Integrative Dynamic Structural Biology with Fluorescence Spectroscopy

Abstract

Förster Resonance Energy Transfer (FRET) studies on the ensemble and single-molecule level probe biomolecular structure and dynamics and identify coexisting conformational states. Integrative structural biology solves biomolecular structures by combining data from different sources, which individually would be insufficient. Here, we combine FRET experiments with computer simulations to overcome their sparsity. I will discuss recent methodological developments in integrative structural modelling by satisfying spatial restraints on networks of FRET pairs (hybrid-FRET). We introduce new procedures for: (i) an automated FRET experiment design tool determining most informative FRET pairs for structural modeling using prior structural knowledge (https://github.com/Fluorescence-Tools), (ii) a protocol for efficient FRET-assisted computational structural modelling at multiple scales (http://nmsim.de), (iii) choice of appropriate models for describing the dye behavior and consequences for designing appropriate labels, (iv) use of all eight characteristic fluorescence parameters as a vectorial local and dynamic structure information, and (v) a new quantitative quality estimate for judging the accuracy of determined structures. I will present applications simulations and real experiments of our techniques to proteins such as T4 Lysozyme (T4L). We used 33 variants with single-FRET pairs to resolve three T4L conformers via their characteristic distance sets. Screening the known T4L structures, revealed that T4L in solution mainly adopts the known open and closed states in exchange at 4 μs. A newly found minor state, undisclosed by at present more than 500 crystal and NMR structures of T4L and sampled at 230 μs, may be actively involved in the product release step in catalysis. Importantly, we released together with the PDB the initial version of the fluorescence (FLR) dictionary extension (https://github.com/ihmwg/FLR-dictionary) on the Integrative/Hybrid Modeling (IHM) working group GitHub site. Now, fluorescence-restrained structural models can be deposited at PDB-Dev.