Conformational Dynamics of large protein Complexes

Abstract

Life on earth is only possible through the enormous capabilities of proteins and their assemblies. Which function a certain protein fulfills is encoded in its amino acid sequence, which gives rise to a defined structure. However, this structure is not static. The thermal energy of the surrounding medium forces the molecule into different conformations. It can be assumed that in most cases these movements play an important role for a protein’s function. Therefore, it is crucial to gain high-resolution 3D information of those movements. For small proteins, Nuclear Magnetic Resonance (NMR) Experiments can provide this information in good detail. For larger proteins and protein complexes single particle Electron cryo Microscopy (cryo EM) is the method of choice, which is used in this thesis. However, analysing the complete conformational landscape is not yet routine for cryo EM. Therefore, several methodological developments were made with this thesis.Since the most crucial prerequisite for any structural analysis is an intact and homogeneous sample, the development of a method, finding stabilizing conditions was the first aim. The new method called ProteoPlex uses a newly developed extended theoretical framework on the existing fluorescence-based stability screen called Thermofluor. Therewith, Thermofluor data obtained from large multidomain proteins and protein complexes can be analyzed. In total, stabilizing conditions could be found for more than 80 complexes from all branches of life. Additionally, the usefulness of ProteoPlex towards assembly and disassembly experiments was demonstrated. Furthermore, two strategies were employed to decrease the number of adopted conformations by decreased temperature. Firstly, a crosslinking strategy at -10 C was successfully used. Secondly, the fermentation of the thermophile fungus Chaetomium thermophilum was established, and the native purification of thermophile protein complexes was successfully demonstrated. To analyze conformational dynamics in practice three model systems were chosen: the 120 kDa single-chain nuclear export factor chromosome region maintenance factor 1 (CRM1), the 3-5 MDa bacterial pyruvate dehydrogenase complex (PDHc) and the icosahedral, 1.5 MDa Acetylcholine binding protein (AchBP) from the snail Biomphalaria glabrata. CRM1 is with 120 kDa rather small for electron microscopic analysis. Nevertheless, it was demonstrated for Chaetomium thermophilum CRM1, that the apo protein cycles between an open superhelical and a closed ring-shaped conformation. Hereby, the full energylandscape of this movement could be described. The energy landscape is in general flat, and CRM1 can change freely between the open and closed conformation. Furthermore, a C-terminal helix is responsible for a slight enthalpic stabilisation of the closed state. The E.coli PDHc could be purified in large quantities from native source. Literature describes the overall structure to be of octahedral symmetry with a cubic core made of the E2 component surrounded by a cubic shell made of the E1 and E3 component. This symmetry could only be partially confirmed. While the core indeed seems to be cubic, no overall cubic model could be obtained. However, also no high-resolution structure could be calculated. It can be hypothesized that the overall structure is very flexible and thus structural investigation is largely hindered. Nonetheless, crystals could be obtained which could lead to structural insights into the complex in the future. For the AChBP, a 3.6 Å resolution structure could be calculated and two conformational substates could be identified. The different states reveal significant differences in loop regions, subunit interfaces and even in -strands. Here, it is the first time that such small fluctuations could be visualized by cryo EM. In summary, this thesis provides new techniques and approaches towards the elucidation of the conformational landscape of large proteins and protein complexes.