smSTRUCT · Fuse smFRET and modeling to a new structural biology method and solve the functional ESCRT assembly structure
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2016-01-01 → 2018-12-31
- EU contribution
- €257,861
- Participants
- 2
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Fuse smFRET and modeling to a new structural biology method and solve the functional ESCRT assembly structure
The project evolved towards a high-risk high-gain fundamental methods development project with the ultimate goal to understand how the ESCRT machinery works. We planned to combine powerful molecular modeling with single-molecule FRET (smFRET) experiments on individual ESCRT subunits. We made great progress towards that goal (Fig 2). At the same time, I realized that the ESCRT system was fundamentally not understood and that a particular experiment could potentially reveal its inner workings. I developed the means necessary to carry out the experiment, including a new protein encapsulation assay in GUVs, UV based ATP uncaging in a microfluidic optical tweezing chamber, installing membrane tweezing capability on campus and finally building the new microscope Confleezer 1.0 and writing the operating software for it. Confleezers 1.0 combines confocal imaging capability with an optical tweezer. It allowed me to study the ESCRT process in unprecedented detail. As a result, I successfully managed to reconstitute the ESCRTs membrane scission reaction, could demonstrate a minimal ESCRT-III scission module and showed that scission is Vps4 and ATP dependent.
Data: CORDIS, © European Union
Project objective
We seek to develop a new structural biology method that is able to overcome barriers to solving very complex functional protein assemblies that are variable enough in their composition and conformation to defeat current methodologies. I intend to combine high-throughput single molecule FRET (smFRET) experiments with computational modeling to achieve this goal. SmFRET will be used to derive individual building block structures as well as distances between these blocks on a molecule-to-molecule level. Computational modeling is used to fuse this information into a full atomistic model of the protein assembly.The yeast ESCRT machinery is proposed as a model system to develop the new methodology. The ESCRT machinery is particularly important because of its role in HIV infections: HIV seizes control of the cell’s ESCRTs to get released from infected cells. The ESCRT assemblies’ size and flexibility lead to the fact that their assembled structure on membranes is largely unknown. Individual ESCRT proteins will be labeled by Cy3/Cy5. The ESCRT assembly will then be reconstructed on invaginated supported lipid bilayers and imaged via TIRF microscopy. FRET efficiencies will be recorded and the label-label distance determined. High-throughput biochemistry and labeling technology will allow us to generate > 100 distinct labeling sites, resulting in overdetermined structures. Stepwise photobleaching will reveal the stoichiometry within full assemblies. Alterations in FRET efficiency due to local contact formations within the assembly will reveal these local contacts. Based on the experimental data of the individual complexes, their copy number in the assembly and their local contacts, the full assembly will be determined computationally, based on replica exchange Monte Carlo simulations.
Original text from CORDIS.
Participants
- MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV · MUNCHENCoordinatorGermany
- THE REGENTS OF THE UNIVERSITY OF CALIFORNIA · OaklandUnited States
Links
Data: CORDIS, © European Union