Project

potassium channel; 2D crystals; membrane protein structure; electron crystallography; electron tomography

Lead
Membran Proteine gehören zu den wichtigsten molekularen Maschinen, welche wir in unseren menschlichen Körpern haben. Ihr korrektes Funktionieren ist eine Voraussetzung für gesunde menschliche Zellen. Dafür ist die Kenntniss der atomaren Struktur der Membran Proteine essentiell, so wie sie in der Regel durch Röntenstrukturuntersuchungen bestimmt werden kann.
Lay summary
In diesem Projekt werden wir die 3D Struktur von ausgewählten Membran Proteinen untersuchen, während die Proteine in biologischen Lipid Membranen eingebaut sind. Interessant dabei wird sein, dass wir die Struktur der Proteine in Membranen bestimmen wollen, welche lebensnahe Bedingungen erleben, indem auf beiden Seiten der Membranen unterschiedliche flüssige Lösungen vorherschen. Um dies zu erreichen, werden wir die Proteine in die Membranen von Lipid Vesikeln einbauen, was kleine Membran-Bläschen sind. Diese Vesikel werden im inneren und im äusseren unterschiedliche Lösungen haben, so wie dies in Membranen in natürlichen Zellen auch der Fall ist. Die Membran Proteine, welche wir untersuchen werden, sind Kaliumkanäle, Natrium-Zitrat Symporter, sowie ein Komplex aus einem bakteriellen Protein mit einem Virus Dock-Protein.

Direct link to Lay Summary

Last update: 05.05.2013

Lead
Membrane proteins in our human body are among the most important molecular machines for human health. A detailed mechanistic understanding of their function requires not only a high-resolution structure as usually obtained by X-ray diffraction methods, but also information about the structure of these membrane proteins under life-like conditions.
Lay summary
Here, we will study the 3D structure of membrane proteins, while they are embedded in biological lipidic membranes. Importantly, we will seek to study the structure of these proteins under life-like environmental conditions, by placing the membrane proteins into the membrane of lipid vesicles (tiny bags) that have different buffer solutions on the inside than on the outside. This should allow studying the reaction of the membrane proteins to gradients in the buffer solutions on both sides of the membranes.  Systems to study are potassium channels, sodium-citrate symporters, and a complex from a bacterial protein with a virus docking protein.

Direct link to Lay Summary

Last update: 05.05.2013

Self-organised

Number	Title	Start	Funding scheme

Membrane proteins are central to health and disease. A detailed understanding of the function of membrane proteins requires access to high-resolution structural data and mechanistic insight into conformational changes occurring when the proteins are active. We here propose to employ the recently established cryo-electron microscopy imaging capabilities in C-CINA, the Center for Cellular Imaging and NanoAnalytics, and our newly developed image processing software, to study the dynamic structure of selected membrane protein systems in liposomes, a close-to-native environment: Vesicles containing excellently ordered two-dimensional (2D) crystalline membrane protein arrays will be vitrified in aqueous buffer solutions, whereby different buffer solutions will be established on the inside and outside of the liposomes. The resulting buffer gradient will impose an electric potential, pH and/or ligand concentration gradient across the membranes, which will be experienced by the reconstituted proteins. Further, 2D crystals grown with a 180-degree screw-axis symmetry will place adjacent membrane proteins in alternating orientations in the membranes, allowing us to determine the 3D structures of oppositely regulated states in one experiment.We will employ a combination of electron crystallography (using our 2dx software), electron tomography (using our Dynamo software), and single particle processing of 2D crystals (using our new module in the 2dx software), to determine the 3D structure at 7Å resolution or better of selected systems. We already have excellently ordered 2D crystals of the bacterial iron-ferrichrome transporter FhuA decorated with the bacteriophage T5 tip protein pb5, of the cAMP-modulated potassium channel MloK1 from M. loti, and of the sodium/citrate symporter CitS from K. pneumonia, and we will grow 2D crystals of the voltage gated potassium channel KvAP from A. pernix. The exceptional quality and contrast of the FhuA-pb5 crystals will yield a high-resolution structure and allow our new methodology to be finely tuned. Analysis of the CitS crystals will reveal the active site and document a new, unknown fold of an important class of membrane proteins. We will expose MloK1 crystals to varying cAMP concentrations on both sides of the lipid membrane, and KvAP crystals to a Nernst electrical potential difference to trigger voltage gating, and document the conformational changes. The produced data will complement existing high-resolution X-ray structural studies with information about the dynamic functioning of these classes of membrane proteins. Importantly, this work will, for the first time, allow the direct structural observation of the voltage gating mechanism of a voltage-gated potassium channel.