Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer

•DMD and smFRET were used to describe the conformational ensemble of the CLYs.•Intramolecular interactions predominated over polymer properties in linker extension.•FRET in the CLYs showed sensitivity to macromolecular crowding and solvent quality.•FRET in the CLYs was the same in cuvettes and at the plasma membrane of live cells.•Distances estimates based on FP-FRET vary widely depending on assumptions about dynamics.