A large number of cellular proteins are located in or on a membrane. Dr. Dirk Schneider from the University of Freiburg believes that biochemists who investigate such proteins must be a little crazy, as the methods required to isolate the molecules from their exotic environment, i.e. from the lipid bilayer, are extremely difficult and complicated. Research has long focused on water-soluble proteins. Schneider and his team have now taken on the challenge of finding out how proteins fold and assemble into complexes.
About one quarter to one third of all cellular proteins are located in a biological membrane. They serve as transporters for substances that are either taken up or excreted by the cells and receptors for cellular communication. Key processes of photosynthesis and cellular respiration also take place in or at membranes inside cells. And that is why around one third of drugs target such proteins. A classical example of such drugs is aspirin, which blocks a membrane-associated protein, thereby preventing the synthesis of signalling substances that trigger inflammatory reactions and the perception of pain. “But it is extremely tricky to investigate membrane proteins,” said Dr. Dirk Schneider from the Institute of Biochemistry and Molecular Biology at the University of Freiburg. It is very difficult to produce them in high quantities in bacteria and it is even more difficult to purify them. Membrane proteins need to be hydrophobic in order to be able to exert their designated function in/at the lipid bilayer. Therefore they clump together when biochemists try to solubilise them.
Despite all the difficulties associated with the investigation of membrane proteins, Schneider and his team of researchers have nevertheless decided to take on the task of investigating them. The linear chain of amino acids generated in the cells from the DNA blueprint is a long way from the membrane-bound three-dimensional protein complex. Membrane proteins are normally directly synthesised into the membrane. How are they able to form the required shape in the lipid environment? How do they fold? How do they form the channels through which the ions flow? Scientists assume that certain protein regions, which are referred to as alpha helices, are important for these functions. The individual amino acid components orient themselves such that the lipophilic regions of the membrane-bound proteins extend into the extra-/intracellular space – something that is perfect for proteins located in a lipid bilayer. The majority of membrane proteins have several alpha helices that approach each other inside the membrane. This causes more distant protein sections to come into contact with each other, thereby forming a three-dimensional structure.
“Why do alpha helices interact,” asks Schneider. “Which amino acids interact with each other? Are there other molecules that maybe support the interaction of certain amino acids?” In order to investigate such questions, Schneider developed an Escherichia coli test system during his post-doctoral period in the USA. Using this system, the Freiburg researchers are able to introduce two alpha helices of a precisely defined amino acid sequence into the lipid bilayer of the bacterial cell membrane. On their intracellular side, the two helices have two complementary molecules attached, which function like a key and a lock. Once they unite, they are able to activate the DNA inside the cell. The researchers have manipulated the DNA so that the activated bacterial cell alters a previously produced colour signal. This helps the biochemists to determine how close the two alpha helices come to each other. The colour signal only changes if the “key” and the “lock” fit perfectly. The system enables the researchers to carry out clever experiments. For example, they can exchange individual amino acids in the helices and check whether this leads the two chains to bind more tightly or more loosely. This enables them to assess the contribution of any particular individual amino acid to the binding of the alpha helices.
The Freiburg researchers have used this method to investigate the glycophorin A membrane protein that spans the cell membrane of red blood cells (erythrocytes). This molecule has one alpha helix that spans the membrane. Two such helices congregate, thereby combining two glycophorins. The congregation between the two helices becomes tighter when the very small amino acids are present at specific sites in the helix. It is assumed that the exchange of amino acids reduces the distance between the two helices. When the two helices come very close to each other, they establish bonds with one another. Schneider and his team found that this congregation can be achieved by glycine, which is a particularly small amino acid, as well as by other small amino acids. They obtained similar results for receptor tyrosine kinases, membrane proteins that transmit external signals into the cell. Several diseases, including cancer, are known to be associated with mutations in the alpha helices of receptor tyrosine kinases. Such mutations lead to the permanent activation of the proteins, thereby communicating the presence of signals that are not actually present. “It was previously assumed that the alpha helices of the receptor tyrosine kinases only anchored the molecules in the membrane,” said Schneider adding “but we were able to show with our test system that true interactions occur between the helices. In addition, we also believe that these interactions are important for the correct function of the proteins.”
Proteins such as glycophorin A or receptor tyrosine kinases seem to be able to interact with each other without requiring molecular assistants. For cytochrome b6, for example, another membrane protein that has a key role in the photosynthetic electron transport, the Freiburg researchers were able to elucidate the function of the heme cofactor in the protein folding process. Heme is a complex with an iron atom at its centre and which can be bound to a range of proteins, including hemoglobin. Cytochrome b6 has two heme groups. It also has four alpha helices that are anchored in the membrane. Artificial amino acid exchanges in different areas of the protein also provided the researchers with information about the individual folding steps and the amino acids that are responsible for the folding of the protein. In future, the researchers hope to find out whether and how the four alpha helices interact with each other. “I would really like to be able to understand every individual step that leads to the folding of membrane proteins,” said Schneider, “from the beginning to the end.”
Once the researchers have obtained detailed insights into how the alpha helices of membrane proteins interact with each other, they might eventually be able to manipulate faulty signalling systems in the cell. Investigations by other research groups have already shown that certain isolated alpha helices can integrate between the alpha helices of receptor tyrosine kinases, thereby disrupting the structure of the molecules and in turn also switching off the proteins’ activity. This might be of great advantage for the treatment of certain diseases. “We are still in the very early days of our research,” said Schneider highlighting that it is important not to neglect the problem children of protein biochemistry.