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Updated: Aug 31, 2026

Measuring Protein Binding to F-actin by Co-sedimentation
Published on: May 18, 2017
FHL2 production enabled new insight into its flexibility and unexpected binding stoichiometry with β-catenin
Tina Logonder1, Uroš Prešern1, Aljaž Gaber1
1University of Ljubljana, Faculty of Chemistry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana, Slovenia.
Abstract:
Four-and-a-half LIM domains protein 2 (FHL2) is a modular adaptor composed entirely of LIM domains that mediate protein-protein interactions linking the cytoskeleton to transcriptional regulation. Here, we report a streamlined preparation of recombinant tag-free full-length FHL2 in bacteria and describe its biophysical characterization using size-exclusion chromatography coupled to static light scattering (SEC-SLS), small-angle X-ray scattering (SEC-SAXS), and cross-linking coupled with mass spectrometry (XL-MS). Soluble expression was enhanced by zinc supplementation and low-temperature induction. Purified FHL2 eluted from SEC-SLS as a monomeric species consistent with a flexible, elongated multidomain architecture. Integrative modeling based on SAXS and XL-MS confirmed that FHL2 exists in an extended and a compact bent conformation in solution. SEC-SLS and Isothermal titration calorimetry (ITC) measurements confirmed a direct 2:1 interaction between FHL2 and β-catenin with submicromolar affinity, supporting FHL2's scaffolding role within the Wnt/β-catenin signaling pathway. These results establish the groundwork for structural and functional analyses of FHL2 and related LIM-domain proteins.
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