The protein-labeling reagent FLASH-EDT 2 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 alpha1s), an alpha1s 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-EDT, 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.

A general approach for chemical labeling and rapid, spatially controlled protein inactivation

“…The absence of background staining in control cells permits useful and accurate staining of proteins expressed at low levels, such as the Lyn-FKBP12(F36V) fusion (Figs. 3 and 4A), whereas other labeling systems have noted problems with proteins which are expressed at low levels (6,7). The ability to label proteins that are expressed at or below physiologic levels is significant because overexpression can alter biological phenotypes (1).…”

“…The biarsenical compound approach developed by Roger Tsien and colleagues (3) is an elegant solution that uses a small peptide epitope for labeling. However, background staining and potential toxicity may limit its use to a subset of cell types (3,6,7). Recently, a novel method was presented in which a human alkyltransferase covalently modifies its fusion partner with a fluorescent substrate (29).…”

“…These techniques include labeling cysteine-containing peptides with biarsenical compounds, binding of fluorescein to an antifluorescein single chain antibody (scFv), and labeling an avidin fusion protein with a biotin conjugate (3)(4)(5). Background labeling, toxicity, and suitability for only a subset of target proteins and cell types may limit the broad use of these systems (6,7). Despite the difficulties faced by these techniques, chemical labeling is of great interest because it permits novel types of experiments by targeting chemicals with a wider range of functionality than that of fluorescent proteins.…”

“…To circumvent this, Marek and Davis (13) recently used a fluorescein-biarsenical compound to inactivate Drosophila Synaptotagmin I that was engineered to contain a particular biarsenical-binding cysteine peptide. Although this approach has been used in Drosophila and mammalian systems (14), several reports observe toxicity or background staining (6,7).…”

Chemical tags: Applications in live cell fluorescence imaging

“…Although there have been further improvements to the tetracysteine-biarsenical system [31], it can show problems with unspecific background labeling and toxicity. The tag motif CCXXCC can not be found in the genome, but similar cysteine rich sequences are present in other proteins and can lead to non-specific labeling [35]. Although nonspecific interactions can be minimized by the addition of dithiols, the background labeling and the unspecific binding to hydrophobic sites in cellular components limits the potential of tetracysteine-biarsenical probes.…”