A relay FRET event in a designed trichromophoric pentapeptide containing an: O -, m -aromatic-amino acid scaffold

Article abstract of DOI:10.1039/c8cc04429e

The concept of a relay FRET event is established in a designed trichromophoric pentapeptide containing an o-,m-aromatic amino acid scaffold in the backbone as a novel β-turn mimetic β-sheet folding nucleator. This system would find application in studying fundamental processes involving interbiomolecular interactions in chemical biology.

Full text of DOI:10.1039/c8cc04429e

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Relay FRET Event in a Designed Trichromophoric Pentapeptide
Containing o-, m-Aromatic-Amino Acid Scaffold
Received 00th June
018,
2
Subhendu Sekhar Bag* and Afsana Yashmeen
Accepted 00th April 2018
DOI: 10.1039/x0xx00000x
www.rsc.org/
5
The concept of relay FRET event is established in a designed FRET between three distinct fluorescent molecules. As for an
trichromophoric pentapeptide containing o-,m-aromatic amino example,
a
sequential energy transfer system in
acid scaffold in the backbone as a novel a β-turn mimetic β-sheet oligonucleotides containing three chromophores in the three
6
a
folding nucleator. This system would find applications in studying separate strands has been reported in 1999. Turro and
fundamental processes involving interbiomolecular interactions in coworkers have synthesized trichromophoric DNA that can
6
b
chemical biology.
serve the purpose of multiplex biological assays and other
applications such as biological labeling and imaging. Few more
4
a, 5d, 6c
Förster resonance energy transfer (FRET), between a donor
and an acceptor chromophore in a close proximity of 0-10 nm,
1
occurs through non-radiative dipole–dipole interation. The
examples exist in literature in the field of DNA.
FRET is extremely sensitive to small changes in distance and is
used as
a research tool to investigate molecular level
interactions in chemistry and biology. Advanced research now
resulted in the design of many FRET pairs aiming at uncovering
the structure, function, dynamics and interactions involving
2
-3
biomolecules inside a cell. To date, a myriad of FRET pairs
and FRET based strategies have been developed to investigate
inter-biomolecular interactions and in several other
4
chemical/biochemical applications. However, most of these
examples restricts FRET occurrence among two chromophors
or two unit of biomolecules having one chromophore in each
and with a limited distance of 10 nm. However, it is a matter of
realization that inside a cell the inter-biomolecular interaction
is not restricted to only two molecules rather occurs among
other large biomacromolecular complexes and often at
4
a, 5
distances more than 10 nm.
Therefore, study of long range
multimolecular interactions in a cell or for multiplex biological
assays has fueled efforts to expand traditional two-dye FRET
system to multichromophoric interacting partners. Recently,
several investigations have been reported that focused on
Figure 1. (A) Three chromophores in a peptide and the interactions of two
coupled FRET pairs-the concept of relay FRET. (B) (i-ii) The absorption and
emission spectra of shown FRET pairs with shaded regions showing overlap
in spectra of donors and acceptors.
It is thus clear that most of the reported triple-dye-FRET
systems are based on DNA scaffolds. Though, several bi-
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chromophoric-traditional-FRET based peptides/proteins have process. Thus, both the non-fluorescent Leu-enkephalin
been reported only very little attention has been paid to analogue peptide as well as fluorescent pentapeptide adopted
expand the two-dye FRET system into three. Only recently, the β-strand structure induced by the scaffold’s turn shape. The
three-dye systems on
a flow cytometer and a three- relay FRET was also established wherein the FRET-source is the
o,m-Ar
fluorophore FRET in protein for live cell imaging have been scaffold (
TAA), the interim chromophore is the triazolyl
TPy
6
d
reported. The triple-dye FRET system exhibiting relay FRET pyrene (TPy) of
Do
Ala and the ultimate emitting (FRET
TPer Do
Ala . Such a
process could enable to study the long range energy transfer terminus) is the triazolyl perylene (TPer) of
that is not allowed by the constraints of one-step FRET relay FRET in a short designed peptide is conceptually efficient
process. Moreover, such sequential energy-transfer in shedding light for newer design of probe for studying
mechanism has been used to elucidate the efficient energy complex inter-biomolecular interaction beyond the limit of
6
c
4a, 5-6
transfer in the photosynthetic light-harvesting arrangements
and can be utilized in a multiplex biological assay. Though
traditional FRET dimension inside a cell.
o,m-Ar
(
The synthesis of the scaffold
1
TAA)started with
t
there are few reports on triple-FRET system in complex preparation of m-[( butyloxycarbonyl)amido]-phenyl acetylene
biological ansembles, novel and simpler design in a short from m-bromo aniline followed by 1,3-azide-alkyne dipolar
peptide is worthwhile for understanding relay FRET event in a cycloaddition reaction between and m-azidomethyl benzoate
6
5
6
o,m-
particular conformation.
7. The scaffold amino acid with free acidic group, BocNH
Ar
As a part of our ongoing research activity toward the
design of constrained turn mimetic molecular scaffold, we
TAA-OH (
2
) was then obtained via hydrolysis of
1
using LiOH
into a
(
Scheme 1). Next, we incorporated the scaffold
1
TPer
Do
–
thought that, o,o- or o,m-aromatic triazolyl amino acid could trichromophoric fluorescent pentapeptide
3
, [BocNH- Ala
TAA–Leu– Ala -CONMe(OMe)] to demonstrate
structure and for spatially orienting terminal chromophores our design concept of FRET relay process (Scheme 1). We also
o,m-Ar
TPy
Do
serve as interesting scaffold for inducing a peptide secondary Leu–
7
very close in a peptide for showing interesting photophysics.
In particular, we, envisioned that a pentapeptide with the analogous to Leu-enkephalin [BocNH-Tyr–
scaffold in the backbone if decorated with triazolylperylene OMe] to test the conformational induction by the scaffold. All
incorporated the scaffold into a tetrapeptide sequence 4,
o,m-Ar
TAA–Phe–Leu-
TPer
Do
amino acid ( Ala ) at the N-terminus and triazolylpyrene intermediate and final peptides were synthesized following a
TPy
amino acid ( Ala ) at the C-terminus, we could achieve a solution phase peptide coupling protocol and were purified by
Do
o,m-Ar
TPy
Do
TAA to Ala to column chromatography using silica-gel (60-120 mesh) and
FRET relay process from the scaffold
TPer
Do
Ala upon exciting the peptide at the absorption (λmax
90 nm) of scaffold wherein no or negligible absorption is
exhibited by other two chromophore (Fig. 1).
=
characterized via NMR, IR and HR-mass spectrometry.
2
o,m-Ar
Figure 2. (A) CD spectra of
and tetrapeptide
Scheme 1. (A) Synthesis of o,m-aromatic triazolyl amino acid scaffold, chain interactions as reflected from ROESY spectra of peptide
TAA (1), fluorescent pentapeptide 3
4
in methanol (50 μM; rt). (B) Backbone and side
. (C-D)
) and its fluorescent pentapeptide, 3 and (B) Leu- Clustering of structures (within 21 kJ/mole global minima) obtained
3
o,m-Ar
(
1
TAA
Enkephalin analogue tetrapeptide,
4
.
from molecular dynamics simulation for tetrapeptide
pentapeptide , respectively.
4
,
and
3
With this light we want to report herein the design (a) of
triazolyl o,m-aromatic amino acid scaffolded tetrapeptide, a
The
secondary
structure
elucidation
by
CD
Leu-enkephalin analogue to study the conformational spectropolarimeter revealed a hairpin turn structure (-196,
7
c, 8
induction by the scaffold and (b) of trichromophoric +200 and +207 nm)
of the scaffold 1 with negative induced
fluorescent pentapeptide to establish a two steps FRET relay CD signals corresponding to its absorption wavelengths at 250
2
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o,m-Ar
and 283 nm in polar protic solvent MeOH (Fig. 2A). On the photophysical properties of scaffold
TPy Do
and Leu- triazolyl amino acid monomers, Ala and
TAA, fluorescent
TPer
Do
Ala indicated
other hand both the fluorescent pentapeptide
3
o,m-Ar
enkephalin analogue tetra peptide 4 containing
TAA a possibility of FRET process from donor scaffold to acceptor
TPy
Do
Ala and then to the end acceptor
TPer Do
Ala (Fig. 1b and
scaffold in the backbone, showed predominantly turn induced
1
β-strand like structures (Fig. 2A) indicating that the scaffold in ESI†, Section 4, Fig. S13). Thus, upon excitation at the scaffold
the turn conformation acted as a β-turn mimetic β-sheet (λex = 290 nm) we observed three emission bands at 320, 405
7
b, 9
folding nucleator.
That the β-strands are associated with intramolecular H-
and 495 nm corresponding to the emission from the scaffold
o,m-Ar
TPy
Do
Ala
TPer
Do
Ala ,
TAA
,
TPy of
and from TPer of
bonding interactions among the two strands was evident from respectively (Fig. 3A). Comparative intensities revealed a
the amide -NH stretching absorptions in FTIR spectra for both negligible emission from the scaffold compared to its free
7
b, 10
the peptides (ESI†, Section 2.2).
The C=O stretching at state, while an increase in intensities by about 2.6 and 1.4
722/1654 and 1743/1658 cm , respectively, for peptide times were observed from TPy and TPer respectively,
and which did not depend on the sample concentration compared to their corresponding free monomer emissions in
supported β-sheet structures in both the peptides. The acetonitrile indicating two consecutive FRET events (Fig. 3A).
support for moderate intramolecular hydrogen bonding Furthermore, upon excitation at 347 nm (absorption of TPy),
interactions in both the peptide and came from VT-NMR the decrease in TPY and increase in TPer emission intensities
analysis in d -DMSO showing moderate ꢀδ/ꢀT (3-6 ppb/k) evidenced the 2 FRET from TPy to TPer in peptide 3 (Fig. 3B).
-1
1
3
,
4
1
0
5
3
4
nd
6
values for the amide NH’s and supported the predominant The time resolved fluorescence study follows the same trend
turn induced β-sheet like structure of the peptides (ESI†, as steady state fluorescence. Thus, a decrease in average life
7
b, 11
Section 2.3).
protons in the solution conformations was revealed from
NOESY and ROESY spectra in d -DMSO of both the peptides
Furthermore, the interaction of various time was observed for the donor scaffold from 4.8 to 3.9 ns
o,m-Ar
(
TAA, λex = 290 nm, λem = 350 nm, in ACN) with an
3
increase in interim acceptor life time from 15.2 ns to 16.2 ns
6
TPy
Do
TPer
Do
and
D NMR supported the close proximity of the two terminal λem = 495 nm) from 3.8 ns to 4.7 ns (Fig. 3C-D, Table 1). All
fluorescent unnatural amino acids and thus the possibility of a observations suggested a consecutive two FRET processes
4
(ESI†, Fig. S6-S7) revealing spatial H-H interactions. The
(
Ala ; λem = 405 nm) and increase in end acceptor ( Ala ;
2
TPer
Do
TPy
Do
st
nd
photophysical interaction between
Ala and
(Fig. 2B). Next, MD simulations using an OPLS
005 force field in Schrodinger Macromodel (Maestro vs. 9.1) Table 1. Summary of time resolved fluorescence in ACN.
Ala in from scaffold to TPy (1 FRET) and then to TPer (2 FRET).
7
pentapeptide
3
2
software package supported the turn induced β-sheet
1
conformationin in both the peptides (Fig. 2C-D).
Entry
Φ
f
λ
em
τ
1
[ns]
τ
2
[ns]
<τ>
[ns]
4.8
0b, 12
[nm]
o,m-Ar
TAA
0.25
0.14
0.65
350
405
495
350
405
350
405
495
2.3 (59 %)
8.3 (41 %)
---
TPy
Ala
Do
15.2 (100 %)
3.8 (100 %)
3.62 (100 %)
3.8 (3 %)
15.2
3.8
TPer
Ala
Do
---
0
0
.01
.17
---
3.6
Tripep. 11
17.3 (97 %)
6.5 (42 %)
16.9 (94 %)
9.5 (17 %)
16.9
3.9
0
.001
0.12
.33
1.9 (58 %)
2.9 (6 %)
PentaPep. 3
16.2
4.7
0
3.67 (83 %)
For lifetimes of the fluorescent amino acids and peptides λex = 290 nm;
Concentration of each fluorescent amino acids and peptides = 10 μM; <τ>,
are weighted means from the bi-exponential fits: <τ> =1/(α
/τ
1 1
+ α
2 2
/τ ).
st
Further evidence of 1 FRET from scaffold to TPy came
separately from the decrease in intensity as well as life time of
the scaffold and increase in intensity and life time of the TPy
upon excitation at 290 nm in the interim tripeptide 11 (ESI†,
Fig. S14-S15, Table S10-11). The second FRET was also
separately supported both from steady state as well as from
time resolved fluorescence (Fig. 3B, D and ESI†, Table S10-
2
o,m-Ar
Figure 3. Fluorescence emission spectra of (A) donor
TPer
TAA, interim
S12). Using the values of
κ
efficiencies which were found to be 89 % and 30 % for FRET
= 2/3, we calculated the FRET
TPy Do
acceptor Ala , end-acceptor
Do
Ala and the pentapeptide
3
and
in
TPy
Do
TPer
Do
(
B) donor
acetonitrile. (C-D) Time resolved fluorescence of
pentapeptide (10 μM each, rt; ex = 290 nm).
Ala , acceptor
Ala and the pentapeptide
3
o,m-Ar
pairs
.2). Thus, we established a relay FRET process with excellent
to good efficiency in our designed fluorescent pentapeptide
TAA/TPy and TPy/TPer, respectively (SI, Section
o,m-Ar
TPy
Do
Ala and
TAA
,
4
3
λ
3
o,m-Ar
with the novel scaffold,
TAA, in the middle of the
To test our hypothesis of FRET relay process, we, next,
proceeded to study the photophysical properties of
backbone inducing an overall β-hairpin structure.
pentapeptide
3 in details. The UV-visible and fluorescence
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As an application the interaction of the fluorescent suggested the binding process is exothermic in nature (ESI†,
7
pentapeptide with only protein BSA was tested (Fig. 4). Our Section 5.12).The binding isotherm was fitted to a sigmoidal
aim was just to see whether in a protein microenvironment curve involving one binding site mode (Fig. 4D). The binding is
-1
5
the FRET photophysics is maintained by the pentapeptide or favoured enthalpically with binding constant (K) 1.1 X 10 M
not. The absorption increases as a solution of the probe and free energy of binding (∆G) -6.9 kcal/mol both of which
peptide 3 was titrated with BSA (Fig. 4A). The overlapping of are found to be closer to the experimental values (ESI†,
7
emission spectra of BSA protein and absorbance of Section 5.11 ).
pentapeptide
3
indicated that there might be a possibility of
In conclusion, we successfully introduced a FRET relay
energy transfer from Trp unit of BSA to pyrene unit of system peptide with a β-sheet inducing turn mimetic o,m-
o,m-Ar
pentapeptide
3
(ESI†, Section 5.6, Fig. S18a). Thus, as we triazolyl aromatic scaffold (
TAA) which acts as FRET origin.
excited at absorption of BSA (280 nm) we observed a decrease The FRET enegy from the scaffold then transferred to the
in BSA emission intensity as well as an increase in pyrene interim acceptor, TPy and then to terminal acceptor, TPer
emission intensity indicating a visual FRET from BSA protein to which ultimately emits fluorescence light. The FRET relay
pentapeptide
3 (Fig. 4B). Time resolved fluorescence study also peptide was also able to interact with BSA leading to the same
supported this FRET phenomenon (ESI†, Section 5.7). The FRET process. This newly designed fluorescent peptide might
4
association constant was found to be 5.3 x 10 M with free find wider application in chemical biology.
-1
energy of binding (∆G) -6.5 kcal/mol (ESI†, Section 5.8).
The authors thank the Department of Biotechnology (DBT:
BT/PR16620/NER/95/223/2015), New Delhi, Govt. of India for generous funding.
Notes and references
1
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(a) K. E. Sapsford, L. Berti, I. L. Menditz, Angew. Chem., Int. Ed., 2006,
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(a) S. Kawahara, T. Uchimaru, S. Murata, Chem. Commun., 1999,
Figure 4. Titration of probe pentapeptide
3 with increasing amount of
BSA (a) UV visible and (b) fluorescence emission spectra (λex = 280 nm;
peptide = 5 µM in 20 mM phosphate buffer, pH 7.0, rt]. (C) Docking
pose of pentapeptide
3 in presence of BSA showing interaction with
5
63. (b) A. K. Tong, S. Jockusch, Z. Li, H.-R. Zhu, D. L. Akins, N. J.Turro,
the hydrophobic pocket of BSA. (D) Plot from isothermal titration
calorimetry.
J. Ju, J. Am. Chem. Soc., 2001, 123, 12923. (c) Q.-H. Xu, S. Wang, D.
Korystov, A. Mikhailovsky, G. C. Bazan, D. Moses, A. J. Heeger, Proc.
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Vámosi, G. Vereb, J. Szöllősi, Cytometry A. 2013, 83A, 375.
To have much information on the interaction a docking
7
(a) S. S.Bag, A. Yashmeen, Bioorg. Med. Chem. Lett., 2017, 27, 5387.
1
3
calculations was carried out using Autodock4 (Fig. 4C).
Docking pose clearly indicated the close proximity of TPy of
probe and Trp of BSA and hence the possibility of occurrence
of FRET process was supported. The probe peptide was
(
b) S. S.Bag, S. Jana, M. K. Pradhan, S. Pal, RSC Adv., 2016,
(c) S. S.Bag, S. Jana, A. Yashmeen, S. De, Chem. Commun., 2015, 51
242;
6, 72654.
,
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and D. J. Pochan, Biochemistry, 2008, 47, 4597.
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3
located in the vicinity of tryptophan (Trp-134) and remained
surrounded by other hydrophobic amino acids of the
hydrophobic pocket of site I of BSA protein. Hydrophobic
44, 1285. (b) A. Fujii, Y. Sekiguchi, H. Matsumura, T. Inoue, W.–S.
Chung, S. Hirota and T. Matsuo, Bioconjugate Chem., 2015, 26, 537.
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MacroModel, Version 9.9 Schrodinger, LLC, New York, NY, 2012.
1
0
binding without perturbing the α-helix secondary structure of
BSA (ESI†, Section 5.9) was also supported from an anisotropy
study (ESI†, Section 5.11).
1
1
1
2
We also carried out Isothermal titration calorimetric
measurement
to
investigate
the
thermodynamic 13 (a) G.M. Morris, R. Huey, W. Lindstrom, et al. J. Comput. Chem.,
2009, 16, 2785. (b) J. S. Hudson, L. Ding, V. Le, E. Lewis, D. Graves
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characterization of the interaction of pentaeptide 3 with BSA
in phosphate buffer (pH 7.0) at 298 K. The negative deflections
4
| Chem. Commun., 2018, 00, 1-4
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DOI: 10.1039/C8CC04429E
Relay FRET Event in a Designed Trichromophoric Pentapeptide
Containing o-, m-Aromatic-Amino Acid Scaffold
Subhendu Sekhar Bag* and Afsana Yashmeen
Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology
Guwahati, North Guwhati-781039, Assam, India. Fax: +91-361-258-2349; Tel: +91-361-258-
2
324.E-mail: ssbag75@iitg.ernet.in