LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins are important mediators of cell specification, proliferation and differentiation. These transcription factors all contain two tandem LIM domains that interact with other proteins. Many LMO and LIM-HD co-factors contain LIM interacting domains (LIDs), with competition between different LIM and LID transcription factors playing an important role in several biological processes including neural development, hematopoiesis and several cancers. Estimating affinities for these interactions would help define the molecular basis of LMO and LIM-HD function; however, LIM domain aggregation prevents the use of standard binding methods.

We have designed Förster Resonance Energy Transfer (FRET)-based approaches to study LIM:LID interactions. We have expressed LID and LIM domains fused with a flexible linker to prevent aggregation, with each domain in turn fused to fluorescent proteins optimised for FRET. Proteolytic cleavage of this linker has allowed us to study binding using homologous competition and dilution-based approaches. These techniques show LIM:LID interactions have higher affinities than previously estimated by ELISA, as well as expanding the range of interactions that can be studied. These approaches provide insights into the kinetic and thermodynamic control of LIM transcription complex formation and competition, as well as providing a guide for aggregation-prone protein interaction studies.