Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

58-POS Board 30 CONFORMATIONAL DYNAMICS OF RAF KINASE COMPLEXES IN SOLUTION AND UPON RAS BINDING REVEALED BY SINGLE-MOLECULE FRET AND SINGLE-MOLECULE IMAGING Rebika Shrestha 1 ; Matt Drew 1 ; Kelly Snead 1 ; Ashley Mitchell 1 ; Hannah Ambrose 1 ; Rodrigo E Cáceres-Gutiérrez 1 ; Que N Van 1 ; Andrew G Stephen 1 ; Dwight V Nissley 1 ; Thomas J Turbyville 1 ; 1 Frederick National Laboratory for Cancer Research, RAS Initiative, Frederick, MD, USA The RAS–RAF–MEK–ERK signaling cascade is a central regulator of cellular proliferation, differentiation, and survival, and its dysregulation is implicated in numerous cancers. Activation of RAF by membrane-associated RAS is a key initiating step in this pathway. Cryo-EM studies have revealed that RAF exists in an autoinhibited complex with MEK and a 14-3-3 dimer, in which the cysteine-rich domain (CRD) and kinase domain are maintained in a cradle-like conformation through intramolecular interactions between the regulatory and catalytic domains, together with binding of the 14-3-3 dimer to two phosphorylated serine residues on BRAF. Upon binding GTP-loaded RAS at the plasma membrane, RAF undergoes structural rearrangements that relieve autoinhibition, promote CRD engagement with the membrane, and facilitate kinase domain dimerization, ultimately leading to RAF activation. Although structural studies have provided high resolution snapshots of RAF complexes, the conformational dynamics underlying the release of autoinhibition and kinase activation remain poorly understood. Here, we combine single-molecule Förster Resonance Energy Transfer (smFRET), and single-molecule Total Internal Reflection Fluorescence microscopy (smTIRF) to directly probe the conformational landscape of BRAF complexes in solution and during interaction with soluble and membrane bound RAS. smFRET measurements between BRAF and 14-3-3 dimer reveal FRET populations consistent with the autoinhibited BRAF–MEK–14-3-3 complex, with only modest conformational changes upon binding soluble RAS. In contrast, membrane-associated RAS induces a pronounced redistribution of FRET populations, consistent with the release of autoinhibition and increased conformational heterogeneity. Furthermore, single-molecule imaging on supported lipid bilayers directly visualizes BRAF homodimerization in the presence of active membrane-bound KRAS, demonstrating that RAS and the membrane cooperatively drive RAF activation and dimer formation. Together, these findings provide direct single molecule evidence that RAF activation proceeds through a redistribution among pre-existing conformational states rather than a simple two-state transition, providing new mechanistic insight into MAPK pathway activation and RAF regulation.

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