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Michael Z. Lin and Lei Wang
Physiology 23:131-141, 2008. doi:10.1152/physiol.00007.2008
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REVIEWS
PHYSIOLOGY 23: 131–141, 2008; doi:10.1152/physiol.00007.2008
Selective Labeling of Proteins with
Chemical Probes in Living Cells
Selective labeling of proteins with small molecules introduces novel chemical and
Michael Z. Lin1 and Lei Wang2
1
Department of Pharmacology, University of California at
San Diego, La Jolla; and 2The Jack H. Skirball Center for
Chemical Biology & Proteomics, The Salk Institute for
Biological Studies, La Jolla, California
[email protected]
physical properties into proteins, enabling the target protein to be investigated
or manipulated with various techniques. Different methods for labeling proteins
in living cells have been developed by using protein domains, small peptides, or
single amino acids. Their application in cells and in vivo has yielded novel insights
into diverse biological processes.
Protein Domain Tags
Many protein domains, as part of their natural functions, interact with small molecules in a highly specific
manner (FIGURE 1). For example, enzymes catalyze
reactions by mediating the precise positioning of substrates in their active sites. Similarly, small molecule
enzyme inhibitors such as natural toxins and therapeutic drugs exert their effects by binding tightly to active
sites. Specialized proteins are also chemically modified
in detoxification processes. Each of these types of interactions—enzyme-substrate, target-inhibitor, and scavenger-toxin—has been exploited to attach chemical
probes to proteins of interest with high specificity.
Covalent attachment of chemical probes has the
advantage of greater irreversibility compared with
noncovalent binding. A modified 33-kDa bacterial
dehalogenase domain has recently been developed to
perform efficient covalent self-labeling. Dehalogenases
break down haloalkanes in a two-step process involving alkyl transfer to an active site Asp side chain (and
the loss of the halide ion) followed by hydrolysis of the
alkyl-enzyme bond. The hydrolysis step requires an
active site His residue (59), mutation of which preserves the alkyl-enzyme conjugate. An optimized
hydrolysis-deficient halogenase (marketed as Halotag,
Promega) can be specifically labeled using cell-permeable haloalkanes bearing fluorophores or biotin
(39). Since eukaryotes lack dehalogenases,
background staining in cells is very low.
Before the use of dehalogenase, Johnsson and colleagues (32) were the first to react small molecules
with a protein domain for labeling purposes, using the
21-kDa O6-alkylguanine-DNA alkyltransferase (AGT).
AGT recognizes guanine bases alkylated on the O6
position and transfers the alkyl group to one of its
reactive cysteine side chains, regenerating guanine.
Cell-permeable O6-benzylguanine derivatives bearing
fluorophores, biotin, haptens, and even other protein
ligands have been used to label AGT fusions (32). A
mutant AGT with increased reactivity for free benzylguanine derivatives and decreased binding to DNA
has been designed (25) and is available along with a
variety of probes from Covalys. The 50-fold faster reactivity of the mutant vs. wild-type AGT allows specific
visualization of fusion proteins even in the nuclei of
cells expressing endogenous AGT (31). Also, although
wild-type AGT changes conformation upon alkylation
to allow its degradation (13), mutant AGT fusions have
been followed for days without unexpected signal
decay, implying that degradation may not occur in
fusions or with mutant AGT (31). AGT has proven to be
a reliable labeling method in time-lapse studies of
centromere and sarcoplasmic reticulum protein
movements in living cells (31, 69).
Enzymes and irreversible active-site inhibitors, i.e.,
suicide substrates, can serve as protein-ligand pairs
for covalent labeling without further protein engineering as long as the inhibitors do not react with endogenous enzymes. In the only example of this concept to
date, fluorophore-conjugated analogs of the suicide
substrate p-nitrophenyl phosphonate (pNPP) were
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The fusion of a fluorescent protein to target proteins
has revealed a wealth of information on many biological systems over the last decade. Compared with fluorescent proteins, small molecule probes offer a
wider range of property and functionality, since they
can be derivatized with fluorophores, haptens, or
reactive chemical groups. To attach small molecule
probes to proteins, new methods have emerged in
recent years involving fusion of protein or peptide
tags followed by either noncovalent or covalent binding of small molecule probes to the tags. These probes
are designed with functional groups at positions that
will not interfere with binding to the tag. A different
set of techniques allow specific protein labeling with
functional groups with single amino acid precision.
These techniques include chemical modification,
native chemical ligation, and genetic incorporation of
unnatural amino acids. These new approaches differ
from earlier biochemical techniques in allowing
greater precision of labeling, either in location on the
protein or in time, and in allowing use in or on live
cells. In this review, we will describe and compare
these new methods for labeling proteins with chemical probes and highlight recent reports applying these
methods in live cells.
131
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A protein domain that allows labeling specifically in
vesicles or the cell surface is a single-chain antibody,
either selected for binding to a small molecule that can
be conjugated to a fluorophore (18) or selected for the
ability to increase the quantum yield of fluorescent
probes upon binding (71). The latter tag, like fluorescent proteins, is restricted to optical readouts but
allows for fluorophores with superior optical characteristics or for membrane-impermeant fluorophores
for selective labeling of surface proteins.
The ever-growing list of enzyme-inhibitor pairs
characterized by the pharmaceutical industry may
serve as a source for future labeling systems. Suitable
pairs are those that exhibit orthogonality and versatility, i.e., neither enzyme nor drug has effects in mammalian cells, and the inhibitors tolerate functional
derivatization while retaining reactivity. Although
most interactions are noncovalent, some well established drugs such as certain ␤-lactam antibiotics are
suicide substrates and may serve as the basis for future
covalent labeling systems. Another route to finding
new protein-chemical pairs may be the engineering of
FIGURE 1. Classes of protein domain or peptide tags used for chemical labeling of fusion proteins
A: domain tags self-label either covalently or noncovalently. B: peptide tags either self-label with varying degrees of
reversibility or utilize enzyme-mediated covalent attachment. The standard methods of fluorescent protein or epitope
tags are shown for comparison. Examples of tags of each class are listed to the right.
132
PHYSIOLOGY • Volume 23 • June 2008 • www.physiologyonline.org
antibodies tha
This provides
interactions, s
react with liga
methods for
variable doma
tively small (1
tion in the cyt
Small Pep
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used to label proteins fused to the fungal cell wall
enzyme cutinase (5). However, because pNPP is cellimpermeant, this system is only suitable for labeling
surface proteins.
Although less desirable for long-term studies, protein domains can also be labeled noncovalently with
chemical ligands. This was first demonstrated using
fluorescent derivatives of methotrexate to visualize
fusions to mammalian dihydrofolate reductase
(DHFR) in DHFR-deficient cells (29). Recently,
Cornish and colleagues created a variety of cell-permeant derivatives of the antibiotic trimethoprim for
visualizing fusions with the 18-kDa E. coli DHFR (7).
Trimethoprim shows high affinity to E. coli DHFR (Kd
= 1 nM) and minimal (Kd = 3 ␮M) binding to mammalian DHFR (66) and, unlike methotrexate, is not
cytotoxic. A similar method arose from research on the
immunosuppressant FK506 and its 12-kDa protein
target FKBP12. The FK506 analog SLF’ binds with 94
pM affinity to the FKBP12(F36V) mutant but not
endogenous FKBP12, allowing specific labeling of
FKBP12(F36V) fusions in cells by SLF’ derivatives (43).
Protein domai
specificity, bu
generally restr
mini of prote
potential to di
certain cases c
in proteins. T
chemical labe
small space, i
tight and spec
able within a f
The first an
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sequence CCX
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ReAsH (1), wi
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n proteins
g degrees of
ein or epitope
antibodies that recognize a desired chemical structure.
This provides for covalent as well as noncovalent
interactions, since antibodies can be engineered to
react with ligands (10, 78). The recent availability of
methods for selecting camel antibody single-chain
variable domains makes possible the isolation of relatively small (13 kDa) domains, which may retain function in the cytosol in some cases (63).
fluorophore with more widely spaced arsenic atoms
and a peptide with sequence CCKAEAACC (8). These
components do not interact with those of the traditional tetracysteine-biarsenical systems, and therefore
the two systems can be used in the same cell in an
orthogonal manner.
“Cysteine is the most frequently used residue for selec-
Small Peptide Tags
tive chemical modification due to its relatively low
Protein domains mediate chemical labeling with high
specificity, but their size and folding requirements
generally restrict their use to the NH2 and COOH termini of proteins of interest. Smaller tags have less
potential to disrupt protein folding or function and in
certain cases can be placed in loops or domains within proteins. The inherent challenge with peptide
chemical labeling pairs is molecular recognition in a
small space, i.e., the designing of an interface that is
tight and specific and yet on the protein side is encodable within a few amino acids.
The first and most extensively used system for specific labeling of a peptide by functionalized small molecules is the tetracysteine-biarsenical system. The
basic components, rationally designed by Griffin and
colleagues (24), are peptide tags with the consensus
sequence CCXXCC and chemical probes, such as the
green fluorophore FlAsH or the red fluorophore
ReAsH (1), with two arsenic atoms positioned so that
each arsenic atom bonds with two thiol groups on
adjacent cysteines. These arsenic-sulfur bonds are
covalent but mediated by outer valence orbitals and
are reversible in the presence of excess thiols. The
bidentate nature of the complex results in extremely
high (~10 pM) affinity vs. interactions that involve only
one arsenic atom (1). Recently, sequences surrounding the CCXXCC core have been optimized for
improved ReAsH and FlAsH affinity, with their effects
presumably being mediated by increased hydrophobic contacts between peptide and ligand outside of the
arsenic-sulfur bonds (45). In being isolated from peptide library screen for a specific binding activity, these
sequences may be considered to have characteristics
of aptamers (i.e., a short peptide sequence evolved for
a specific binding activity; see below).
The tetracysteine-biarsenical system has been used
with a wide array of biarsenical-conjugated functional
groups. Aside from their usual use as fluorophores for
visualization, FlAsH and in particular ReAsH can generate reactive oxygen species for light-assisted protein
inactivation in functional studies (42, 75) or
diaminobenzidine precipitation in electron microscopy (21). The tetracysteine-biarsenical system has also
been extended to attach chemical cross-linkers (38)
and calcium sensors (74) to particular sites on
proteins. Recently, a variation of the tetracysteinebiarsenical system has been developed that uses a
abundance in proteins and the increased nucleophilicity of the thiol group relative to other amino
acid side chains.”
The primary drawbacks of the tetracysteine-biarsenical system are background labeling and toxicity.
CCXXCC motifs are not found in the genome, but
multiple proteins contain motifs that differ from
CCXXCC by only one cysteine, and substantial labeling
of cytoplasmic proteins can occur to various degrees
depending on cell type (23, 41, 70). Typically, high
micromolar concentrations of dithiols are used during
labeling to reduce background accumulation and afterward as a destaining step (23, 41), although these
reagents reduce the effective affinity of the desired
interaction to the nanomolar range (1). FlAsH and
ReAsH may also bind to cellular components through
nonspecific hydrophobic interactions (23). The system
is therefore most useful for overexpressed or highly concentrated proteins of interest, such as actin concentrated on polysomes (62) or viral proteins during assembly
(64). Furthermore, potential toxicities from residual offtarget binding of arsenic or the dithiol antidotes themselves remain a concern (2, 35). These drawbacks, as
well as the need for long labeling and destaining times,
are likely to make use of the tetracysteine-biarsenical
system in thick tissues or animals difficult.
Following the introduction of the tetracysteinebiarsenical system, other strategies for specifically
attaching chemical probes to peptides have been
developed. These fall into three categories: rationally
designed peptide-probe pairs, peptide aptamers
evolved for binding to chemical probes, and peptides
from natural biosynthetic pathways that serve as specific sites for covalent attachment of chemical groups
by enzymes. In analogy to the tetracysteine system,
Tsien and colleagues developed a method for labeling
polyhistidine peptides with a complex of two zinc ions
and a zinc-chelating dye called HisZiFit (26). This
interaction exhibits moderately tight affinity (~40 nM)
but is currently limited by suboptimal dye characteristics. In a similar concept, the zinc chelator DpaTyr
binds to a string of four Asp residues with 1.4 ␮M affinity and can be attached to various fluorophores (54).
This low-affinity interaction can be converted into a
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nhibitor pairs
industry may
tems. Suitable
y and versatilfects in mamate functional
ity. Although
me well estabantibiotics are
basis for future
ute to finding
engineering of
REVIEWS
133
REVIEWS
covalent one by placing appropriate reactive chemical
groups in the peptide tag and in the chelator probe
(51). However, applicability inside eukaryotic cells has
yet to be demonstrated for these zinc-chelating probes
and may be difficult given their large sizes and therefore poor membrane permeability, especially for
DpaTyr derivatives. Furthermore, zinc is required at
micromolar levels but is maintained in the cytosol at
only picomolar levels (6).
As an alternative to the rational design, other groups
have turned to in vitro selection of aptamers from
randomized libraries. A 14-aa aptamer has been evolved
to bind lanthanide atoms (20), which can be visualized
by luminescent or spectroscopic methods. One study
took advantage of the small size of the lanthanide-binding tag to estimate distances between insertion sites on
extracellular regions of proteins by measuring resonance energy transfer (65). A potentially more generalizable aptamer is TR512, recently isolated by Nolan and
colleagues with impressively strong (25 pM) affinity to
the central three-ring core of the dye Texas Red (44). This
structure can be derivatized while retaining binding, as
Table 1. Comparison of protein and peptide tags
Size (aa)
Mechanism
Kd
Probe Type
Demonstrated
Functionalities
Notes
Dehalogenase
297
Ester linkage
~0
Alkylhalides
Fluorescence,
biotinylation
Sold as Halotag,
Promega
Alkylguanosine
transferase
182
Thioether linkage
~0
Alkylguanosines
Fluorescence, biotin, and
methotrexate handles
Sold as SNAP tag,
Covalys
Cutinase
200
Phosphoester
linkage
~0
Alkyl-p-nitrophenylphosphonates
Fluorescence,
quantum dots
EC only
DHFR
157
Noncovalent
1 nM
Trimethoprim
conjugates
Fluorescence
FKBP12(F36V)
108
Noncovalent
0.094 nM
SLF' conjugates
Fluorescence
Single-chain
antibody
230-300
Noncovalent
5 nM
phOx conjugates,
fluorophores
Fluorescence
Tetracysteine
12
Thiol-arsenic bond
0.01-1 nM
Biarsenical conjugates
Fluorescence, singlet
oxygen generation,
chemical crosslinking,
calcium sensing
Polyhistidine
6
Noncovalent
40-1400
nM
Zinc chelator conjugates Fluorescence, chemical
reactivity with nucleophiles
EC only
Lanthanidebinding tag
14
Noncovalent
220 nM
Lanthanides
Phosphorescence,
spectroscopy
EC only
TR514
38
Noncovalent
0.025 nM
Texas Red derivatives
Fluorescence, calcium sensing
Biotin acceptor
peptide
15-75
Amide linkage,
enzymatic
~0
Biotin and analogs
Biotin handle, click chemistry Requires ATP
for multiple functionalities
Lipoic acid
acceptor peptide
22
Amide linkage,
enzymatic
~0
Lipoic acid conjugates
Click chemistry for multiple
functionalities
Requires ATP
Phosphopantetheinyl
15
Phosphoester
linkage, enzymatic
~0
CoA conjugates
Organic fluorescence, biotin
handle
EC only
Sortag
5
Amide linkage,
enzymatic
~0
Pentaglycine conjugates Organic fluorescence, biotin
handle
EC only
Q-tag
4-7
Amide linkage,
enzymatic
~0
Primary amines
Organic fluorescence, biotin
handle
EC only
Farnesylation tag
4
Thioether linkage,
enzymatic
~0
Prenyl diphosphate
conjugates
Click chemistry for multiple
functionalities
EC only
Aldehyde tag
6
Enzymatic cysteine ~0
oxidation
None
Chemical reactivity with
nucleophiles
EC, lumenal only
134
PHYSIOLOGY • Volume 23 • June 2008 • www.physiologyonline.org
Downloaded from physiologyonline.physiology.org on June 19, 2008
Tag
demonstrated
the Texas RedLabeling with
performed, an
hydrophobic T
binding, but th
cally feature d
flexibility, selfIn a differen
of enzymes f
have been ada
cules to specif
solves the issu
requiring that
the peptide su
jugating enzym
enzymes is bi
the biotin ca
decarboxylase
sequence supp
plasmic biotin
lation of secre
despite the lac
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specifically co
lysine residue
aa biotin acce
library screen
eukaryotic bio
pletely depen
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when inserted
affected by va
(12). Long-ter
ed by the gen
BirA and BAP
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be detected in
of streptavidin
Assuming e
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BirA biotinyla
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able amount o
by the equat
[substrate] <<
are in the hun
action with BA
measured o
ATP-biotin) an
micromolar p
of several mic
initial conjuga
lar per minut
enough to lab
peptides over
Biotinylatio
lack of endoge
REVIEWS
s been evolved
n be visualized
ds. One study
nthanide-bindertion sites on
easuring resomore generalizby Nolan and
pM) affinity to
s Red (44). This
ng binding, as
es
d as Halotag,
mega
only
only
only
uires ATP
uires ATP
only
only
only
only
lumenal only
and by access to streptavidin-conjugated reagents for
downstream assays (28, 56). Intracellular use, on the
other hand, requires probes that can be conjugated to
target peptides preferentially over native biotin. Biotin
analogs containing chemically reactive groups such as
ketones, azides, and alkynes, which are not present in
mammalian cells, may allow specific chemical reaction with a variety of appropriately activated
compounds in the cytoplasm. However, several
parameters must be improved to make this labeling
pathway robust enough for routine use. For one, adequate orthogonality with endogenous biotinylation
mechanisms needs to be achieved. Ting and colleagues have characterized an alkyne-containing
biotin analog that is not utilized as a substrate by
mammalian biotin ligases but is accepted by yBL with
only a modest loss in catalytic efficiency (68) and
therefore can be attached to a yBL-specific acceptor
peptide (9). Likewise, an azide analog is only utilized
by Pyrococcus horikoshii biotin ligase (PhBL),
although this proceeds at a 1,000-fold slower rate than
biotin, and no small acceptor peptide is yet available
(68). Still, neither the yBL-alkyne nor PhBL-azide system is currently orthogonal as endogenous biotin in
the cell would still serve as a superior substrate, so further mutation may be desired to reduce cross-utilization of native biotin. It is also not clear to what extent
biotin analogs are able to enter cells through the same
transport mechanisms utilized by biotin. A final limitation is that the subsequent steps of performing
chemistry on azides and alkynes in the cytoplasm of
living cells using currently available reagents are
rather inefficient (3). Improving and adapting biotinylation for intracellular protein tagging will therefore
continue to be an interesting challenge for
biochemists and chemists.
A similar system using the E. coli lipoic acid ligase
LplA, which is structurally related to biotin ligase and
functions analogously to attach a lipoic acid prosthetic group to pyruvate dehydrogenase, has also been
developed by Ting and colleagues and may provide an
easier path to orthogonality (19). Impressively, both a
22-aa LplA acceptor peptide (LAP) and an azide-containing lipoic acid probe were successfully engineered
through rational design, although LAP is not as efficient a substrate as the native LplA substrate protein
E2p, and the azide probe shows a relatively high Km for
LplA (127 ␮M vs. 1.7–4.5 ␮M for lipoic acid). In the
presence of high probe concentrations, attachment of
the azide probe to extracellularly presented LAP proceeds as quickly as biotinylation. Because mammalian
cells confine pyruvate dehydrogenase to the mitochondrial matrix (57), LplA-mediated labeling in the
cytoplasm should be possible with minimal background labeling or toxicity. Indeed, some lipoic acid
derivatives, including the azide probe, are able to
enter cells (A. Y. Ting, personal communication). But
intracellular probe concentrations are still lower than
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d as SNAP tag,
alys
demonstrated inside cells with a cell-permeable form of
the Texas Red-based calcium indicator X-Rhod-5F (44).
Labeling with other Texas Red derivatives has yet to be
performed, and there is a possibility that the rather
hydrophobic Texas Red core will exhibit nonspecific
binding, but the TR512-Texas Red system does theoretically feature desirable characteristics of high affinity,
flexibility, self-labeling, and intracellular compatibility.
In a different approach from self-labeling, a variety
of enzymes from microbial biosynthetic pathways
have been adapted as tools to conjugate small molecules to specific peptide sequences. Conceptually, this
solves the issue of specificity encoding in a peptide by
requiring that specificity determinants reside on both
the peptide substrate and in the active site of the conjugating enzyme. The best characterized among these
enzymes is biotin ligase, which attaches biotin onto
the biotin carboxyl carrier subunit of acetyl-coA
decarboxylase. A 67-aa mammalian biotin acceptor
sequence supports biotinylation by endogenous cytoplasmic biotin ligases (55) and may also allow biotinylation of secreted proteins by endogenous processes
despite the lack of known biotin ligases in the secretory pathway (73). The E. coli biotin ligase BirA can also
specifically conjugate biotin or biotin analogs onto a
lysine residue in the natural 75-aa substrate or in a 15aa biotin acceptor peptide (BAP) identified by peptide
library screening (4, 12). BAP is not an acceptor for
eukaryotic biotin ligases, so its biotinylation is completely dependent on BirA coexpression. As expected
for its small size, BAP can function as an acceptor
when inserted into protein domains, but efficiency is
affected by variability in access or recognition by BirA
(12). Long-term lack of toxicity has been demonstrated by the generation of transgenic mice expressing
BirA and BAP-tagged protein (15). Transplanted cells
expressing biotinylated proteins on their surfaces can
be detected in living mice after intravenous injection
of streptavidin-conjugated imaging reagents (73).
Assuming efficient access of biotin ligase to its small
molecule co-substrates ATP and biotin, the kinetics of
BirA biotinylation of BAP are acceptably fast for labeling a large proportion of tagged proteins in an acceptable amount of time. Conjugation rates are governed
by the equation [enzyme][substrate]kcat/Km when
[substrate] << Km, a safe assumption given Km values
are in the hundreds of micromolar for the BirA interaction with BAP or intact protein substrates (4). When
measured on BirA (preloaded with activated
ATP-biotin) and the BAP substrate, kcat/Km is ~1 per
micromolar per minute (4). Assuming concentrations
of several micromolar for both enzyme and peptide,
initial conjugation rates will also be several micromolar per minute. Therefore, this reaction is efficient
enough to label a substantial fraction of unreacted
peptides over minutes under ideal conditions.
Biotinylation on cell surfaces is made feasible by the
lack of endogenous extracellular biotinylated proteins
135
REVIEWS
FIGURE 2. Principle of native chemical ligation
Peptide A with a COOH-terminal thioester group reacts with peptide B
with an NH2-terminal Cys residue through thiol/thioester exchange,
resulting in a thioester-linked initial product. This product then undergoes an intramolecular rearrangement and gives the final polypeptide
that is linked by a native peptide bond. Multiple modifications can be
introduced into either peptide through chemical synthesis.
136
PHYSIOLOGY • Volume 23 • June 2008 • www.physiologyonline.org
COOH terminus to pentaglycine peptide derivatives
(58), but the large polar surface areas of these peptide
analogs prevent cellular entry. Transglutaminase catalyzes amide bond formation between Gln side chains
within Gln-rich substrate sequences and primary
amines of chemical probes but shows poor specificity
and can only function outside cells due to ion requirements (19, 37). Farnesyltransferases can mediate
attachment of a variety of farnesyl derivatives to the
cysteine residue in the COOH-terminal sequence
CVIA, but the presence of endogenous targets again
precludes use inside cells (17).
especially wh
NH2 and COO
where protein
rescent protein
(2, 27). Howe
tags may alte
linker sequenc
Single-Res
Side Chai
Protein and Peptide Tag
Comparisons
The covalent labeling reactions mentioned above have
made possible some unique applications. One such
application is multicolor pulse-chase labeling of proteins synthesized at different times, as has been demonstrated using tetracysteines, AGT, and dehalogenases
(21, 33). Other applications include stoichiometric
purification of fusion protein complexes from lysates
under stringent conditions (25, 39) and long-term
visualization of single protein molecules, either on cell
surfaces by biotinylation and visualization with streptavidin-conjugated quantum dots (28), or on cell surfaces by PPTase-mediated Cy5 labeling of an ACP tag
(30), or in vitro by dehalogenase-mediated
fluorophore labeling (60).
Each of the discussed methods has unique advantages and disadvantages (Table 1). Dehalogenases and
AGT mediate specific and irreversible self-labeling
with sensitive chemical probes, allowing detection of
protein expressed within endogenous levels, but are
large. Tetracysteines are capable of stable self-labeling
with a large variety of chemical probes at various locations within proteins, but toxicity and background are
common issues. Aptamers allow self-labeling and flexible insertion sites in proteins, but they have yet to be
evaluated in multiple contexts. Enzymatically mediated conjugation is highly specific and irreversible but
requires that the tag be in a location on the protein that
is accessible to enzyme, at least transiently. However,
requisite co-expression of an enzyme may be a feature
rather than a complication depending on the experimental design, e.g., it could allow confinement of
labeling to particular times or tissues via restricted
enzyme expression. Each system also has a different
set of requirements in terms of extracellular or lumenal or intracellular environments and a different set of
compatible ligands. Currently, none of the characterized enzyme-peptide conjugation systems functions
efficiently in the cytosol, leaving AGT, dehalogenase,
and tetracysteine as the only covalent labeling methods suitable for intracellular use. Finally, the fusion of
tags requires testing that structure or function is not
perturbed. Larger tags are more likely to be a problem,
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can be achieved extracellularly, so efficient labeling
inside cells may require further evolution of small
peptide substrates and/or the LplA enzyme.
Other transferases capable of attaching chemical
groups to peptides are limited to extracellular uses for
the foreseeable future. The Bacillus subtilis phosphopantetheinyl transferase (PPTase) Sfp catalyzes the
transfer of a phosphopantetheinyl group from the
metabolic intermediate CoA to bacterial acyl carrier
proteins (ACPs) or the related B. subtilis carrier protein
(PCP). Another PPTase, E. coli AcpS, functions similarly on a subset of ACPs. Both PPTases tolerate the presence of various functional substituents on the terminal
thiol of phosphopantetheinyl and have been successfully used to mediate the covalent of functionalized
phosphopantetheinyl ligands from CoA derivatives to
ACPs and PCPs, e.g., for multicolor sequential fluorophore labeling or for attaching fluorophores to extracellular regions of proteins (46, 76). Yin and Walsh
have identified 12-amino acid sequences that serve as
specific substrates for Sfp and AcpS with kcat/Km values
of 0.19 and 0.015 per micromolar per minute, respectively, in saturating CoA concentrations (86). The former rate is about 12 times slower than the native
substrate protein PCP (85) and several times slower
than biotinylation by BirA but is simpler in not requiring ATP as a co-substrate. It is currently limited to
extracellular tagging because CoA is cell impermeant,
and intracellular use would require proper conjugation
of functional groups to CoA by biosynthetic pathways.
Other systems for extracellular tagging include the
bacterial enzyme sortase, which cleaves the Thr-Gly
amide bond in LPXTG sequences and ligates the new
To investigate
inside target p
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label on targe
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then modified
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dehalogenase,
abeling methy, the fusion of
unction is not
be a problem,
especially when inserted at locations other than the
NH2 and COOH termini (5), and there are examples
where protein localization is altered by a 30-kDa fluorescent protein but not by the smaller tetracysteine tag
(2, 27). However, even COOH-terminal tetracysteine
tags may alter protein localization depending on
linker sequences (64).
Single-Residue Labeling on Reactive
Side Chains
To investigate the role of a specific amino acid residue
inside target proteins, it is desirable to modify the protein on selected single residues. Residue-specific
protein labeling can minimize perturbation by the
label on target protein folding and function, and provide greater locational flexibility in attaching biophysical probes, e.g., to report protein conformation
changes or protein-protein interactions. Chemical
modification, native chemical ligation, and genetic
incorporation of unnatural amino acids have been
used to label proteins on single residues.
Cysteine is the most frequently used residue for
selective chemical modification due to its relatively
low abundance in proteins and the increased nucleophilicity of the thiol group relative to other amino
acid side chains. Cysteine can be introduced at any
desired sites or removed from unwanted sites through
site-directed mutagenesis, provided such mutations
do not affect the protein under study. The residue is
then modified through disulfide bond exchange or
alkylation reactions. This method has been widely
used to study the structure and function of proteins in
vitro (80). For intracellular research, the labeling procedure can be carried out in vitro, and the modified
protein is introduced into cells via microinjection. For
instance, Hahn et al. labeled the p21-binding domain
with fluorescent dye Alexa 546, which is injected into
fibroblasts to image the Rac activation (34). Similarly,
labeling of a WASP domain, which binds to activated
Cdc42 only, with a dye sensitive to polarity followed by
microinjection enables the visualization of the Cdc42
activation inside living cells (50). This approach has
even been extended to whole animal studies. For
instance, an agonist linked by an azobenzene moiety
is attached to an introduced Cys on the ligand-binding
domain of ionotropic glutamate receptor directly in
HEK293 cells (77). Upon photoisomerization, the agonist is reversibly presented to the binding site to control the channel activity. This strategy was later
applied in neurons in zebrafish larvae to manipulate
the firing of neurons through light (72).
The Cys side chain can be exploited to attach a variety of tags, biophysical probes, or analogs of posttranslational modifications. A recent study installed
methyl-lysine analogs into recombinant histones, so
that the site and degree of methylation could be specified (67). These analogs preserve the function of
methylated lysines and thus provide convenient
means to study how lysine methylation affects chromatin structure and function. Nonetheless, chemical
modification relies on the unique reactivity of an
amino acid side chain. The intrinsic selectivity is low
unless no Cys is present or unwanted Cys can all be
mutated in the target protein. To label buried residues,
the protein may need to be denatured to increase the
accessibility and then renatured for functional assay.
For intracellular and in vivo labeling, a reduced environment is necessary for Cys labeling, and careful controls need to be performed to exclude complications
resulting from labeling other proteins. Thus the overall
selectivity and efficiency are limited.
Introduction of Chemical
Modifications by Native Chemical
Ligation
Downloaded from physiologyonline.physiology.org on June 19, 2008
ed above have
ns. One such
beling of pros been demondehalogenases
stoichiometric
s from lysates
nd long-term
, either on cell
on with strepor on cell surof an ACP tag
ase-mediated
REVIEWS
Native chemical ligation and its extension, expressed
protein ligation, chemically couple two or more peptide fragments into a full-length protein (14, 47). The
COOH-terminal ␣-thioester of one peptide reacts with
the NH2-terminal Cys of another peptide to form a
FIGURE 3. Genetically encoding unnatural amino acids (UAAs)
in live cells
A new set of components consisting of an orthogonal tRNA, an orthogonal aminoacyl-tRNA synthetase, and a unique codon is required. The
synthetase specifically aminoacylates the UAA onto the orthogonal
tRNA only. During protein translation, the acylated tRNA delivers the
UAA in response to a unique codon that does not encode any of the
common 20 amino acids. The UAA cannot be a substrate for the
endogenous synthetases and needs to be efficiently transported into the
cytoplasm when added to the growth medium or be biosynthesized by
the host cell. Various modifications can be site-specifically introduced
into proteins in the format of an UAA or by labeling the UAA side chain
via an orthogonal reaction.
PHYSIOLOGY • Volume 23 • June 2008 • www.physiologyonline.org
137
REVIEWS
native peptide link, leaving no chemical artifacts
behind (FIGURE 2). Both the thioester peptide and
the N-Cys peptide can be prepared either recombinantly or synthetically. Desired modifications can be
introduced in the synthetic peptide fragment by using
chemical synthesis and incorporated into the target
protein after ligation. This method has been successfully used to introduce various probes and modifications for studying protein structure and function in
Site-Specific Incorporation of
Unnatural Amino Acids
FIGURE 4. Examples for the application of genetically encoded UAAs
A: p-Carboxymethyl-L-phenylalanine is a nonhydrolyzable mimic for phosphorylated
tyrosine. B: photocaged serine is used to control when the target serine is regenerated
and phosphorylated. C: photocross-linker p-benzoyl-L-phenylalanine covalently locks
interacting proteins. D: O-methyl-L-tyrosine extends the side chain length of tyrsoine by
0.9 Å, which impedes the inactivation peptide of Kv1.4 from threading through its side
portal, abolishing the fast inactivation of the ion channel.
138
PHYSIOLOGY • Volume 23 • June 2008 • www.physiologyonline.org
Site-specific incorporation of unnatural amino acids
(UAAs) into proteins is another powerful technique to
selectively modify and label proteins. Schultz et al.
developed a general in vitro biosynthetic method in
which an amber suppressor tRNA is chemically acylated with an UAA (52). The tRNA and the target gene
containing an amber stop codon at the desired mutation site are then added to cell extracts that support
transcription and translation. The tRNA recognizes
the amber codon and incorporates the attached UAA.
A variety of UAAs have been incorporated into proteins using this approach, regardless of the incorporation site or protein size, and have been applied to a
large number of problems in protein chemistry (11).
An extension of this method involves the microinjection of the chemically acylated tRNA and amber
codon-containing mutant mRNA into Xenopus
oocytes (53). The endogenous oocyte protein synthesis machinery supports translation and incorporation
of the UAA. This change enables the structure-function studies of integral membrane proteins, which are
generally not amenable to in vitro expression systems
(16, 40). In practice, chemical acylation of the suppressor tRNA is technically demanding. In addition,
acylated tRNA is consumed stoichiometrically and
cannot be regenerated in cells or cell extracts, which
leads to low expression of the target protein.
Downloaded from physiologyonline.physiology.org on June 19, 2008
vitro (48). In one elegant intracellular study, a palmitoylated (PalFar) and a hexadecylated (HDFar) Nras
protein were prepared using native chemical ligation
and microinjected into MDCK cells (61). The hexadecyl group mimics the properties of the palmitoyl group
but is not cleavable by enzymes catalyzing de- and repalmitoylation. Although the PalFar-Nras rapidly traffics from the plasma membrane to the Golgi, the
HDFar-Nras accumulates nonspecifically in cellular
membranes, suggesting that depalmitoylation is
directly involved in Nras trafficking.
The native chemical ligation method has the power
to introduce diverse structures that are synthetically
accessible, and multiple modifications can be incorporated in one synthetic peptide. Nonetheless, because
peptides longer than 50 residues are difficult to synthesize using solid-phase peptide synthesis, appropriate
sites for cleavage and ligation must be chosen carefully, and modifying internal sites in large proteins is
cumbersome. It is also possible to ligate the peptides in
living cells. A synthetic fragment can be injected into
cells to react with an endogenously produced protein
fragment (22). For cell biology and in vivo studies,
microinjection of either the in vitro ligation product or
the synthetic fragment for intracellular ligation can be
a drawback. New methods for protein delivery in cells
are awaited to circumvent this issue (49).
Genetically
that of commo
ed mutagenes
method to exp
involves the g
thetase set th
cross talk with
80) (FIGURE
charge specifi
tRNA recogni
common amin
ed codon). W
synthetase pai
incorporated
high fidelity an
Genetically
modifications
of biological p
For instance,
analog for pho
by protein tyr
replace Tyr701
tor of transcrip
tein is constitu
to the same D
STAT1 (84). In
tion in yeast is
dimethoxy-2-n
blocks phosph
phosphoserine
Upon photode
ed and subse
nuclear export
acting protein
cells. UAA p-b
tocross-linker
positions prox
light exposure
linked to the ep
probe the ina
Kv1.4, UAAs w
into Kv1.4 expr
K+ currents of
the bulkiness
essential for fa
not been possi
In addition
UAAs with un
ed as a chemic
tion, providing
onal to those o
azide, acetyle
genetically en
vitro (83). The
ed into the m
directly labele
However, intr
chemical reac
REVIEWS
tudy, a palmi(HDFar) Nras
mical ligation
. The hexadealmitoyl group
ng de- and reas rapidly trafhe Golgi, the
lly in cellular
mitoylation is
of
l amino acids
ul technique to
Schultz et al.
tic method in
hemically acyhe target gene
desired mutas that support
NA recognizes
attached UAA.
ated into prothe incorporan applied to a
hemistry (11).
he microinjecA and amber
nto Xenopus
rotein syntheincorporation
tructure-funcins, which are
ession systems
n of the supg. In addition,
metrically and
xtracts, which
tein.
occur efficiently under mild cellular conditions and
are fully orthogonal to cellular components.
Over 40 UAAs have been genetically incorporated
into different proteins in bacteria, yeast, and mammalian cells, and the list is growing (81, 83). These
amino acids contain chemical reactive groups, photoreactive groups, biophysical probes, or moieties mimicking posttranslation modifications. It should be possible
to generate stable cell lines or transgenic animals capable of inheriting UAA alterations for long-term studies.
However, this method cannot be used to incorporate
UAAs that are toxic or incompatible with the protein
biosynthesis machinery (e.g., D-amino acids). Encoding
the UAA with a stop codon may lead to UAA incorporation into other proteins whose genes end with the same
stop codon, which may bring about complications and
needs to be investigated. To fully exploit the power of
these UAAs, efforts are required to improve their incorporation efficiency in eukaryotic cells, to develop
extended codons or unnatural base codons for the
incorporation of multiple UAAs simultaneously, and to
expand the method to multicellular organisms.
Downloaded from physiologyonline.physiology.org on June 19, 2008
has the power
e synthetically
an be incorpoeless, because
cult to synthes, appropriate
hosen carefulge proteins is
the peptides in
e injected into
duced protein
n vivo studies,
ion product or
igation can be
elivery in cells
.
Genetically encoding an UAA in a manner similar to
that of common amino acids would enable site-directed mutagenesis in living cells with UAAs. A general
method to expand the genetic code to include UAAs
involves the generation of a new tRNA-codon-synthetase set that is specific for the UAA and does not
cross talk with other sets for common amino acids (79,
80) (FIGURE 3). The new synthetase is evolved to
charge specifically an UAA onto the new tRNA. This
tRNA recognizes a codon that does not encode any
common amino acids (e.g., a stop codon or an extended codon). When expressed in cells, the new tRNAsynthetase pair enables the UAA to be site-specifically
incorporated into proteins at the unique codon with
high fidelity and efficiency.
Genetically encoded UAAs can directly introduce
modifications in proteins to facilitate the investigation
of biological processes in native settings (FIGURE 4).
For instance, p-carboxymethyl-L-phenylalanine is an
analog for phosphotyrosine but is resistant to hydrolysis
by protein tyrosine phosphatases. When it is used to
replace Tyr701 in human signal transducer and activator of transcription-1 (STAT1), the resulting mutant protein is constitutively active, which dimerizes and binds
to the same DNA sequences as phosphotyrosine701
STAT1 (84). In a second example, protein phosphorylation in yeast is controlled by a photocaged serine, 4,5dimethoxy-2-nitrobenzylserine (DMNB-Ser). This UAA
blocks phosphorylation and is incorporated at different
phosphoserine sites in the transcription factor Pho4.
Upon photodecaging (␭ = 405 nm), serine is regenerated and subsequently phosphorylated, triggering the
nuclear export of Pho4 (36). In another example, interacting proteins have been photocross-linked in live
cells. UAA p-benzoyl-L-phenylalanine carries the photocross-linker and is incorporated into human GRB2 at
positions proximal to the ligand-binding pocket. Upon
light exposure (␭ = 365 nm), the protein is covalently
linked to the epidermal growth factor receptor. Lastly, to
probe the inactivation mechanism of the K+ channel
Kv1.4, UAAs with extended side chains are incorporated
into Kv1.4 expressed in HEK239 cells. By comparing the
K+ currents of the mutant channels, it was found that
the bulkiness of residues in the inactivation peptide is
essential for fast channel inactivation, a finding that had
not been possible using conventional mutagenesis (82).
In addition to introducing modifications directly,
UAAs with unique reactive groups can be incorporated as a chemical handle for further protein derivatization, providing new labeling reactions that are orthogonal to those of endogenous amino acids. The ketone,
azide, acetylene, and thioester groups have all been
genetically encoded and used to modify proteins in
vitro (83). The ketone group has also been incorporated into the membrane protein LamB in E. coli and
directly labeled with membrane-impermeable dyes.
However, intracellular labeling is still challenging, as
chemical reactions have not been well developed that
Conclusion
The last few years have seen an explosion in new techniques for site-specific chemical labeling of proteins.
These techniques span a wide range in terms of submolecular precision, temporal control, labeling specificity,
and ease of implementation. No one system excels in all
characteristics, and the choice of system involves finding
the best fit for the experimental problem. The techniques with the longest history, AGT, tetracysteines,
biotinylation, cysteine modification, native protein ligation, and UAA incorporation, have already demonstrated their utility in cultured cells in a variety of questions.
For methods that are completely genetically encodable,
we expect it will not be too long before some are applied
to study proteins in their physiological contexts through
the generation of transgenic animals. Meanwhile, ongoing work promises to increase the quality and number of
protein chemical labeling techniques.
M. Z. Lin acknowledges the support of the Jane Coffin
Childs Foundation. L. Wang acknowledges the support of
the Searle Scholar Program and the Beckman Young
Investigator Program. „
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