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

Abstract.

FLASH-EDT₂ – 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)₂ – has been reported to fluoresce only after binding with high affinity to a specific tetracysteine motif (CCXXCC, "Cys₄") and thus to provide 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-specific label of the II-III loop of the dihydropyridine receptor (DHPR) in skeletal muscle. Upon expression in dysgenic myotubes (which lack endogenous α_1S), an α_1S mutated to contain CCRECC in the II-III loop was able to produce L-type calcium currents and to mediate skeletal-type excitation–contraction (EC) coupling, but FLASH-EDT₂ labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys₄-containing calmodulin were significantly more fluorescent than non-transfected cells, whereas the difference between transfected and non-transfected cells was less apparent for CHO-K and HEK 293 cells. Because the fluorescence of non-transfected cells increased substantially after treatment with FLASH-EDT₂, it suggested the possibility that FLASH binds to endogenous cysteine-containing proteins. This finding was confirmed in cuvette experiments in which FLASH-EDT₂ fluorescence was observed after FLASH-EDT₂ was added to protein homogenates from myotubes or cell lines. The enhanced fluorescence was abolished by pretreatment of cells or cell homogenates with coumarine maleimide (CPM), which modifies cysteine residues covalently. Thus, enhanced FLASH fluorescence appears to occur both after binding to an introduced Cys₄ 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.