Determination of the Rab27-Effector Binding Affinity Using a High-Throughput FRET-Based Assay.

Thus far, two Rab27 isoforms (Rab27a and Rab27b) have been identified that interact with their eleven downstream effectors proteins, preferentially in their GTP-bound state. In recent years, a number of studies has suggested roles for Rab27-effector protein interactions in the development of cancer cell invasion and metastasis, and immune and inflammatory responses. Here we develop an in vitro fluorescence resonance energy transfer (FRET)-based protein-protein interaction assay to report Rab27 protein interactions with their effectors. We particularly focus on determining the interaction of mouse (m) Synaptotagmin-like protein (Slp)1 and mSlp2 effector proteins with human (h)Rab27. Green fluorescent protein (GFP)-N-terminus Rab27 binding domains (m-Slp1 and m-Slp2) recombinant proteins were used as donor fluorophores, whereas mCherry-hRab27a/b recombinant proteins were used as acceptor fluorophores. The conditions of this assay were validated and optimized, and the specificity of the assay was confirmed. Accordingly, this assay can be used to assess and identify key determinants and/or candidate inhibitors of Rab27-effector interactions.