Identification of an Orthogonal Peptide Binding Motif for Biarsenical Multiuse Affinity Probes

Abstract

Biarsenical multiuse affinity probes (MAPs) complexed with ethanedithiol (EDT) permit the selective cellular labeling of proteins engineered with tetracysteine motifs, but are limited by the availability of a single binding motif (i.e., CCPGCC or PG tag) that prevents the differential labeling of coexpressed proteins. To overcome this problem, we have used a high-throughput peptide screen to identify an alternate binding motif (i.e., CCKACC or KA tag), which has a similar brightness to the classical sequence upon MAP binding, but displays altered rates and affinities of association that permit the differential labeling of these peptide sequences by the red probe 4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufin (ReAsH-EDT₂) or its green cognate 4‘,5‘-bis(1,3,2-dithoarsolan-2-yl)fluorescein (FLAsH-EDT₂). The utility of this labeling strategy was demonstrated following the expression of PG- and KA-tagged subunits of RNA polymerase in E. coli. Specific labeling of two subunits of RNA polymerase in cellular lysates was achieved, whereby ReAsH-EDT₂ is shown to selectively label the PG-tag on RNA polymerase α-subunit prior to the labeling of the KA-tag sequence of the β-subunit of RNA polymerase with FlAsH-EDT₂. These results demonstrate the ability to selectively label multiple individual proteins with orthogonal sequence tags in complex cellular lystates with spectroscopically distinct MAPs, and indicate the absolute specificity of ReAsH to target expressed proteins with essentially no nonspecific binding interactions.