Single Molecule FRET analysis and modeling
DFG Proposal. Joint Project by Uli Nienhaus (Experiments), Helmut Grubmüller (Simulation+Modeling) and Frank Noe (Modeling+Theory).
Goals / Program
Experimental
- Establish synthesis and measurement process for the system of choice
- Record Single-Molecule FRET data of immobilized molecules.
- Several Conditions (Mutants, Ions, …)
- Perhaps Multi-Color FRET?
- Perhaps Vesicle Immobilization?
Simulation
- Molecular Dynamics Simulations of the system in different conformations → Modeling the Structure and Flexibility of these conformations.
- Conformational Flooding / Metadynamics simulatinos in order to explore new conformations.
- Simulation of the system with fluorescent dyes in order to obtain a orientational distribution of the dyes to be used in the theory.
- FEP calculations to estimate barriers between conformations.
Theory
- Development of statistical methods to compute the most likely dye-distance trajectory from the experimental photon count trajectory, taking dye orientation into account.
- Development of statistical methods to identify the conformations that are hidden in the experimental data, their flexibility in terms of the dye distance, and their transition rates.
- Suggestion of Distance constraints to be used for the dye positions in the simulation, so as to explore different conformations.
- Combination of structural information from MD simulation + transition rates from experiment into a joint multi-state model.
- Suggestion of new experiments in order to optimally refine the model.
TODOs
- 31 July: Helmut and Frank should suggest a number of interesting systems to work with. Uli will decide whether that is feasible in experimental practice. Ideas:
- Simple test system with few conformations: 2 or 3 Polyproline segments with flexible linkers (e.g. Glycin) in between. This can probably have only a few conformations and will have large distance changes (good for FRET).
- A biologically interesting large System.
- 15 August: Agree on a detailed list of goals and the interfaces between the three groups. Agree on how to sell the proposal (Is the goal mainly biological interest in the used system, methodological/technical advance, or understanding the energy landscape?)
- 30 August: Write Proposal