Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

34-POS Board 18 LOCK AND KEY: SM-FRET MAPS HOW FARNESYLATION SUPPRESSES VITAL 4 STATE DYNAMICS OF HUMAN GUANYLATE-BINDING PROTEIN 1 UNTIL ACTIVATION Paul Lauterjung 1,2 ; Julian Folz 1 ; Christian Herrmann 2 ; Claus A. M. Seidel 1 ; 1 Heinrich-Heine-Universität Düsseldorf, Molecular Physical Chemistry, Düsseldorf, Germany 2 Ruhr-Universität-Bochum, Physical Chemistry I, Bochum, Germany The human Guanylate-Binding Protein 1 (hGBP1) is a human immune protein and GTPase, which acts against viral and microbial attacks within the cell. This protein can be posttranslationally modified with a farnesyl moiety near its C-terminal helix 13, which allows the protein to perform its most observed nucleotide induced activities: polymerisation and membrane binding. As both activities require major conformational changes after activation with a suitable nucleotide (GTP or GDP-AlF x ) [1], we measured and analysed the underlying protein dynamics with quantitative single-molecule FRET (sm-FRET) methods.Considering non-farnesylated hGBP1, we found extensive dynamic behaviour in its monomeric form. By measuring of a set of 12 distinct FRET pairs, only two unique conformational protein states were resolved [2] although at least 4 states would be expected from the three kinetic relaxation time observed by filtered FCS. By adding 11 new FRET pairs to our initial set - bringing the total to 23 FRET pairs - we were able to elucidate another important dynamic mode of helix 13. This allowed us to resolve a complex exchange between 4 conformers of hGBP1 as expected from FCS. Additionally, we investigated the farnesylated hGBP1 to study the impact of the farnesylation on the protein dynamics. We observed that the farnesyl moiety acts as a suppressor for dynamics in the monomeric state. However, upon activation with GTP or GDP-AlF x and in the presence of dimer binding partners, the farnesyl anchor is released and the predefined dynamics resurface. Notably, the dynamics detected during the dimerization of farnesylated hGBP1 resemble that of monomeric non-farnesylated hGBP1. The encoded controlled conformational flexibility is indeed essential for dimerization and in turn for its activity.[1] M. Kutsch and J. Coers, The FEBS Journal 288 (2021) 20: 5826-5849.[2] T.-O. Peulen and C. S. Hengstenberg et al., eLife 12 (2023) e79565.

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