The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins

FLASH-EDT2 - 4 ' ,5 ' -bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethane dithiol)(2) - has been reported to fluoresce only after binding with high a ffinity to a specific tetracysteine Motif (CCXXCC, "Cys(4)") and thus to pr ovide a technique for labeling recombinant proteins in vivo (Griffin et al. Science 281:269-272). We have attempted to use FLASH-EDT2 as a site-specif ic label of the II-III loop of the dihydropyndine receptor (DHPR) in skelet al muscle. Upon expression in dysgenic myotubes (which lack endogenous alph a (1S)), an alpha (1S) mutated to contain CCRECC in the II-III loop was abl e to produce L-type calcium currents and to mediate skeletal-type excitatio n-contraction (EC) coupling, but FLASH-EDT2 labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys (4)-containing calmodulin were significantly more fluorescent than non-tran sfected cells, whereas the difference between transfected and non-transfect ed cells was less apparent for CHO-K and HEK 293 cells. Because the fluores cence of non-transfected cells increased substantially after treatment with FLASH-EDT2, it suggested the possibility that FLASH binds to endogenous cy steine-containing proteins. This finding was confirmed in cuvette experimen ts in which FLASH-EDT2 fluorescence was observed after FLASH-EDT2 was added to protein homogenates from myotubes or cell lines. The enhanced fluoresce nce was abolished by pretreatment of cells or cell homogenates with coumari ne maleimide (CPM), which modifies cysteine residues covalently. Thus, enha nced FLASH fluorescence appears to occur both after binding to an introduce d Cys(4) motif and to endogenous, cysteine-containing proteins. Therefore, FLASH-EDT2 may be useful only for labeling those recombinant proteins that express at a very high level.