Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging

Detection of protein-protein interactions is a fundamental step to understa nding gene function. Here we report a sensitive and rapid method for assayi ng protein-protein interactions at the single-molecule level. Protein molec ules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The int eraction of proteins thus prepared was visualized using a single-molecule i maging technique. As a demonstration of this method, a motor protein, kines in, was labeled with Cy5-puro at an efficiency of about 90%, and the proces sive movement of kinesin along microtubules was observed by using total int ernal reflection microscopy. It took only 2 h from the synthesis of protein s to the functional analysis. This method is applicable to the functional a nalysis of various kinds of proteins. (C) 2001 Federation of European Bioch emical Societies. Published by Elsevier Science B.V. All rights reserved.