Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

25-POS Board 13 INVESTIGATING NUCLEOTIDE-DEPENDENT CONFORMATIONS OF SECA BY SINGLE-MOLECULE FRET AND MD SIMULATIONS Dulcie Havers 1 ; Joel A Crossley 2 ; Sofia Oliveira 3 ; Ian Collinson 1 ; 1 University of Bristol, Biochemistry , Bristol, United Kingdom 2 University of Leeds, Astbury Centre for Structural Molecular Biology, Leeds, United Kingdom 3 University of Bristol, Chemistry, Bristol, United Kingdom Protein translocation across membranes is an essential process common to all organisms. The Sec machinery is the ubiquitous system by which the majority of this transport occurs. The cytosolic motor protein SecA is essential for efficient pre-protein transport via the Sec machinery in bacteria. SecA is known to undergo large conformational changes throughout the transport cycle. The proposed mechanism for transport relies on the passive diffusion of the majority of pre-protein substrate through the Sec machinery, biased by certain cellular factors, and the stimulation of SecA's ATP hydrolysis activity by 'difficult to transport' regions — i.e. bulky or positively charged residues — eliciting a response throughout the translocon to drive their transport across the membrane. How the conformation of SecA changes throughout the transport cycle to facilitate these different behaviors remains to be understood. Here we use a combination of diffusion-based smFRET and molecular dynamics simulations to unpick the conformations of SecA throughout the nucleotide cycle. We demonstrate that SecA has distinct dynamic profiles and conformations at different stages of the nucleotide cycle. This diffusion-based smFRET approach enables us to extract dynamics on a timescale which we can then compare to motions observed in molecular dynamics simulations, and non-equilibrium simulations reflecting stages of the ATP hydrolysis cycle, to build a more complete picture of the motor dynamics responsible for efficient protein transport in bacteria. During the meeting I will share my progress in the application of singe molecule technologies in protein translocation and also seek to engage with the conference to help further advance our understanding.

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