The proteins in the membranes of the mitochondria are complex machines in the nanoworld that mediate the oxygen-coupled generation of energy in cells. Their role in this process is not yet understood in detail. However, the proteins’ intractability has not discouraged Prof. Dr. Carola Hunte’s research group at the University of Freiburg. The group is using state-of-the-art methods to look deep into the proteins’ active centres and decipher the functions of these huge protein complexes, which – as research has shown – can do more than just generate energy.
It all seems quite simple to us, you do a bit of exercise, eat a bar of chocolate, take a few deep breaths to recover and carry on with whatever you want to do next. However, providing us with the energy we need takes the form of highly complex processes in our cells. Such processes have long intrigued cell biologists. It is a well-known fact that the membrane-enclosed organelles, called mitochondria, are the powerhouses of cells. The protein complexes that withdraw energy-rich electrons from glucose in a process during which oxygen is reduced are also well known. But how do these protein complexes transfer the electrons to complexes further along what is known as the respiratory chain? And what leads to energy being freed up? “In order to understand the nanomachines in the membrane of the mitochondria, we first need to elucidate their atomic structure,” said Prof. Dr. Carola Hunte from the Institute of Biochemistry and Molecular Biology and the BIOSS (Centre for Biological Signalling Studies) excellence centre at the University of Freiburg.
The unravelling of the three-dimensional structure of membrane proteins involved in cellular respiration has become more interesting and more pressing over the last few years since research has shown that these protein complexes are involved in a greater number of cellular processes than previously assumed. In addition to providing the cells’ energy, they have been shown to be involved in the transduction of signals in processes such as cell division and programmed cell death (apoptosis). These protein complexes might therefore also be involved in the pathogenesis of cancer. They have also been shown to be involved in the generation of so-called reactive oxygen species, i.e. free radicals that damage vital proteins and lipids and cause cells to age. Not so long ago, researchers also found that the protein complexes play a role in the pathogenesis of neurodegenerative diseases such as Parkinson’s and Alzheimer’s. “Due to growing evidence that mitochondrial membrane proteins can do more than just produce energy, unravelling the function of these proteins in cellular processes is of great importance. To do this, we will need to look at the structure of these protein machines, which will also provide us with information on potential targets for therapeutic drugs,” Hunte said.
Hunte started to study membrane proteins when she was doing her doctoral thesis around twenty years ago at the Institute of Agricultural Biology at the University of Bonn. Three years ago, Hunte and her team started studying complex I of the respiratory chain in greater detail. The proton-pumping respiratory complex I is the first stage of the mitochondrial energy generation process and the largest and most complicated membrane protein complex known to date. The researchers used X-ray crystallographic analyses to unravel the three-dimensional structure of the complex. This project was carried out with partners from Frankfurt and took the researchers nine years to complete. They were able to show that the transfer of electrons and the associated pumping process (which translocates free proteins across the membrane, resulting in an energy-rich gradient across the membrane) are somehow coupled to each other by way of molecular rods, which means that some of the energy stored in the molecules is transferred by way of a mechanical process.
It is still difficult for biochemists to determine the structure of membrane proteins. This is due to the fact that the molecule complexes embedded in the lipid bilayer of a membrane can only be removed with great difficulty. Care must be taken to choose a solvent that does not disintegrate the protein complexes into their individual parts. “Even though chemists have made huge progress in finding detergents that do not destroy the proteins, there are still many problems related to the subsequent step, i.e. crystallisation,” Hunte said explaining that the structure of protein complexes can only be analysed with X-rays when present as perfectly ordered crystals. It requires a great deal of trial and error to find the right parameters and takes rather a long time to find temperatures, concentrations and precipitants that are suitable for the supermolecules under investigation. Therefore, it comes as no surprise that the structure of only 308 of the 5000 to 7500 or so membrane proteins is known.Hunte’s team is one of only a handful of research groups worldwide who use a special method to optimise the crystallisation process in order to minimise the difficulties as much as possible. They use antibodies that bind to specific areas of the protein complex, thereby increasing the hydrophilic surface of the otherwise largely lipophilic membrane proteins. This makes it easier for the molecules to assemble into symmetric crystal structures in the water phase.
”In order to find out why reactive oxygen species are generated in the mitochondria and promote the ageing of cells or why the energy generation process or cell division is defective, we need to gain a detailed understanding of how the mitochondrial membrane protein complexes work,” said Hunte. And this is why the Freiburg researchers are planning to focus in even greater detail on the structure of complex I, which also requires looking at the atomic level. The researchers are also working on the analysis of the cytochrome bc1 complex which is located further downstream in the respiratory chain. The cytochrome bc1 complex is mainly involved in transferring the electrons, but has also been shown to produce a rather high number of reactive oxygen species. Moreover, a well-known malaria drug targets the cytochrome bc1 complex of the electron transport chain of malaria parasites and Hunte and her team are working to optimise the drug with the aim of preventing the parasites from becoming resistant too quickly.
In addition to investigating membrane proteins of the respiratory chain, Hunte and her team have broadened their focus of interest and are now also investigating membrane proteins in human cells that transport sodium ions and proteins across the cell membrane. These so-called Na/H pumps interact with numerous cellular signalling proteins. They are thus closely coupled with cellular signalling and play a role in the regulation of important cellular processes, including vesicular transport, cell division and volume adjustment. These are highly regulated processes, which can get out of control and lead to the development of tumours. “This project is part of the cooperative research centre 746, and we are planning to use it to find the interaction partners of the Na/H pumps, determine the structure of the membrane proteins and their bound interaction partners, reproduce the system synthetically in Petri dishes and obtain a detailed understanding of the underlying processes,” said Hunte whose research clearly shows that mitochondrial membrane proteins can do a lot more than just generate energy.
Prof. Dr. Carola HunteInstitute of Biochemistry and Molecular BiologyBIOSS Centre for Biological Signalling StudiesUniversity of FreiburgStefan-Meier-Str. 17D-79104 FreiburgE-mail: carola.hunte(at)biochemie.uni-freiburg.deTel.: +49 (0)761/ 2035 279Fax: +49 (0)761/ 2035 284