Proteins as molecular machines perform numerous tasks in the cell. So far, understanding fundamental mechanisms of their functions has relied on the analysis of their three-dimensional structure. "But that alone is not enough," says Thorben Cordes, Professor of Physical and Synthetic Biology at LMU.
"We also need to understand their movements and structural changes within a reaction." Cordes and his group specialize in making such dynamics visible. In cooperation with scientists from Imperial College, London, and the University of Groningen, he has now for the first time succeeded in demonstrating how membrane transport proteins move in real time. The scientists report on their findings in the EMBO Journal.
The ABC transporters studied by scientists are essential membrane proteins involved in numerous cellular processes, including nutrient import, detoxification, and immune responses. All ABC transporters consist of two modules: the so-called transmembrane domain forms the actual sluice through which substrate is exported, while the nucleotide binding domain generates the necessary energy by splitting ATP, the cell's energy currency.
To make visible how ABC transporters transport substrate through the membrane, the scientists used single-molecule FRET method for the first time.
With this method, they can analyze both changes in the spatial structure and interactions between different protein units, as sensitive microscopes capture distance-dependent changes in the fluorescence light of two dyes. "In this way, we were able to show that transport across the membrane requires large conformational changes," says Cordes.
Both modules of the transport protein have an open and a closed form. In the initial state, the nucleotide binding domain is opened inwards. If ATP diffuses in, this module closes. At the same time, if the substrate is in the transmembrane domain - and only then - does its sluice open outwards, release the substrate and close again. Subsequently, the ATP is cleaved and the energy gained is used to reopen the nucleotide binding domain.
The scientists hope that single-molecule FRET will assert itself as a method for further analysis of ABC transporters. "This transporter class is also involved in the development of various diseases - such as cystic fibrosis - and tumor resistance," says Cordes. "A better understanding of transport cycles could also open up new treatment options."
This article has been republished from materials provided by LMU Munich. Note: material may have been edited for length and content. For further information, please contact the cited source.
Husada, F., Bountra, K., Tassis, K., Boer, M. D., Romano, M., Rebuffat, S., . . . Cordes, T. (2018). Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET. The EMBO Journal. doi:10.15252/embj.2018100056