220 11 Small Functional Peptides and Their Application in Superfunctionalizing Proteins
11.3 Functional Peptides
11.3.1 Functional Peptides that Act as Binders
Peptides can specifically bind to small molecules, metals, or proteins. The ability
of peptides to bind small molecules is often exploited for optical purposes. One
of the most widely applied peptide tags and the first described alternative to the
color labeling by protein fusions with fluorescent proteins was the small 6 resi-
due‐long tetracysteine tag (TC‐tag) with the sequence CCPGCC. The TC‐tag
was rationally designed to covalently bind the arsenic green fluorescent dye
FlAsH (fluorescein arsenical helix binder) whose fluorescence increases 1000‐
fold upon binding to the polypeptide tag. By now, a number of different bisarsen-
ical fluorophores and corresponding tags have been developed [33–35]. Redesign
of the FlAsH binding motif CCPGCC to bind the cyan dye AsCy3 furthermore
allows for simultaneous multiple‐color labeling [35]. The AsCy3 binding motif
has the sequence CCKAEAACC, and discrimination between the two dyes is
based on the larger interatomic distance between the two arsenics in AsCy3
(14.5 Å) than in FlAsH (6 Å). Due to its small size, the TC‐tag and its derivatives
have already resulted useful as a tool for in vivo imaging in bacterial [36] as well
as eukaryotic cells [37], enabling experiments not possible with large fluorescent
protein reporters. However, the method suffers from high background labeling
by binding of the arsenic dyes to thiol‐rich biomolecules, and extensive washing
steps need to be applied to gain highly specific labeling [38].
Besides the TC‐tag, other fluorophore‐binding peptides have been developed:
generally known as affinity tags for protein purification, the 6x histidine tag
(6xHis‐tag) was shown to bind metal–nitrilotriacetate (NTA)–chromophore
conjugates [39] and a zinc‐chelating membrane‐impermeable fluorophore
called HisZiFit [40]. This enabled the site‐specific labeling and tracking of
the stromal interaction molecule STIM1, a membrane protein for which an
N‐terminal fluorescent protein fusion had been shown to interfere with surface
exposure [40], exemplifying again the advantage of peptide tags over protein
fusions. However the binding affinity of the mentioned dyes to the 6xHis‐tag
was only moderate and restricted the application to extracellular labeling of cell
surface proteins.
Next to these rational approaches for the design of labeling tags, directed evo-
lution was shown to yield peptides with binding properties. Phage display was
used to evolve a peptide tag that binds the dye Texas Red (called “Texas Red
aptamer”) and its calcium‐sensing derivative X‐rhod with high affinity. This way,
the authors developed a 28‐residue‐long calcium sensor that can be “hijacked” to
various cell compartments depending on the cellular localization of the protein
to which the Texas Red aptamer is fused [41].
The same approach was used to evolve a lanthanide binding peptide (LBT),
specifically a terbium(III)‐binding peptide, of 15 residue lengths for lumines-
cence studies [42, 43]. LBTs that bind different lanthanide ions had already been
employed before for NMR studies [44] or X‐ray crystallography [45]. Interestingly
it was shown that the insertion of LBTs into internal loops of a protein helped in
rigidifying the peptide. This made internal fusions superior to terminal fusions