Fluorescent IGF-II analogues for FRET-based investigations into the binding of IGF-II to the IGF-1R

Article abstract of DOI:10.1039/c5ob02110c

The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two non-native IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners.

Full text of DOI:10.1039/c5ob02110c

Organic &
Biomolecular Chemistry
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Fluorescent IGF-II analogues for FRET-based
investigations into the binding of IGF-II to the
Cite this: DOI: 10.1039/c5ob02110c
IGF-1R†
a
b,c,d
a
e,f
J. M. Cottam Jones,* P. W. R. Harris,
M. A. Brimble
D. B. Scanlon, B. E. Forbes,
b,c,d
a,g
and A. D. Abell*
The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor
IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Under-
(
standing the interactions of IGF-II with these receptors is required for the development of potential anti-
cancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two non-
native IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These
fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluore-
scence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou
IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the
IGF-1R and its other high affinity binding partners.
Received 12th October 2015,
Accepted 26th January 2016
DOI: 10.1039/c5ob02110c
www.rsc.org/obc
1
6,17
metastasis.
While a structure of insulin bound to a frag-
Introduction
1
8,19
ment of the IR has been reported,
detailed structural infor-
Insulin-like growth factor II (IGF-II) is a 67-residue regulatory mation on the interaction of IGF-II with either the IR-A or
peptide that binds with high affinity to three receptors; the IGF-1R remains elusive.
insulin receptor (IR), type 1 insulin-like growth factor receptor
A comparison of IGF-II and IGF-II analogues with the
IGF-1R), and type 2 insulin-like growth factor receptor related ligands, IGF-I and insulin in competition binding
IGF-2R). The binding of IGF-II to these receptors promotes a assays on soluble receptors or cells expressing either the IR-A
(
(
range of responses, including cell growth, proliferation, differ- or the IGF-1R, does however, provide some information on the
1
–6
19–32
entiation and apoptosis.
mechanisms of interaction of IGF-II with IGF-1R and IR-A.
Deregulation of the IGF system results in elevated levels of Alanine mutagenesis of IGF-II has identified two separate
circulating IGF-II with an associated increase in binding to the binding sites (defined as sites 1 and 2) that are critical for
6
–15
IGF-1R and IR-A (insulin receptor isoform A).
Activation of binding to the IGF-1R and IR and are analogous to the two
3
3
both receptors can then promote cancer cell growth and insulin binding sites that bind the IR. However, precise
molecular details of the contact residues within the receptors
remain ill-defined. X-ray crystallographic analysis of insulin
a
Department of Chemistry, The University of Adelaide, Adelaide 5001, SA, Australia
School of Chemical Sciences, The University of Auckland, 23 Symonds Street,
18,19
bound to a fragment of the IR
suggests that the site 1 resi-
b
dues of IGF-II most likely contact the leucine-rich domain 1
and α-CT peptide.
Auckland 1010, New Zealand
c
Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland,
Private Bag 92019, Auckland 1010, New Zealand
Here we present the convergent synthesis of F19Cou IGF-II
and F28Cou IGF-II and their use in a fluorescence resonance
energy transfer (FRET) based study to define the interactions
of IGF-II with the IGF-1R. A coumaryl glycine acceptor (see
d
Institute for Innovation in Biotechnology, The University of Auckland, 3A Symonds
Street, Auckland 1010, New Zealand
e
School of Molecular and Biomedical Sciences, The University of Adelaide,
Adelaide 5005, SA, Australia
2
8
19
f
School of Medicine, Flinders University of South Australia, Bedford Park, 5042 SA,
Scheme 1) was incorporated into IGF-II at Phe and at Phe
Australia
to probe interactions with sites 1 and 2, respectively. The cou-
maryl glycine probe has advantages of a strong quantum yield
and a large Stokes shift, while also exhibiting appropriate spec-
g
ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), The University of
Adelaide, South Australia, 5005, Australia
†
Electronic supplementary information (ESI) available: Synthesis and character-
3
4–41
tral overlap with tryptophan (Trp) as a FRET donor.
Trp
isation of peptide fragments, experimental conditions, analysis of ligations and
fluorescent experiments. See DOI: 10.1039/c5ob02110c
was deemed a suitable donor as these residues were identified
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Scheme 1 One-pot, three fragment ligation approach to the synthesis of IGF-II analogues. Inset: coumaryl glycine acceptor. NCL conditions: 6.0 M
GnHCl, 200.0 mM Na HPO , 20.0 mM TCEP, 200.0 mM MPAA, pH of 6.7–7.0. Folding conditions: 2.50 M urea, 0.70 M Tris, 12.5 mM glycine, 2.0 mM
EDTA, 0.5 mM DTT, 1.25 mM 2-hydroxyethyl disulphide at a pH of 9.1 and protein concentration of <0.10 mg mL
2
4
−1
.
as being positioned nearby likely IGF-II binding sites through fluorescent IGF-II analogues (F19Cou IGF-II and F28Cou
analysis of the X-ray crystal structure of the Insulin:IR complex IGF-II) was investigated as summarised in Scheme 1.
1
8,42,43
as well as IR and IGF-1R mutagenesis data.
The IGF-II peptides were assembled from three fragments
There are three reports on the chemical synthesis of IGF-II of 20, 26 and 21 amino acids in length respectively, using itera-
2
0
21
46
47
and all utilise a similar linear approach that requires multi- tive Val to Cys and Cys to Cys ligations as depicted in
step post-cleavage deprotections and lengthy multi-step purifi- Scheme 1. Specific ligation sites were selected based on the
4
4–47
cation protocols.
Furthermore, none are suitable for the size of the respective fragments and the predicted reactivity of
5
1
20
incorporation of unnatural amino acids. The yield of IGF-II is the C-terminal residue bearing the thioester. Thus the Val –
2
1
8
9
presented in only two of these syntheses and this is somewhat Cys junction (rather than Leu –Cys ) was selected as the
problematically reported to be of the order of 2% (based on N-terminal ligation site, where disconnection here gives rise to
the starting resin). Thus a more modular approach to the peptide fragments of similar size (approx. 20 residues)(see
coumaryl glycine-containing IGF-II analogues was deemed ESI† for IGF-II sequence).
necessary. With this in mind we have developed an efficient
The peptide thioesters (IGF-II (1–20) and IGF-II (Thz-46))
convergent synthesis of IGF-II and its application to the prepa- were prepared using an in situ neutralisation Boc-SPPS proto-
4
9
ration of two specific fluorescent IGF-II analogues; F19Cou col, and the C-terminal IGF-II (47–67) fragment was syn-
IGF-II and F28Cou IGF-II (see Scheme 1).
thesised using standard Fmoc-SPPS (see ESI†). The use of a
manual in situ neutralisation Boc-SPPS protocol for the prepa-
ration of the N-terminal IGF-II (1–20) thioester prevented the
formation of deletion by-products, which resulted from on-
Results and discussion
4
7
resin aggregation using an Fmoc-SPPS approach. The use of
9
21
46
47
51
60
The six Cys residues (Cys , Cys , Cys , Cys , Cys and Cys ) TFA in the deprotection of the Nα-Boc amino functionality was
5
2,53
of the IGF’s provide suitable sites for a native chemical ligation key in disrupting on-resin aggregation.
4
8
(
NCL) approach to the synthesis. The viability of such an
The one-pot three fragment synthesis of the native IGF-II
approach to IGF-II and its analogues was first investigated peptide (summarised in Fig. 1) began with ligation of the
with a two fragment-based synthesis of native IGF-II. This IGF-II (Thz-46) thioester with the C-terminal IGF-II (47–67)
5
0,54
began with the synthesis of a C-terminal IGF-II (47–67) frag- fragment under standard NCL conditions.
LCMS analysis
ment by standard Fmoc-SPPS and the N-terminal IGF-II (1–46) of the reaction mixture, after 60 min, revealed complete con-
thioester using an in situ neutralisation Boc-SPPS protocol (see version to the IGF-II (Thz-67) fragment (see Fig. 1b). Treatment
4
9
ESI†). Ligation of the N-terminal IGF-II (1–46) thioester and of this ligation mixture with methoxyamine hydrochloride
5
5
the C-terminal IGF-II (47–67) fragment, in the presence of (0.2 M) at pH 3, for 8 h gave complete conversion (as deter-
.0 M GnHCl, 200 mM Na HPO
, 20 mM TCEP, 200 mM mined by LCMS) of the thiazolidone (Thz) to cysteine (refer to
MPAA, at a pH of 6.8, gave the desired native IGF-II peptide Fig. 1c). The ligation mixture was then returned to pH 6.8 and
see ESI†). However, access to sufficient quantities of the the N-terminal IGF-II (1–20) thioester was added. Complete
6
2
4
5
0
(
required 46-residue fragment, or unnatural amino acid- conversion to the desired native IGF-II peptide was apparent
containing derivatives thereof, proved impractical due to low after 36 h, based on LCMS analysis as shown in Fig. 1d,
5
1
yields associated with the assembly and purification of such a despite the expected slow reactivity of Val thioester. Native
long peptide sequence. Therefore, a more convergent, three IGF-II peptide was isolated by solid phase extraction and puri-
fragment-based approach to synthesis of the native IGF-II and fied by RP-HPLC. This material was then folded using the
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Fig. 3 Competitive binding of the synthetic native IGF-II, F19Cou IGF-II,
and F28Cou IGF-II proteins to immunocaptured IGF-IR. Immuno-
captured IGF-1R was incubated with europium-labelled IGF-II (EuIGF-II)
in the absence or presence of increasing concentrations of recombinant
native IGF-II (black, dashed line), synthetic native IGF-II (two fragment:
grey squares; three fragment: brown diamonds), F19Cou IGF-II (orange
circles) or F28Cou IGF-II (green triangles). Results are expressed as a
percentage of binding in the absence of competing ligand (Bo). Graphs
show data pooled from three separate experiments and each data point
is measured in triplicate per experiment, except data from the native
IGF-II protein (two fragment) was derived from a single experiment per-
formed in triplicate. Data is shown as the mean ± S.E. Error bars are
shown when greater than the size of the symbols.
Fig. 1 LCMS analysis of the one-pot three fragment synthesis of the
native IGF-II peptide. Analysis of the cysteine-based ligation between
the IGF-II (Thz-46) thioester and C-terminal IGF-II (47–67) fragment
after (a) 2 min and (b) 60 min; (c) analysis of the thiazolidone de- thioester and C-terminal IGF-II (47–67) fragment. Folding of
protection of the IGF-II (Thz-67) fragment after 8 h; (d) analysis of the the fluorescent IGF-II peptides was essentially as described
valine-based ligation between the N-terminal IGF-II (1–20) thioester and
IGF-II (21–67) fragment after 36 h.
above for the native IGF-II protein, and resulted in a single
folded product (see ESI†). RP-HPLC was used to confirm the
56
correct folding of the synthetic IGF-II analogues, which was
consistent with folding profiles observed for other IGF-II ana-
3
3,57
logues.
Both the F19Cou IGF-II and F28Cou IGF-II proteins
were isolated in excellent purity (>98% based on RP-HPLC, see
Fig. 2c and d), and in moderate to low overall yields of 1%
(
from the IGF-II (Thz-67) thioester fragment) and 0.1% (from
the F28Cou IGF-II (Thz-67) thioester fragment) respectively.
A real advantage of the ligation methodology is that it is robust
and highly reproducible. Sufficient quantities of peptide were
obtained for the study, however the yield from protein folding
could likely be improved if conducted on a larger scale.
Competition binding assays of the synthetic IGF-II ana-
logues with the IGF-1R were conducted using solubilized
5
8
immunocaptured IGF-1R and the resulting binding curves
Fig. 2 Purified synthetic IGF-II proteins synthesised using NCL are depicted in Fig. 3. The IC50 values for the binding of the
approach. (a) Native IGF-II (2 fragment); (b) native IGF-II (3 fragment); (c)
F19Cou IGF-II; (d) F28Cou IGF-II.
synthetic native IGF-II (two fragment synthesis) and synthetic
native IGF-II (three fragment synthesis), to the IGF-1R were
determined to be 2.1 ± 1.6 nM and 2.0 ± 1.2 nM respectively,
see Table 1. These values are essentially identical to the IC50
5
6
optimised folding conditions described by Delaine et al. (see values determined and those reported for the recombinant
5
6
ESI†) to give synthetic native IGF-II in an overall yield of 0.3% native IGF-II protein (2.1 ± 1.2 nM). The F19Cou IGF-II and
from the IGF-II (Thz-67) thioester fragment) and importantly the F28Cou IGF-II proteins gave IC50 values of 7.0 ± 1.3 nM
in improved purity (>98% based on RP-HPLC, see Fig. 2b) com- and 6.5 ± 1.5 nM respectively. A small decrease in affinity com-
(
4
4,45
pared to previous reports.
pared to the native IGF-II protein was not unexpected since
1
9
28
The methodology was next applied to the synthesis of the both Phe and Phe are reported to be important residues for
3
3
two fluorescent IGF-II analogues, F19Cou IGF-II and F28Cou IGF-II binding. Importantly, the observed three-fold decrease
IGF-II. The F19Cou IGF-II peptide was synthesised by ligation in binding affinity is consistent with reports for other IGF-II
3
3,56
of the N-terminal F19Cou IGF-II (1–20) thioester, IGF-II (Thz- analogues with substitutions at Phe19 and Phe28.
6) thioester and C-terminal IGF-II (47–67) fragment. The
A FRET analysis of the binding of both fluorescent IGF-II
4
59
F28Cou IGF-II peptide was similarly synthesised by ligation of analogues to a soluble form of the IGF-1R (sIGF-1R) was next
the N-terminal IGF-II (1–20) thioester, F28Cou IGF-II (Thz-46) investigated. Native tryptophan fluorescence from the IGF-1R
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Table 1 IC50 values derived from competitive binding assays of the syn-
thetic IGF-II analogues binding to immunocaptured IGF-1R. Where the
affinity relative to IGF-II is the IC50 relative to that of IGF-II binding to
the IGF-1R (IC50 IGF-II/IC50 IGF-II analogue) and is expressed as a per-
centage of IGF-II binding. IGF-II ± S.E is derived from at least three sep-
arate experiments performed in triplicate
Affinity relative
to recombinant native
IGF-II (%)
Protein
IC50 (nM)
Fig. 5 Summary of fluorescence emission spectra of the F19Cou IGF-II
analogue after excitation at 280 nm (A) and 320 nm (B). Fluorescence
emission spectra in the absence (orange solid line) and presence (black
solid line) of the sIGF-1R, the emission of the sIGF-1R (red solid line) and
the summed emission spectrum resulting from the sum of the individual
donor (sIGF-1R) and acceptor (F19Cou IGF-II) spectra (blue solid lines)
are displayed. Spectra were collected when the protein and receptor
were present in an equimolar ratio at concentration of 0.20 µM, in 0.1 M
sodium phosphate buffer at pH 7.2 after excitation at 280 nm or
Recombinant native IGF-II
Native IGF-II (2 fragment)
Native IGF-II (3 fragment)
F19Cou IGF-II
F28Cou IGF-II
2.1 ± 1.2
2.1 ± 1.6
2.0 ± 1.2
7.0 ± 1.3
6.5 ± 1.5
100
100
105
30
a
32
a
Results derived from a single experiment performed in triplicate.
320 nm. Spectra are derived from a single experiment, where each spec-
trum is averaged from three consecutive scans and have not been cor-
rected for background fluorescence.
Fig. 4 Summary of fluorescence emission spectra of the native IGF-II
analogue and sIGF-1R after excitation at 280 nm (A) and 320 nm (B).
Fluorescence emission spectra in the absence (purple solid line) and
presence (black solid line) of the sIGF-1R, the emission of the sIGF-1R
Fig. 6 Summary of fluorescence emission spectra of the F28Cou IGF-II
analogue after excitation at 280 nm (A) and 320 nm (B). Fluorescence
emission spectra in the absence (green solid line) and presence (black
solid line) of the sIGF-1R, the emission of the sIGF-1R (red solid line) and
the summed emission spectrum resulting from the sum of the individual
donor (sIGF-1R) and acceptor (F28Cou IGF-II) (blue solid lines) are dis-
played. Spectra were collected when the protein and receptor were
present in an equimolar ratio at concentration of 0.19 µM, in 0.1 M
sodium phosphate buffer at pH 7.2 after excitation at 280 nm or
320 nm. Spectra are derived from a single experiment, where each spec-
trum is averaged from three consecutive scans and have not been cor-
rected for background fluorescence.
(
red solid line) and the summed emission spectrum resulting from the
sum of the individual donor (sIGF-1R) and acceptor (native IGF-II)
spectra (blue solid lines) are displayed. Spectra were collected when the
protein and receptor were present in an equimolar ratio at concentration
of 0.20 µM, in 0.1 M sodium phosphate buffer at pH 7.2 after excitation
at 280 nm or 320 nm. Spectra are derived from a single experiment,
where each spectrum is averaged from three consecutive scans and
have not been corrected for background fluorescence.
was used as the donor, with the coumarin contained within
synthetic F19Cou IGF-II and F28Cou IGF-II proteins acting as
an acceptor. Binding was investigated using a sensitized emis- (acceptor) in the absence and presence of the donor (sIGF-1R),
sion approach, where the presence of a FRET signal requires the emission of the donor (sIGF-1R) and the summed emis-
the tryptophan fluorescence of the IGF-1R to be quenched and sion spectrum resulting from the sum of the individual donor
the coumarin fluorescence of the IGF-II analogue is sensitized. and acceptor spectra are displayed. The spectra reveal two key
The experiment involved titration of an IGF-II analogue (accep- emissions, one at 332 nm resulting from excitation of the
tor) into a solution of sIGF-1R (donor) until the protein and sIGF-1R (donor) and a second at 455 nm that arises from exci-
receptor were present in an equimolar ratio. After each tation of the coumarin within the synthetic F19Cou IGF-II and
addition of an IGF-II analogue, the sample was excited at F28Cou IGF-II analogues (acceptor).
2
80 nm and 320 nm and the fluorescence emission spectrum
The fluorescence emission spectra for the native IGF-II ana-
for each wavelength was recorded (see ESI†). Control experi- logue are shown in Fig. 4. As expected, excitation at 280 nm of
ments for the IGF-II analogues (acceptor) and sIGF-1R (donor) native IGF-II (control) alone (Fig. 4A: purple solid line) gave no
were also analysed using the same methodology.
fluorescence emission at 332 nm. This is because native IGF-II
The resulting fluorescence emission spectra for the native lacks endogenous Trp or a fluorescent probe. Similarly, exci-
IGF-II, F19Cou IGF-II, and F28Cou IGF-II, after excitation at tation at 320 nm of native IGF-II alone (Fig. 4B: purple solid
2
80 nm and 320 nm are shown in Fig. 4–6 respectively. Specifi- line) did not result in an increase in fluorescence emission at
cally fluorescence emission spectra of the IGF-II analogues 455 nm as it lacks the coumarin fluorophore present in
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F19Cou IGF-II and F28Cou IGF-II. However, excitation at
2
80 nm of the sIGF-1R (donor) alone gave an intense emission
at 332 nm (Fig. 4A: red solid line), which is due to the presence
of endogenous Trp. Surprisingly, excitation at 280 nm of the
sIGF-1R (donor) in the presence of native IGF-II resulted in
fluorescence emission at 332 nm that was less intense than
the fluorescence emission at 332 nm for the sIGF-1R alone
(Fig. 4A: red solid line). This emission at 332 nm was also less
intense than the sum of the individual emission spectra of
native IGF-II and sIGF-1R (Fig. 4A: blue solid line). This large
decrease in fluorescence at 332 nm indicates that the receptor
Fig. 7 Sensitized fluorescence emission spectra for F19Cou IGF-II in
complex with the sIGF-1R after excitation at 280 nm (A) with expansion
(sIGF-1R) is extremely sensitive to ligand binding. The
decrease is likely the result of extensive Trp quenching within between 390–540 nm (B). Fluorescence emission spectra of the
the sIGF-1R, or perhaps a decrease in the quantum yield of the complex (sIGF-1R and F19Cou IGF-II) uncorrected (black solid line),
extracted acceptor emission (black dotted line) which was obtained by
subtracting the donor (sIGF-1R) emission from the complex (sIGF-1R
and F19Cou IGF-II) emission, acceptor (F19Cou IGF-II) emission (orange
solid line), fluorescence emission spectrum resulting from FRET (blue
endogenous Trp within the sIGF-1R. This is an interesting
result that suggests a change in the local environment of the
IGF-1R Trp residues upon IGF-II binding, and supports the
idea of a structural change in the receptor upon ligand dotted line), which was obtained by subtracting the donor (sIGF-1R) and
binding.
An analysis of the fluorescence emission spectra for the
F19Cou IGF-II analogue alone and in complex with the
sIGF-1R is shown in Fig. 5. Excitation at 280 nm of the F19Cou
acceptor (F19Cou IGF-II) spectra from the complex (sIGF-1R and
F19Cou IGF-II) are displayed. Spectra were collected when the protein
and receptor were present in an equimolar ratio at concentration of
0
.2 µM, in 0.1 M sodium phosphate buffer at pH 7.2 after excitation at
280 nm. Spectra are derived from a single experiment, where each spec-
IGF-II analogue alone (Fig. 5A: orange solid line) resulted in trum is averaged from three consecutive scans.
no fluorescence emission at 332 nm and a small intensity
emission at 455 nm, and excitation of the sIGF-1R alone
(
3
donor) at 280 nm produced an intense emission peak at
32 nm (Fig. 5A: red solid line). Excitation at 280 nm of cally, the extracted acceptor emission (Fig. 7: black dotted line)
F19Cou IGF-II (acceptor) in the presence of the sIGF-1R and the extracted FRET signal (Fig. 7: blue dotted line) are
donor) (Fig. 5A: black solid line) resulted in fluorescence shown. Where the extracted acceptor emission (Fig. 7: black
(
emissions at 332 nm and 455 nm. The fluorescence emission dotted line) was obtained by subtracting the donor (sIGF-1R)
of the complex at 455 nm was increased compared to the fluo- emission from the complex (sIGF-1R and F19Cou IGF-II)
rescence emission for the acceptor (F19Cou IGF-II) alone emission, and the FRET signal (Fig. 7: blue dotted line) was
(
Fig. 5A: orange solid line). Whereas the fluorescence emission obtained by subtracting the donor (sIGF-1R) and acceptor
of the complex at 332 nm was unchanged compared to the (F19Cou IGF-II) spectra from the complex (sIGF-1R and
fluorescence emission spectrum for the donor (sIGF-IR) alone F19Cou IGF-II) spectra. The extracted acceptor emission
(
Fig. 5A: red solid line), and was unchanged compared the (Fig. 7: black dotted line) shows a fluorescence emission at
sum of the individual emission spectra of the sIGF-1R and 455 nm, which is consistent with coumarin emission. The
F19Cou IGF-II (Fig. 5A: blue solid line). This unaltered fluo- emission was more intense than the fluorescence emission at
rescence emission at 332 nm suggests a lack of Trp quenching 455 nm for the acceptor (F19Cou IGF-II) alone (Fig. 7: orange
in the IGF-1R upon F19Cou IGF-II binding. Excitation at solid line). This increase in fluorescence emission at 455 nm is
3
20 nm of the F19Cou IGF-II (acceptor) alone (Fig. 5B: orange also visible in the extracted FRET signal shown in Fig. 7 (blue
solid line) gave rise to fluorescence emission at 455 nm. The dotted line). Together these results suggest that the quantum
same fluorescence emission at 455 nm was also observed for yield of the Trp residues, within the IGF-1R, is enhanced by
the F19Cou IGF-II in complex with the sIGF-1R (acceptor & F19Cou IGF-II binding but is simultaneously and equally
donor) (Fig. 5B: black solid line), after excitation at 320 nm. quenched by FRET. This results in the unaltered Trp emission
Importantly the fluorescence emission at 455 nm for F19Cou seen in Fig. 5.
IGF-II in complex with the sIGF-1R (acceptor & donor) was
Fig. 6 depicts the FRET analysis of the F28Cou IGF-II alone
identical in intensity to the emission for the F19Cou IGF-II and in complex with the sIGF-IR. Excitation at 280 nm of
alone (acceptor). These results demonstrate that fluorescence F28Cou IGF-II alone (acceptor) (Fig. 6A: solid green line) gave a
emission at 455 nm is enhanced upon F19Cou IGF-II binding low intensity fluorescence emission at 455 nm, and excitation
to the receptor (sIGF-1R), and direct excitation (Ex 320 nm) of at 280 nm of the F28Cou IGF-II in complex with sIGF-IR
the coumarin residue is unaffected after ligand binding. (donor & acceptor) (Fig. 6A: black solid line), gave fluorescence
Together these results confirm the F19Cou IGF-II coumarin emissions at 332 nm and 455 nm. The fluorescence emission
fluorescence is sensitized.
at 332 nm was less intense than the emission at 332 nm for
Fig. 7 shows the acceptor emission data from the FRET the sIGF-IR alone (Fig. 6A: red solid line). While the fluo-
interaction between F19Cou IGF-II and the sIGF-1R. Specifi- rescence emission at 455 nm for the complex was more
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intense than the emission at the same wavelength for the and 8 (blue dotted lines) shows the FRET signal for F19Cou
acceptor (F28Cou IGF-II) alone (Fig. 6A: green solid line). IGF-II is stronger than the FRET signal for the F28Cou IGF-II
The relative decrease in fluorescence at 332 nm between the analogue. This suggests that binding of F28Cou IGF-II to the
complex and receptor alone is consistent with Trp quenching sIGF-1R causes a decrease in the quantum yield of the receptor
within the sIGF-1R upon F28Cou IGF-II binding, albeit to a (sIGF-1R), leading to a decrease in fluorescence emission at
lesser extent than with the native IGF-II (Fig. 4). While the 332 nm, which results in the reduced emission at 332 nm
increase in fluorescence emission at 455 nm for the complex shown in Fig. 6.
compared to the acceptor (F28Cou IGF-II) alone, is consistent
A FRET interaction was expected for both site 1 (F28Cou
with a FRET interaction between the sIGF-1R and F28Cou IGF-II) and site 2 (F19Cou IGF-II) interactions as there are
IGF-II. Furthermore excitation at 320 nm gave fluorescence several naturally occurring Trp residues located adjacent to the
emission at 455 nm for both the F28Cou IGF-II in complex putative IGF-II binding sites (including Trp residues 79, 127,
with the sIGF-1R (acceptor & donor) (Fig. 6B: black solid line) 176, 244 for site 1 and 402, 404, 479, 519 601 and 618 for site
1
9,27,28,43
and F28Cou IGF-II alone (acceptor) (Fig. 6B: green solid line). 2) (see ESI†).
Of these, the most likely donor candi-
This emission was only slightly more intense for the F28Cou dates, identified from the X-ray crystallographic data of the
1
9,31
IGF-II in complex with the sIGF-1R than for the F28Cou IGF-II insulin:IR interaction
alone, and suggests direct excitation (Ex 320 nm) of the these residues are surface exposed and located approximately
coumarin residue is relatively unaffected after ligand binding. 18–25 Å from the proposed ligand binding site. As expected,
These results are supported by the acceptor emission data an increase in fluorescence emission was not observed for the
shown in Fig. 8. native IGF-II protein in the presence of the sIGF-1R, as it lacks
are Trp79, Trp519 and Trp544, as
The extracted acceptor emission shown in Fig. 8 gave fluo- a fluorescent probe (Fig. 4). However surprisingly binding of
rescence emission at 455 nm, which is consistent with accep- native IGF-II to the sIGF-1R causes a significant decrease in
tor emission. This extracted emission was more intense than the Trp fluorescence of the sIGF-1R. The site 2, F19Cou IGF-II
the fluorescence emission at 455 nm for the acceptor (F28Cou analogue (Fig. 5) displayed fluorescence emission that was sen-
IGF-II) alone (Fig. 8: orange solid line). This emission is also sitized, and Trp emission was unaltered. Finally, the F28Cou
shown in the extracted FRET signal (Fig. 8: blue dotted line). IGF-II analogue displayed Trp fluorescence which was lower in
Together this data confirms the increase in the fluorescence the presence of the sIGF-1R and the coumarin fluorescence of
emission at 455 nm is likely the result of a FRET interaction the F28Cou IGF-II analogue was sensitized. These results
between the Trp residues of the IGF-1R and the coumarin of confirm that the coumarin probes of the F19Cou IGF-II and
the F28Cou IGF-II analogue. A comparison of F19Cou IGF-II F28Cou IGF-II bind in close proximity (10–100 Å) to a Trp
and F28Cou IGF-II acceptor emission spectra shown in Fig. 7 residue(s) within the IGF-1R and in turn Phe19 and Phe28 are
appropriate sites within IGF-II for the incorporation of a fluo-
rescent probe/FRET acceptor. The strong binding affinities and
positive FRET results demonstrate these are good analogues
for further FRET binding studies.
Conclusions
In conclusion, we report an efficient, modular synthesis of the
native IGF-II protein and two fluorescent IGF-II analogues. A
three fragment approach using a Val20–Cys21 ligation site,
gave complete ligation in less than 47 h. The IGF-II proteins
were isolated in comparable yields (0.1–1%) and importantly
Fig. 8 Sensitized fluorescence emission spectra for F28Cou IGF-II in
in higher purity compared to previously reported IGF-II syn-
complex with the sIGF-1R after excitation at 280 nm (A) with expansion
4
4–46
between 390–540 nm (B). Fluorescence emission spectra of the thesises (ca. 2%).
complex (sIGF-1R and F28Cou IGF-II) uncorrected (black solid line),
The native IGF-II and two fluorescent
IGF-II analogues bind with nanomolar affinity to the IGF-1R.
Both the F19Cou IGF-II and F28Cou IGF-II proteins displayed
a FRET interaction with the IGF-1R, with binding of the native
IGF-II protein causing the largest quenching of IGF-IR fluo-
extracted acceptor emission (black dotted line) which was obtained by
subtracting the donor (sIGF-1R) emission from the complex (sIGF-1R
and F28Cou IGF-II) emission, acceptor (F28Cou IGF-II) emission (green
solid line), fluorescence emission spectrum resulting from FRET (blue
dotted line), which was obtained by subtracting the donor (sIGF-1R) and rescence. These results demonstrate that the Trp residues
acceptor (F28Cou IGF-II) spectra from the complex (sIGF-1R and
F28Cou IGF-II) are displayed. Spectra were collected when the protein
and receptor were present in an equimolar ratio at concentration of
within the sIGF-1R are extremely sensitive to ligand binding,
and these coumarin probes bind in close proximity to Trp resi-
dues within the IGF-1R and as such are ideal analogues for
0
.19 µM, in 0.1 M sodium phosphate buffer at pH 7.2 after excitation at
investigating the interaction of IGF-II with its high affinity
280 nm. Spectra are derived from a single experiment, where each spec-
trum is averaged from three consecutive scans.
binding partners.
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2
0 T. P. J. Garrett, N. M. McKern, M. Lou, M. J. Frenkel,
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