Contribution of the interdomain linker to dmDcr1-binding affinity. (A) GST-pulldown assay of Loqs-3 and dsRBD3 constructs and quantification of the dmDcr1 signal normalized to the Loqs-3 construct. (B) Overview of the FCCS setup. Two laser lines illuminate an overlapping microscopic detection volume (1). Molecules entering/leaving the detection spot give rise to a fluctuation curve (2). Auto- and cross-correlation of the fluctuation curves gives specific information on various biophysical data (3). (C) Fluorescently labeled Loqs-3AF647 was bound to GFP-dmDcr1 and subsequently competed with different amounts of unlabeled Loqs-3 or dsRBD3. IC50 values were obtained and Ki values were calculated according to Cheng-Prusoff.
