Tetracysteine-biarsenical system biarsenicals

History and Design Concepts of the Tetracysteine-biarsenical System... 

Work in progress on future applications of the tetracysteine-biarsenical system includes targeting fluorescent indicators, such as Ca2+ sensors to voltagegated Ca2+ channels on the plasma membrane (unpublished results [22]). 

Fig. 12.3. Some expression systems involving noncovalent (binding) linkages. Numbers correspond to entries (methods) in Table 12.2 (5) labeling with methotrexate derivatives that recognize a dihydrofolatereductase segment, and (11) labeling with biarsenical derivatives binding to a 6-12 tetracysteine...

The biarsenical-tetracysteine method was first introduced more than 7 years ago, and further refinements and development of novel applications are still appearing. Within the last few years, biologists have started to exploit the unique features of this system for probing protein trafficking, turnover, localization, and dynamics. This review aims to describe the conception and development of this protein tag and its applications in the biological sciences. 

The small size of the biarsenical-tetracysteine tag has made it useful in biological studies requiring a genetically encoded fluorescent tag and GFP is not tolerated or has deleterious effects because of its size. FlAsH binding occurs rapidly, does not require any protein secondary structure to generate fluorescence (unlike GFP and its variants that can take minutes to days to become fully fluorescent) and should therefore be a more faithful reporter of the initiation of protein synthesis. The following examples include studies in viruses, bacteria, yeast, and mammalian cells and also indicate the broad applicability of the biarsenical-tetracysteine system. 

One noticeable feature of the biarsenical-tetracysteine system is the high stability of the complex when formed, with off-rates up to weeks in vitro [8]. 

The picomolar affinity of biarsenicals for a tetracysteine peptide and its quick reversal by millimolar concentrations of dithiols make it a useful system... 

An alternative approach to incorporate spectroscopic probes can be accomplished by the use of biarsenical tetracysteine system (Adams et al, 2002) or enzymatic alkylation (Keppler et al, 2003). A mutant 0 -alkylguanine-DNAalkyltansferase can efficiently transfer a benzyl group to itself when presented with 0 -benzylguanine derivatives. This enables the labeling of spectroscopic probes on to the transferase, which is fused to the target protein under investigation. 

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