Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

54-POS Board 28 NO EASY WAY OUT: CONFORMATIONAL DYNAMICS REGULATE PRODUCT RELEASE IN PHOSPHOGLYCERATE KINASE David Scheerer 1 ; Dorit Levy 1 ; Alon Wenger 1 ; Yuval Mor-Scheerer 1 ; Mariia Shaulskaia 1 ; Anastasia Venets 1 ; Inbal Riven 1 ; Gilad Haran 1 ; 1 Weizmann Institute of Science, Chemical and Biological Physics, Rehovot, Israel Large-scale conformational transitions are often essential to protein function, yet how individual conformational states and transitions contribute to catalysis remains poorly understood. Phosphoglycerate kinase (PGK) is an attractive model because it positions its substrates for phosphoryl transfer by bringing two domains together through a large interdomain motion known as hinge-bending. Here, we combine microsecond-resolved single-molecule FRET (smFRET) measurements with photon-by-photon hidden Markov analysis to dissect how conformational dynamics contribute to enzymatic turnover. Using multiple inter- and intradomain labeling schemes, we resolve three sequentially connected conformational states— open, half-closed, and closed—with no direct transitions between the open and closed states. The conformational transition is intrinsically multidimensional, involving both domain closure and a pronounced rotational component. Transition kinetics reveal how different conformational transitions govern individual stages of the catalytic cycle. Formation of the half-closed state is readily promoted by ligands matching the substrate's charge, whereas progression to the catalytic closed state requires specific substrate interactions. However, efficient access to the catalytically competent state must be balanced with rapid reopening after catalysis, allowing tightly bound products to escape. Consistent with this view, turnover correlates strongly with the opening rate of the closed state across a broad range of conditions. Remarkably, despite reopening occurring on a much faster timescale than turnover, the observed changes in enzymatic velocity cannot be explained by the steady-state population of conformational states. Instead, the probability of successful product release and completion of an enzymatic cycle depends on the time spent in the release-incompetent closed state. These findings highlight how direct measurements of transition kinetics by smFRET can reveal mechanistic insights inaccessible from ensemble averaged measurements.

90