11.3 Functional Peptides 221for phase determination in X‐ray crystallography and potentially also for the
structure determination of large protein complexes by NMR [13].
Peptides can exhibit high affinity for metals, other peptides, tags, or proteins.
Such affinity tags are already widely applied for protein purification, immobiliza-
tion, and pull‐down studies [46]. Monoclonal antibodies for most affinity tags
are commercially available, making (parallel) detection of tagged proteins possi-
ble, thus circumventing the need for protein‐specific antibodies. Internal tagging
expands these applications to proteins with functionally relevant or inaccessible
termini. An affinity tag, which is considered to be particularly suited for inser-
tion into internal permissive sites, is the small, uncharged Strep‐tag. The nine‐
residue peptide sequence exhibits intrinsic affinity toward Strep‐Tactin, a
specifically engineered streptavidin [47]. Due to the highly specific but non‐
covalent binding, proteins can be purified under physiological conditions in one
step from crude cell lysates, without the need for high salt concentrations or
other additives [48].
11.3.2 Peptide Motifs that are Recognized by Labeling Enzymes
Peptides can serve as specific substrates for enzymes. Highly specific binding of
small molecules to peptides is often hard to achieve. Like this, enzyme‐mediated
labeling has received attention as an alternative methodology to tag cellular pro-
teins with chemical probes. Here, enzymes selectively act on a specific peptide
sequence to covalently add their cognate substrate. One such enzyme is lipoic
acid ligase (LpIA) from E. coli. Naturally responsible for attaching lipoid acid to
proteins involved in oxidative metabolism [49], LplA was rationally redesigned
to specifically attach useful small molecule probes – such as alkyl azides [50] and
photo‐cross‐linkers [51] – onto an engineered 22 aa’s long LplA acceptor peptide
(LAP1). The authors used this technology to label cell surface proteins and to
map protein–protein interactions in vitro. Using yeast display for affinity selec-
tion, the originally used engineered 22‐residue acceptor peptide LAP1 could be
resized to only 13 residues (LAP2) while at the same time showing a 70‐fold
higher catalytic efficiency (kcat/Km) as a substrate for LplA [52]. By structure‐
guided mutagenesis, LplA was then further evolved to accept a fluorescent cou-
marin derivative instead of a lipoic acid derivative as substrate [53]. The resulting
variant LplAW37V – together with the optimized acceptor peptide LAP2 – made
the method suitable for in vivo labeling of proteins in eukaryotic cells. In contrast
to the previously used fluorescent lipoic acid derivatives, the employed coumarin
derivatives were orthogonal to eukaryotic metabolism. The original LAP1 pep-
tide, which had been employed in cell surface and in vitro assays, was then revis-
ited to develop an in vivo protein–protein interaction assay: the relatively low
affinity but good catalytic activity of LplA for LAP1 allowed to render fluoro-
phore attachment protein–protein interaction dependent [54]. Attachment of
LAP1 and LplA to potential dimerizing partners allowed to sufficiently discrimi-
nate between an interacting and a noninteracting protein pair according to the
labeling efficiency [54]. Altogether, two powerful alternative methods for the
highly specific but unobtrusive labeling of proteins for imaging and interaction
studies in vivo were introduced: PRIME (PRobe Incorporation Mediated