Exploring dynamic protein systems with two- and three-color single-molecule FRET

Bilgen, Ecenaz (2026): Exploring dynamic protein systems with two- and three-color single-molecule FRET. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

The single-molecule fluorescence resonance energy transfer (smFRET) approach has emerged as a fundamental technique to resolve the structure, interactions, and dynamics of biomolecules. This thesis provides a comprehensive investigation of dynamic protein systems using two- and three-color smFRET, with a main focus on solution-based, multiparameter fluorescence detection combined with pulsed interleaved excitation (MFD-PIE) experiments. By using advanced analysis tools including fluorescence lifetime analysis, photon distribution analysis (PDA) and fluorescence correlation spectroscopy (FCS), sub-millisecond conformational dynamics can be quantitatively characterized. The first part of this work investigates the conformational flexibility of the AMPylase, “filamentation induced by cyclic-AMP” (Fic) domain containing protein FicD, a regulator of the endoplasmic reticulum (ER)-resident Hsp70, Binding immunoglobulin Protein (BiP). Dual-color smFRET and fluorescence lifetime analyses revealed three distinct conformational states of the tetratricopeptide repeat (TPR) domain, including dynamically interconverting TPR-in and TPR-out states and a static TPR-in conformation. A global dynamic PDA model, supported by molecular dynamics simulations, quantitatively described the transitions between these states and how they are regulated in the presence of nucleotides and BiP. The second project focused on the mitochondrial Hsp70 homolog Ssc1, demonstrating the different timescales of conformational dynamics between the nucleotide-binding and substrate-binding domains. In a third study on the human Hsp90, three conformational states and the dynamics between them as well as their dependence on different nucleotides was studied. Finally, extending the measurements to three-color smFRET enabled direct observation of coordinated domain motions in the metabotropic glutamate receptor 2 (mGlu2). This approach resolved simultaneous, ligand-induced structural rearrangements and identified an intermediate conformational state. Overall, the results and applications presented in this thesis demonstrate the strengths and current capabilities of smFRET spectroscopy in dynamic structural biology.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 29. May 2026
• 1. Referee: Lamb, Don C.
• Signature of the printed copy: 0001/UMC 32112