Confined chromophores in tobacco mosaic virus to mimic green fluorescent protein

Article abstract of DOI:10.1039/c5cc05751e

Due to the nano-confinement effect, grafting green fluorescent protein-like chromophores on the interior surface of tobacco mosaic virus can greatly enhance its fluorescence emission in various solvents.

Full text of DOI:10.1039/c5cc05751e

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DOI: 10.1039/C5CC05751E
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Confined Chromophores in Tobacco Mosaic Virus for the Mimic of
Green Fluorescent Protein†
Quan Zhou,ab Fengchi Wu,ab Man Wu,a Ye Tian,a and Zhongwei Niu*
Received 00th January 20xx,
Accepted 00th January 20xx
a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Due to nano confinement effect, grafting green fluorescent trapping GFP-like chromophore in confined nano pore
protein like chromophores in the interior surface of tobacco structures. For artificial GFP β-barrel mimics, nanoparticles,
mosaic virus can greatly enhance its fluorescence emission in especially viral nanoparticles (VNPs) based on protein
various solvents.
assembly, raise the possibility of structure-driven design of
fluorescent materials by modulating nano pore aperture and
confined microenvironment.
Green fluorescent protein (GFP) is widely used as a biomarker
in genetic and molecular biology, enlightening a new way to
study the function of proteins inside living system.1 The
chromophore in GFP is 4-hydroxybenzylidene imidazolinone
(HBI),2 formed via a post-translational autocatalytic cyclization
and autoxidation,3 surrounded by 11-stranded β-barrel
structure.4 The fluorescent properties of HBI are closely
related to the surrounding environment and molecular
Tobacco mosaic virus (TMV) is a classic rod-like plant virus,
which consists of 2130 capsid proteins stacked in helix around
6395 nucleotides single-strand RNA genome, constituted
monodisperse nanotube with 300 nm long, 18 nm in diameter
and 4 nm diameter of inner channel.15 The well-defined inner
channel structure of TMV may provide an ideal confined space
for mimicking the β-barrel structure of GFP.
conformation.5 The β-barrel structure of GFP offers
a
Herein, we demonstrated a simple method to mimic the
confinement effect of GFP β-barrel structure by grafting GFP-
like chromophores in the inner channel of TMV (Scheme 1).
HBI-ethylamine (HBI-en) was synthesized as GFP-like
chromophore by following literature protocols (Scheme S1, see
hydrophobic environment and restricts intrarmolecular
vibration to enhance the fluorescence emission of HBI.6 The
unconfined chromophore is almost non-fluorescence emission
by partly destroying the β-barrel structure in denatured GFP,7
and the fluorescence emission can be restored by recovering
the β-barrel structure,8 which is suggested that the
confinement effect of protein-chromophore interactions is
very important for the fluorescence emission pathway of
energy release.9 This confined emission property of GFP has
always been an object for mimicking. As a famous RNA mimic
of GFP, the fluorogenic aptamer Spinach10 has immobilized
GFP-like chromophore in a G-quadruplex binding site by
restricting the small molecule to planar conformation.11
Polymer scaffold12 and supramolecular host-guest system13
can also supply a hydrophobic environment for blocking
molecular vibration of GFP-like chromophores. Even the rigid
porous scaffold of metal-organic frameworks14 could be
utilized to mimic the GFP β-barrel behavior by coordinatively
Scheme 1 (a) Coupling reaction of HBI-en chromophore and TMV capsid protein with
glutamate residues 97&106 emphasized (blue). (b-d) Structural illustration of TMV-HBI
conjugates in side view (b), cross section view (c) and close-up top view (d). Images
generated by the PyMOL Molecular Graphics System, Version 1.7, Schrödinger, LLC.
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ESI
).16 The structure of HBI-en was characterized by nuclear
Journal Name
†
magnetic resonance spectrum (NMR) (Fig. S1-S4, see ESI for
†
more experimental details). The interior surface of TMV is
covered with glutamate residues 97&106 (Glu 97&106), which
are easy to be conjugated with the amino group of HBI-en
activated by N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide
(EDC) and 1-hydroxybenzotriazole (HOBt) (Scheme 1a and
ESI†
).17,18 GFP β-barrel has a diameter of 2.4 nm with a height
of 4.2 nm, and HBI chromophore is located in the centre of β-
barrel.19 Similar to GFP, TMV inner channel is 4 nm in diameter
with 300 nm in length, and HBI-en chromophores are
immobilized inside the inner channel, which can simulate the
essential character of GFP β-barrel structure in geometry.
The conjugation of HBI-en chromophores in the inner
channel of TMV was confirmed by UV-Vis spectra. As shown in
Fig. 1A, a new absorption band at 366 nm20 indicated that HBI-
en was successfully conjugated onto TMV. From UV-Vis data,
the grafting efficiency of TMV interior surface modification is
85% according to a reported method21 (Fig. S5 see ESI
more details). The conjugation was also identified by MALDI-
TOF MS (Fig. S6, see ESI ). After conjugation, TMV-HBI still
† for
†
maintains its original structure (Fig. 1C). The photographs of
TMV-HBI solution were shown in quartz cuvette under daylight
(Fig. 1D) and UV lamp at λex=365 nm (Fig. 1E). The fluorescence
intensity at 447 nm of TMV-HBI (Fig. 1B) was significantly
enhanced comparing to TMV and HBI-en chromophore
background. PH value is a key factor in TMV assembly &
disassembly process.22 In harsh pH environment at 2.30 or
9.56, TMV was disassembled. As a result, without nano
confinement effect, the fluorescence emission of TMV-HBI was
dramatically decreased (Fig. S7, see ESI† for more details).
Fig. 2 (A) Fluorescence emission spectra of TMV-HBI in 100% water and various 20%
organic mixed aqueous solution at λex=365 nm, i.e. dimethyl sulfoxide (DMSO),
methanol (MeOH), N, N’-dimethyl formamide (DMF), 2-propanol (IPA), acetonitrile
(MeCN), acetone, 1,4-dioxane (Diox), ethanol (EtOH) and tetrahydrofuran (THF). (B)
Fluorescence emission spectra of TMV-HBI in 100% water, 20% and 50% DMSO
aqueous solution. (C) The bathochromic shift between UV-Vis absorbance and
fluorescence emission spectra of TMV-HBI.
Excited state proton transfer (ESPT) is one of the basic ways
of proton transfer in photochemical transformation and
biological process.23 The ESPT process of GFP-like
chromophores in various solvents is obviously influenced by
intermolecular hydrogen bond.24 We have succeeded in
further enhancing the fluorescence emission of TMV-HBI by
adding different organic solvents into aqueous solution (Fig.
2A). The fluorescence enhancement was consistent with the
increase of dimethyl sulfoxide (DMSO) proportion in the
aqueous solution. As depicted in Fig. 2B, the fluorescence
emission of TMV-HBI in 50% DMSO aqueous solution was
Fig. 1 (A) UV-Vis spectra of HBI-en (5 μg·ml-1), TMV (0.242 mg·ml-1) and TMV-HBI (0.238
mg·ml-1). (B) Fluorescence emission spectra of HBI-en, TMV and TMV-HBI at λex=365
nm. (C) TEM image of TMV-HBI after uranyl acetate stained. The photographs of TMV-
HBI solution (1.0 mg·ml-1) under daylight (D) and UV lamp at λex=365 nm (E).
2 | J. Name., 2012, 00, 1-3
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In summary, we have successfully demonstrated that TMV
inner channel structure can be used to simulate GFP β-barrel
behavior by nano confinement effect with grafting HBI-en
chromophore on TMV interior surface. The observed
fluorescence enhancement of HBI-en in the inner channel of
TMV with high grafting density indicated that the molecular
vibration of HBI-en chromophore was efficiently confined. The
well-defined TMV-HBI conjugates promote the understanding
of protein-chromophore interactions in confined nano tubular
space of protein assembly and expand the application of TMV
in protein-based fluorescent materials for future bioimaging.
22 A. Klug, Philosophical transactions of the Royal Society of
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This work was financially supported by the 973 Program of
China (Grant No. 2013CB933800) and the National Natural
Science Foundation of China (Grant No. 21304103, 51303191,
51173070 and 21474123).
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