LAUSR

Characterizing interactions of proteins is pivotal for understanding their function. Recently, single- molecule imaging based methods have proven useful for directly testing the stoichiometry of multi-subunit protein complexes. A limiting factor is the labeling of proteins with multiple spectrally discernible tags and low background. Here we describe a use of zinc finger-mediated protein labeling for single-molecule imaging studies in living cells. A DNA-binding zinc finger (ZF) is fused to the protein of interest and labeled by a DNA probe carrying the specific ZF binding sequence and an organic dye. Non-specific binding is minimized by injecting the DNA/dye conjugate into the cell. With a reproducible labeling efficiency of 20%, we developed an approach to deduce the multiplicity of the subunits in a protein complex from the overall degree of labeling. We were able to confirm the fixed 2:2 assembly of the NMDA receptor in a 3-color single molecule imaging setup and reject alternative stoichiometries. Due to the modular design and small size of ZF proteins, this approach will allow the analysis of more complicated protein interaction patterns to understand the assembly rules for large protein complexes.