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hybridFRET - deciphering biomolecular structure and dynamics

Periodic Reporting for period 4 - hybridFRET (hybridFRET - deciphering biomolecular structure and dynamics)

Reporting period: 2020-06-01 to 2021-05-31

To understand and modulate biological processes, we need their spatio-temporal molecular models. In view of the recent methodological and technical advances in fluorescence spectroscopy and microscopy as well as in multi-scale modeling of complex biochemical systems, the applicant proposed to build these models by a holistic approach. This goal corresponds to the development of a novel integrative platform for a Molecular Fluorescence Microscope (MFM) to achieve ultimate resolution in space (sub-nanometer) and time (picoseconds) for characterizing the structure and dynamics of proteins. The MFM will combine fluorescence spectroscopy with computer simulations in a hybrid approach, first, to derive a molecular description of all fluorescence properties of the tethered 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 allowed us to tackle the central biophysical question of how intra- and intermolecular domain interactions modulate proteins' overall structure, dynamics, and thus ultimately function by spatio-temporal models (objective 4).
In 2015, no holistic use of fluorescence spectroscopy for structural modeling of proteins was reported. During the hybridFRET project, we established the proposed workflow to generate and deposit FRET-based structural models in the prototype archiving system for integrative structural models, PDB-Dev, so that the models are searchable and accessible for society.
The hybridFRET project has reached all four research objectives (O):

O1: Fluorophore biomolecule interactions and labelling. We developed a more detailed dye model that considers also interactions between the biomolecular surface and the tethered dye. We established fast simulation tools to simulate the properties of tethered dyes on proteins and nucleic acids and compared it with experiments.
O2: Establishing a widely applicable fluorescence spectroscopic measurement platform for data acquisition and analysis. We extended our confocal microscope to three-color excitation pulsed interleaved excitation (PIE) and to three-color polarization-resolved spectral emission channels so that we can probe the correlation of motions. We recognized that FRET data of larger biomolecular assemblies were often very complex due to the presence of several FRET species with distinct dynamic properties. Thus, we developed a quantitative analysis theory with a "FRET-line explorer" as practical tool, so that one can draw theoretical FRET-lines as pathfinders to decipher two-dimensional plots of intensity-based and lifetime-based FRET indicators. As second approach to reduce data ambiguity, we established the joint analysis of a large set of FRET pairs. Notably, data analysis tightly connected with integrative modeling to test and chose appropriate analysis models.
O3: Establishing an integrative analysis platform with multiscale models to describe fluorescence parameters in complex biomolecular systems with single or multiple conformational states. We established the following toolkit: (i) algorithm for the selection of informative FRET pairs; (ii) generating integrative structural models by (a) FRET-guided rigid body docking or (b) coarse grained and all-atom MD simulations in absence and presence of FRET guiding. If the FRET data analysis revealed multiple FRET species with dynamical broadening, we established a new procedure to generate ensembles with a small member number that exploits the first and second moment of the fluorescence lifetime distribution. For less structured proteins, we established procedures to generate ensembles with more members by ensemble reweighting using the maximum entropy method. In close collaboration with the RCSB Protein Data Bank (PDB), we established a dictionary for fluorescence with a corresponding submission template, so that FRET-based integrative structural models can be deposited together with quality parameters in PDB-Dev.
O4: Analyzing the dynamic higher-level structural organization of multi-domain proteins and biomolecular assemblies. Due to the significant complexity of the sample preparation, we decided to study most of them via close collaborations. We showed for several medically relevant systems that their conformational flexibility is essential for their function. Examples: (i) oligomerisation of the guanylate binding protein hGBP1, major player in innate immune defense; (ii+iii) the regulation of the activity of kinases that are important cancer drug targets such as the Abl tyrosine kinase and the Cdk2/Cyclin A complex; (iv) molecular basis for the function of bacterial toxins; and (v-vii) environmental influence on the flexibility of nucleosome assemblies that is a key determinant of genome regulation.
The established hybrid-FRET methods actually allow realizing an integrated MFM combining optical and computational microscopy at a huge spatial and temporal range to display suitably labeled biomolecular systems at unprecedented resolution by detailed 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 FLR dictionary is an important step to enable depositing FRET-based structural models in PDB-Dev, which provides easy worldwide access to the results of FRET-based dynamic structural biology. In the future, we plan to include more information from fluorescence in integrative models. The success of the hybridFRET project makes it possible that FRET-based structural models can make important contributions for understanding the molecular basis of biomolecular function by adding a dynamic perspective.

The good progress allowed us to strive for related next-level goal by realizing the proposed idea of combining FRET spectroscopy with STED microscopy for seamless imaging of molecular assemblies with sub-nanometer resolution. Thus, we established a suitable confocal microscope to realize FRET nanoscopy.
In view of the progress of super-resolution light microscopy, FRET nanoscopy closes the gap between localization and FRET measurements: (i) we apply STED nanoscopy to break the diffraction limit up to a resolution of ~40 nm to distinguish individual molecules; (ii) we combine STED with single-molecule colocalization (cSTED) to utilize the high localization precision for single fluorescent molecules with nanometer accuracy in the range of 5-40 nm; and (iii) we take advantage of FRET between a donor and an acceptor dye at shorter distances as a natural extension of the cSTED approach, so that we resolve distances from 4-12 nm.
Moreover, FRET nanoscopy has additional unique advantages. While FRET provides isotropic 3D distance information, colocalization measures the projected distance onto the image plane. The combined information allows us to directly determine its 3D orientation in the same measurement using Pythagoras's theorem. Using particle-averaging approaches like in cryoEM, structural biology of large complex assemblies by optical imaging becomes feasible.
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