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hybridFRET - deciphering biomolecular structure and dynamics

Periodic Reporting for period 3 - hybridFRET (hybridFRET - deciphering biomolecular structure and dynamics)

Reporting period: 2018-12-01 to 2020-05-31

To understand and modulate biological processes, we need their spatiotemporal molecular models. In this project we propose to build these models by a holistic approach. The recent methodological and technical advances in fluorescence spectroscopy and microscopy as well as in multi-scale modelling of complex biochemical systems set the stage to tackle cross-fertilizing challenges in biophysics, biochemistry and cell biology. The applicant proposes to develop a novel integrative platform for a Molecular Fluorescence Microscope (MFM) to achieve ultimate resolution in space (sub-nanometer) and time (picoseconds) for characterizing structure and dynamics of proteins. MFM will combine Multi-parameter Fluorescence Detection with Computational Microscopy (molecular dynamics and coarse grained simulations) in a hybrid approach, first, to derive a complete molecular description of all fluorescence properties of the tailored dyes in proteins (objectives 1 and 2) and, second, to utilize this information in simulations to report on the protein properties (objective 3). In this hybrid approach high precision FRET measurements are the core experimental technique (hybridFRET). The MFM will allow us to tackle the central biophysical question of how intra- and intermolecular domain interactions modulate proteins' overall structure, dynamics, and thus ultimately function (objective 4). In this proposal we will apply MFM to two prototypic proteins of significant medical relevance. The combination with Multi-parameter Fluorescence Image Spectroscopy will exploit the ultimate resolution of the MFM for molecular protein imaging in live cells. To follow and ultimately understand biological processes, we need their spatiotemporal models of the integrative fluorescence spectroscopy platform. Until now, no holistic use of fluorescence spectroscopy for structural modelling of proteins has been reported.
+Quantitative analysis of FRET experiments
Förster resonance energy transfer (FRET) measurements from a donor (D) to an acceptor (A) fluorophore are frequently used in vitro and in live cells to reveal information on the structure and dynamics of DA-labeled macromolecules. Accurate descriptions of FRET measurements by molecular models are complicated because the fluorophores are usually attached to the macromolecule via flexible long linkers allowing for diffusional exchange between multiple states with different fluorescence properties due to variations in quenching of the fluorophores by their surrounding environment, changes of DA distances and dye mobilities. It is often assumed for analysis of fluorescence intensity decays that DA-distances and D quenching are uncorrelated (homogenous quenching by FRET) and that the exchange between distinct fluorophore states is slow (quasi-static). This allows us to introduce the FRET-induced donor decay, εD(t), a function solely depending on the species fraction distribution of the rate constants of energy transfer by FRET, for a convenient joint analysis of fluorescence decays of FRET- and reference samples by integrated graphical and analytical procedures.
Additionally, we developed a simulation toolkit to model dye diffusion, fluorescence quenching at the protein surface and FRET and demonstrate by a benchmark study with simulated fluorescence decays of 500 protein structures that for single protein conformations average DA-distances are recovered by quasi-static homogenous models with an accuracy of 2 %. For more complex cases where proteins adopt multiple conformations with significantly different dye environments (heterogeneous case) we introduce a general analysis framework and evaluate the power in resolving heterogeneities in DA-distances by time-resolved measurements. The developed fast simulation methods, relying on Brownian dynamics (BD) of a coarse grained dye in its sterically accessible volume, allow us to incorporate structural information in the decay analysis of heterogeneous cases by relating dye states to protein conformers. This theoretical framework paves the way for structure-driven analysis and accurate modeling of fluorescence and FRET experiments for characterizing the structure and dynamics of proteins.
Finally, we present theory and simulations to assess the accuracy and precision of steady-state and time-resolved FRET measurements on the single-molecule and ensemble level in resolving of DA distances and to provide a rigorous framework for estimating approximation, systematic and statistical errors

+Methods and tools for FRET-restrained structural modelling.
FRET experiments can yield state-specific structural information on complex dynamic biomolecular assemblies. However, FRET experiments need to be combined with computer simulations to overcome their sparsity. We introduce (i) an automated FRET experiment design tool determining optimal FRET pairs for structural modeling, (ii) a protocol for efficient FRET-assisted computational structural modeling at multiple scales, and (iii) a quantitative quality estimate for judging the accuracy of determined structures. We tested against simulated and experimental data.
In conclusion, the presented hybrid-FRET methods actually allow realizing an integrated molecular fluorescence microscope combining optical and computational microscopy at a huge spatial and temporal range to display suitably labeled biomolecular systems at unprecedented resolution by atomistic structural models. The wide time range resolved by fluorescence (ps to minutes) opens up many new applications in dynamic structural biology. The quantitative framework for experimental FRET data, presented above, paves the way for structure-driven analysis and accurate modeling of fluorescence and FRET experiments for characterizing the structure and dynamics of proteins and biomolecular assemblies.
The RCSB Protein Data Bank (PDB; archives biological macromolecular structures obtained through classical biophysical methods such as X-ray, NMR, and cryo-EM.
In a complementary effort, the PDB-Dev database ( has the goal to archive structural models of biomolecules obtained through integrative and hybrid modeling. In close collaboration with the group of Helen Berman we developed an extension of the IHM (integrative/hybrid modeling) dictionary containing definitions and rules for the deposition of fluorescence-derived structural models. The fluorescence dictionary represents an important step to allow for depositing fluorescence-based structural modeling in PDB-Dev, which allows for easy access worldwide.