determines the efficiency of energy conversion. This proton-powered turbine is predicted to consist of 12 subunits, based on data for Escherichia coli. The yeast mitochondrial enzyme, however, has only 10 subunits. We have imaged the ATP synthase from leaf chloroplasts by using atomic force microscopy and, surprisingly, find that its turbine has 14 subunits, arranged in a cylindrical ring.

Architecture of Active Mammalian Respiratory Chain Supercomplexes

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 I1III2 and I1III2IV1 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 I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, 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.

Proton-powered turbine of a plant motor

ATP synthases are enzymes that can work in two directions to catalyse either the synthesis or breakdown of ATP, and they constitute the smallest rotary motors in biology. The flow of protons propels the rotation of a membrane-spanning complex of identical protein subunits, the number of which determines the efficiency of energy conversion. This proton-powered turbine is predicted to consist of 12 subunits, based on data for Escherichia coli. The yeast mitochondrial enzyme, however, has only 10 subunits. We have imaged the ATP synthase from leaf chloroplasts by using atomic force microscopy and, surprisingly, find that its turbine has 14 subunits, arranged in a cylindrical ring.

ATP synthase: constrained stoichiometry of the transmembrane rotor

Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation‐powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native‐like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch‐shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.

Function and picosecond dynamics of bacteriorhodopsin in purple membrane at different lipidation and hydration

© 1998 Federation of European Biochemical Societies.

From protons to OXPHOS supercomplexes and Alzheimer's disease: Structure–dynamics–function relationships of energy-transducing membranes

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 Alzheimers dementia (AD) are triggered by perturbation of membranes and bioenergetics as demonstrated by our neutron scattering studies.

Dye removal, catalytic activity and 2D crystallization of chloroplast H+-ATP synthase purified by blue native electrophoresis

The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F(1)) and its subcomplexes CF(O) and CF(1) were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization.

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels.

Due to its unmatched resolution, gel electrophoresis is an indispensable tool for the analysis of diverse biomolecules. By adaptation of the electrophoretic conditions, even fragile protein complexes as parts of intracellular networks migrate through the gel matrix under sustainment of their integrity. If the thickness of such native gels is significantly increased compared to the analytical version, also high sample loads can be processed. However, the cage‐like network obstructs an in‐depth analysis for deciphering structure and function of protein complexes and other species. Consequently, the biomolecules have to be removed from the gel matrix into solution. Several approaches summarized in this review tackle this problem. While passive elution relies on diffusion processes, electroelution employs an electric field to force biomolecules out of the gel. An alternative procedure requires a special electrophoresis setup, the continuous elution device. In this apparatus, molecules migrate in the electric field until they leave the gel and were collected in a buffer stream. Successful isolation of diverse protein complexes like photosystems, ATP‐dependent enzymes or active respiratory supercomplexes and some other bioparticles demonstrates the versatility of preparative electrophoresis. After liberating particles out of the gel cage, numerous applications are feasible. They include elucidation of the individual components up to high resolution structures of protein complexes. Therefore, preparative electrophoresis can complement standard purification methods and is in some cases superior to them.

Detection and analysis of protein-protein interactions of organellar and prokaryotic proteomes by blue native and colorless native gel electrophoresis.

Native gels enable the analysis of protein complexes on a proteome‐wide scale in a single experiment. The protocols described in this unit are based on separation of protein complexes by blue native polyacrylamide electrophoresis (BN‐PAGE), the most versatile native gel system, and the closely related milder colorless native PAGE (CN‐PAGE). Both BN‐PAGE and CN‐PAGE are described on analytical to preparative scales. In addition, methods for subsequent analysis of protein complexes are given, including electroelution from native gels as well as denaturing and native two‐dimensional PAGE. Finally, the removal of Coomassie dye from electroeluted proteins is detailed along with a discussion of fundamental considerations for the solubilization of membrane protein complexes from various biological samples, which are exemplified for mitochondria, chloroplasts (thylakoids), and cyanobacteria. Curr. Protoc. Protein Sci. 51:14.11.1‐14.11.36.

ATP synthase superassemblies in animals and plants: Two or more are better

ATP synthases are part of the sophisticated cellular metabolic network and therefore multiple interactions have to be considered. As discussed in this review, ATP synthases form various supramolecular structures. These include dimers and homooligomeric species. But also interactions with other proteins, particularly those involved in energy conversion exist. The supramolecular assembly of the ATP synthase affects metabolism, organellar structure, diseases, ageing and vice versa. The most common approaches to isolate supercomplexes from native membranes by use of native electrophoresis or density gradients are introduced. On the one hand, isolated ATP synthase dimers and oligomers are employed for structural studies and elucidation of specific protein-protein interactions. On the other hand, native electrophoresis and other techniques serve as tool to trace changes of the supramolecular organisation depending on metabolic alterations. Upon analysing the structure, dimer-specific subunits can be identified as well as interactions with other proteins, for example, the adenine nucleotide translocator. In the organellar context, ATP synthase dimers and oligomers are involved in the formation of mitochondrial cristae. As a consequence, changes in the amount of such supercomplexes affect mitochondrial structure and function. Alterations in the cellular power plant have a strong impact on energy metabolism and ultimately play a significant role in pathophysiology. In plant systems, dimers of the ATP synthase have been also identified in chloroplasts. Similar to mammals, a correlation between metabolic changes and the amount of the chloroplast ATP synthase dimers exists. Therefore, this review focusses on the interplay between metabolism and supramolecular organisation of ATP synthase in different organisms.