Integrative dynamic structural biology of biomolecular assemblies resolved by multi-scale super-resolution FRET spectroscopy and imaging
FRET spectroscopy and imaging can provide state-specific information on the structure and dynamics of complex dynamic biomolecular assemblies under ambient conditions with nanosecond time resolution and single-molecule sensitivity. To overcome the sparsity of FRET experiments, we developed procedures to combine these with computer simulations to map biomolecular dynamics and to resolve quantitative integrative structure models at a precision and accuracy better than 3 Å. The integrative structure models are deposited in the new protein data bank, PDB-dev [1-3]. Moreover, we combined super-resolution microscopy via stimulated emission depletion (STED) and Multi-parameter Fluorescence Image Spectroscopy (MFIS)  to reach molecular resolution with sub-nanometer precision in molecular imaging of biomolecules and their complexes. While STED-MFIS captures the spatial and temporal information of the cellular context with a resolution below 10 nm, the concurrent measurement of Förster resonance energy transfer (FRET) between an excited donor and acceptor provides a zoom with Ångström precision. Thus, integrative super-resolution FRET image spectroscopy exploits these synergies to reach molecular resolution.
I will introduce the concepts of our novel optical tools and demonstrate recent applications: (1) Detection of a so far hidden functionally important conformational state in the enzyme T4 Lysozyme , (2) Resolving the conformational transitions of Guanylate binding proteins (GBPs) during GTP-controlled phase transition to exert their function as part of the innate immune system of mammalian cells . (3) Mapping the dynamic exchange network in chromatin fibers by studying a 12-mer nucleosome array .
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