Download Protein Labeling

General strategy for site-specific chemical
labeling of proteins in vivo.
(a) Living cells are
transfected with DNA
encoding a protein of
interest (POI) fused to a
receptor domain.
(b) Upon expression of
the receptor fusion, a
cell-permeable small
molecule probe
consisting of a ligand
coupled to a detectable
tag is added to cell
growth medium.
(c) Protein function is
analyzed in the living
cells via fluorescent
microscopy or other
detection methods.
Green fluorescent protein (GFP) and other fluorescent proteins have the advantage of
being completely genetically encodable. However, the large size of GFP (25 kDa) can
This approach relies upon the subnanomolar affinity between a short
tetracysteine peptide (CCXXCC, where X is any amino acid except
cysteine) and a biarsenical compound such as 4',5'-bis(1,3,2dithioarsolan-2-yl)fluorescein (FlAsH).
1) biarsenical derivatives
show a dramatic increase
in fluorescence upon
binding to their target,
minimizing background noise
in labeling experiments.
2) the tetracysteine motif is sufficiently
small that it can be fused not only to the N or
C terminus of a protein, but it can also be
incorporated into loops or on the outer
surface of a helices with little chance of the
tag interfering with target protein function.
3) it allows the fluorescence detection to be confirmed
by electron microscopy. ReAsH localized to the
tetracysteine fusion protein photoconverts
diaminobinzidine into an electronrich precipitate.
Recently, it was demonstrated that FlAsH and ReAsH
can be used as mediators of chromophore-assisted
Limitations : 1) the background fluorescence in
biarsenical-labeled cells is high due to the nonspecific labeling of cysteine-rich proteins.
2) the cysteines of the receptor tag must be in
reduced form making labeling of proteins in
Fluorescently labeled O6-benzylguanine
derivatives irreversibly and specifically
label human O6-alkylguanine-DNA
alkyltransferase (hAGT) fused to target
proteins and expressed in mammalian
cells
hAGT, at ca. 25 kDa, is comparable in
size to GFP, and should thus be
similarly non-interfering of target
protein function.
Drawback : it can only be applied for
labeling proteins in hAGT-deficient cell
lines, as the benzyl guanine substrates
fluorescently labeled
DHFR inhibitors
dihydrofolate reductase
(DHFR), an 18 kDa
monomeric enzyme
fusions of the Escherichia coli form of DHFR
(eDHFR) were labeled with fluorescent Mtx.
DHFR-deficient CHO cells transiently
expressing fusions of eDHFR localized to the
plasma membrane or nucleus could be
effectively labeled by incubating the cells
Caged Proteins and Protein Ligation
Caged Protein : activity masked until subjected to illumination with
light
Expressed Protein Ligation : taking advantage of the biosynthesis of
-thioester and N-terminal cysteine protein fragments, EPL readily
enables the addition of unnatural functionality to a recombinant
protein framework, thereby extending the concept of ligation to
proteins of all sizes
A conceptually simple strategy is to carry out chemical modification in
vitro and then to deliver the modified protein to the desired biological
context.
Although microinjection of pre-made protein analogs has been successful
in some situations, in general it will be desirable to carry out chemical
modification directly in living cells because many proteins, such as
membrane proteins, are not easily manipulated in vitro.
non-hydrolyzable
phosphorylated
tyrosine mimetic.
non-hydrolyzable
phosphorylated
Ser/Thr
photocaged
phosphorylated
Serine
Cysteinyl Biotin
Cysteine
thioester
Expressed
Protein Ligation
Characteristic positions of intein motifs and numbering. The inserted intein carries
the N-terminal extein (left shaded box) and the C-terminal extein (right shaded box).
The residues important for the splicing process as well as the conserved segment
blocks (A, B, C, D, E, H, F, G) and some internal intein key amino acids are depicted in
the one letter code within the certain segments (bold black). Numbering of the amino
acids of a precursor protein is made in the following way: the intein's N-terminal amino
acid (Cys, etc.) is numbered as 1 whereas the C-terminal amino acid of the N-terminal
extein is numbered as -1 and the N-terminal residue of the C-terminal extein is
numbered beginning with +1.
Inteins can be divided into four classes: the maxi inteins (with integrated endonuclease
domain), mini inteins (lacking the endonuclease domain), trans-splicing inteins (where
Mechanism of intein-mediated
protein splicing. In the initial
step a thioester intermediate is
formed by an NS-acyl shift at
the N-terminal Cys of the
intein (Cys1).
Transthioesterification by a
nucleophilic attack of the
sidechain of the N-terminal Cys
of the C-extein (Cys+1) on the
thioester is formed in the first
step and results in a branched
intermediate. Peptide bond
cleavage coupled to succinimide
formation of the C-terminal inteinAsp releases the intein. The
knotted exteins undergo a
spontaneous SN-acyl shift and
yield a peptide bond.
Mutation of Cys1 to Ala prevents
splicing at the N-terminus and leads
to a C-terminal extein bonded with
the intein. C-terminal splicing cannot
occur when the C-terminal Asn is
b tit t d b
Al
id
d th
exploits intein-based splicing to yield purified proteins with C-terminal
thioesters. The thioester is then reacted with derivatized cysteines to
append peptides, affinity labels or fluorophores to the protein of interest
expressing a target protein with the first half of the naturally occurring Ssp DnaE split
intein fused to its C-terminus. The second half of the split intein covalently linked to a
small-molecule probe and a protein transduction domain (PTD) peptide is added to
the cell growth media.
Upon entering the cell, the PTD, linked to the probe-derivatized intein half via a disulfide
bond, is released. The split intein halves combine and self-splice, leaving the protein of
interest labeled at its C-terminus with the probe.
Any synthetic molecule
that can enter cell
Cell permeable synthetic
fragment containing an Nterminal Cys
Bulk of the protein of interest
is expressed in cells, fused
to a modified intein
advantage : the reaction between the
recombinant and synthetic pieces is exquisitely
specific, owing to the requirement for intein
reconstitution in vivo.
Effective design criteria for site-specific protein labeling in living systems :
• Receptor moieties must be amenable to genetic encoding as fusions to
the protein of interest.
• The receptors must be relatively small so as to not perturb protein
function — an ideal receptor would be a short peptide sequence that
could be inserted into various locations within the protein.
• Chemical probes should bind receptors with high specificity and
stability so as to enable functional studies over a time-scale of hours
with no background noise.
• Probes should be designed in a modular fashion so that a wide variety
of fluorophores, affinity labels or other functional moieties can be easily
linked.
• The kinetics of cell loading and receptor binding should be fast enough
(on the order of minutes) to facilitate the most time-sensitive biological
assays.
• A variety of complementary probe–receptor pairs will be needed to
enable the simultaneous study of multiple target proteins.
an unnatural amino acid is
inserted site-specifically into
a protein of interest by
infiltration of the protein
biosynthetic pathway. A
nonsense codon in the
mRNA encoding the protein
of interest is
recognized by a suppressor
tRNA charged with an
unnatural amino acid.
In the chemical approach,
the tRNA is aminoacylated in
vitro and subsequently
delivered to cells. In the
biosynthetic approach, an
orthogonal aminoacyltRNA synthetase (aaRS)
that charges only a mutant
suppressor tRNA is
expressed in cells. Either the
unnatural amino acid must be
delivered to the cells, or the
Nonsense suppression
Nonsense suppression enables an unnatural amino acid to be
incorporated site-specifically into a protein, provided that the amino
acid can be accepted by the ribosome The new functionality can be a
Bump-and-hole strategy
A sensitized enzyme is
made by mutagenesis to form
a cavity at or near the active
site. An inhibitor is derivitized
with a bulky substituent that
complements the cavity in the
sensitized enzyme, but does
not bind to the wild-type
enzyme, resulting in selective
inhibition of the sensitized
enzyme.
The bumped kinase inhibitor
NM-PP1 that has been used
to inhibit -Ca2+/calmodulindependent kinase II
(CaMKII) is shown in the
inset
judicious mutations of residues near the active site of a
protein are made to sensitize it to chemically modified
Bump-and-hole strategy
The bump-and-hole strategy
has been also used in an
alternative format to enable the
specific labeling and
identification of enzyme
substrates.
A single phenylalanine to
glycine mutation in the ATPbinding pocket of Cdk1 enables
the kinase to use N6(benzyl)ATP, an analog that is
not accepted by wild-type
kinases. Incubating yeast
extract with N6-(benzyl)ATP
radiolabeled at the -phosphate
and Cdk1-Clb2 resulted in the
specific phosphorylation of
many proteins.
judicious mutations of residues near the active site of a
protein are made to sensitize it to chemically modified
Conditional protein splicing
A protein is split into two fragments (X and Y) that
are inactive on their own. The fragments are
expressed in cells, each fused to one-half of a split
intein and to either FK506-binding protein 12
(FKBP) or FKBP-rapamycin binding protein (FRB)
as shown. On addition of the chemical inducer of
dimerization (CID) rapamycin (inset), the intein
halves are reconstituted, leading to the joining of
fragments X and Y in a traceless fashion
Chemical rescue of protein degradation.
The protein of interest is expressed as a fusion with FRB*, which
is constitutively degraded by the proteasome. Addition of the CID
C20-MaRap (inset) results in the dimerization of FKBP with
Chemically induced protein degradation. An endogenous target
protein is recruited to a ubiquitin ligase complex on the addition of a
bifunctional small molecule (Protac). The target protein is
polyubiquitinated by the ubiquitin ligase complex, leading to its
d
d ti b th
t
P t 5 hi h
b
dt
ACTIVITY-BASED PROTEIN PROFILING combines chemical synthesis with proteomics to
determine the active enzyme complement of a given biological sample. Chemical probes
designed to label covalently the active sites of a whole class of enzymes are synthesized with
an appended reporter tag, such as a fluorophore or biotin. Incubation of an ABPP probe
ith bi l i l
l
lt i th
l t l b li
f th
ti
f th
b
Synthesis of HAUbDerived Probes
The intein-based
chemical ligation
method. Recombinant
HAUb75-intein-chitin
binding domain (CBD)
fusion protein was
bound to a chitin affinity
column; on-column
cleavage of the HAUbintein junction was
induced by the addition of
-mercaptoethane
sulfonic acid (MESNa).
The resulting HAUb75MESNa thioester was
reacted with a desired
C-terminal thiol-reactive
group, generating the
desired HAUb-derived
probe.
Chemistry-based techniques for studying protein function in vivo.
A Universal Strategy for Proteomic Studies of SUMO
and Other Ubiquitin-like Modifiers. Mol Cell Proteomics. 2005;4(1):56-72
A Proteomic Strategy for Gaining Insights into Protein
Sumoylation in Yeast
Mol Cell Proteomics. 2005 Mar;4(3):246-254
SUMO-conjugated
proteins were isolated
by a double-affinity
purification procedure
from a Saccharomyces
cerevisiae strain
engineered to express
tagged SUMO. The
components of the
isolated protein mixture
were then identified by
subsequent LC-MS/MS
analysis using an LTQ
FT mass spectrometer.
By combining the tools of affinity purification and MS, substrates, associated proteins, and,
in many cases, conjugation sites have been determined. Similar strategies have also been
employed to identify other components of these pathways. For example, proteasomalassociated proteins were identified by tagging proteasomal components at the genetic level,
purifying the tagged complexes, and identifying associated polypeptides. A powerful
Proc Natl Acad Sci U S A. 2004 Feb 24;101(8):2253-8
A. Borodovsky, H. Ovaa, N. Kolli, T. Gan-Erdene, K.D. Wilkinson, H.L. Ploegh and B.M.
Kessler, Chemistry-based functional proteomics reveals novel members of the
deubiquitinating enzyme family, Chem Biol 9 (2002), pp. 1149-1159
J. Hemelaar, P.J. Galardy, A. Borodovsky, B.M. Kessler, H.L. Ploegh and H. Ovaa,
Chemistry-based functional proteomics: mechanism-based activity-profiling tools for
ubiquitin and ubiquitin-like specific proteases, J Proteome Res 3 (2004), pp. 268-276
This is a good review of activity-based probes, with an
emphasis on the design of such reagents that target the
DUBs of both Ub and Ubl proteins. Their application in
various cell lines, tissues and cellular states is also
described
Identifying and quantifying in vivo methylation sites
by heavy methyl SILAC
Nature Methods 1, 119 - 126 (2004)
The heavy methyl
SILAC strategy
Mol Cell Proteomics. 2005 Mar;4(3):310-327
Mol Cell Proteomics. 2005 Feb;4(2):144-55
selective isolation of peptides that are N-linked
glycosylated in the intact protein, the analysis of
these now deglycosylated peptides by liquid
chromatography electrospray ionization mass
spectrometry, and the comparative analysis of the
resulting patterns. By focusing selectively on a
few formerly N-linked glycopeptides per serum
protein, the complexity of the analyte sample is
significantly reduced and the sensitivity and
throughput of serum proteome analysis are
increased compared with the analysis of total
tryptic peptides from unfractionated samples
Nat Biotechnol. 2003 Jun;21(6):660-6
Identification and quantification of N-linked
nonreactive
free reactive
cysteine thiols
Isotope-coded affinity tag (ICAT) approach to redox
proteomics: identification and quantitation of oxidantsensitive cysteine thiols in complex protein mixtures.
oxidized
J Proteome Res. 2004 Nov-Dec;
3(6):1228-33