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

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