Researchers of Mainz University and EMBL Hamburg present a new approach to determine the form of disordered proteins by using two different methods simultaneously in a single sample
20 January
Proteins are essential for our human body functions. There are thousands of different proteins responsible for a whole range of various physiological activities and tasks. While some of these are present in our body cells, others act as enzymes in basic metabolic processes, serve as hormones, or take the form of antibodies supporting our immune system. Simply put, proteins consist of long chains of amino acids that are organized in various three-dimensional structures. There is an alpha helix, for example, where the chain of amino acids twists into a right-handed coil, and so-called folded beta-sheet proteins. These formations determine how the proteins interact with other proteins and the tasks they assume. However, not all proteins form orderly arrangements. About 30 percent are in an intrinsically disordered state, which makes it difficult to establish to what extent the chains of such proteins form entanglements or extend themselves in an environment such as an aqueous, cell-like solution. However, these aspects are fundamental to their behavior. The more compact a protein becomes when it is isolated in an aqueous solution, the more readily it will coagulate with other proteins present to form clumps.
Protein aggregation is the first stage in the formation of plaques in the brain
Intrinsically disordered proteins often result in amyloid formations. When these amyloid protein structures clump together in the brain, these deposits – so-called plaques – increase the risk of developing Alzheimer’s disease and other neurodegenerative disorders. Biophysicists are thus particularly interested in the sizes of proteins in solution. "This factor can tell us about the aggregation potential of a protein, which is the key parameter in assessing the probability of falling victim to a neurodegenerative disease. And the aggregation process is a crucial step in the formation of plaques," explained Professor Edward A. Lemke of the Institute of Molecular Physiology of Johannes Gutenberg University Mainz (JGU), who is also Adjunct Director of the Institute of Molecular Biology (IMB). The problem is that there are two popular methods that can be used to measure this key parameter, and they produce inconsistent results. The one technique employs fluorescence to measure the end-to-end distance, i.e., the length of a protein chain from one end to the other. Using small-angle X-ray scattering, on the other hand, it is possible to gauge the size of an entangled protein or – in technical terms – its radius of gyration. "The results of both methods can be used for prognostic purposes, but the incompatibility of the results means that there is still uncertainty concerning this key parameter," said Dr. Dimitri Svergun, former group leader at the European Molecular Biology Laboratory (EMBL) in Hamburg.
New scattering technique: Radius of gyration and end-to-end distance determined in a single sample
The team of researchers managed to resolve this dilemma by bringing together chemical biology and scatter methods. They combined molecular labeling and anomalous scattering techniques to measure the size of protein clumps and end-to-end distance simultaneously in a single sample. "This way we get results for two parameters and can evaluate in what way these two are interdependent," concluded Lemke. The researchers have been able to measure both parameters since 2017, but only in two separate samples. Now they succeeded in determining these two parameters at the same time in a single sample.