Activation of fluorescent protein chromophores by encapsulation

Article abstract of DOI:10.1021/ja908870k

(Figure Presented) Chromophores related to fluorescent proteins, when sequestered into the "octaacid" capsule, recover their fluorescence. The fluorescence recovery is related to the inhibition of torsional motions within the cavity, implicating the single-bond torsion as an important contributor to internal conversion within this important class of chromophores.

Full text of DOI:10.1021/ja908870k

Published on Web 01/19/2010
Activation of Fluorescent Protein Chromophores by Encapsulation
Anthony Baldridge,† Shampa R. Samanta,‡ Nithyanandhan Jayaraj,‡ V. Ramamurthy,*,‡ and
Laren M. Tolbert*,†
School of Chemistry and Biochemistry, 901 Atlantic DriVe, Georgia Institute of Technology, Atlanta,
Georgia 30332-0400, and Department of Chemistry, UniVersity of Miami, Coral Gables, Florida 33146
Received October 19, 2009; E-mail: tolbert@chemistry.gatech.edu
Table 1. Substitution Pattern of BMIs Incorporated in OA
The chromophores in fluorescent proteins (FPs) exhibit a
remarkable difference in emission quantum yield as a function of
sequestration within the ꢀ-barrel. Upon denaturation, the fluores-
cence diminishes by 4 orders of magnitude, the result, presumably,
of disruption of the restraining ꢀ-barrel.1 The effect of sequestration
has been the subject of many studies, revolving mostly around the
inhibition of double bond twisting leading to isomerization through
a “one-bond flip” (OBF) process. However, rotation about the
formal single aromatic bond has been proposed as a deactivation
mechanism.2 The former effect is somewhat belied by both the
fluorescence of some chromophores which also undergo cis/trans
isomerization and the relatively modest viscosity effect. Alterna-
tively, a combination of the two twisting modes (“hula twist” or
HT) has been speculated to account for a volume conserving process
within the ꢀ-barrel (see Scheme 1).3 We were therefore intrigued
with the possibility of encapsulation of the chromophore within a
deep cavity cavitand, the so-called “octaacid” (OA), which forms
a hydrophobic capsule-like container, of dimensions 14 Å × 7 Å
upon dimerization4 and mimics the ꢀ-barrel.
Cavitanda
No.
R1
R2
No.
R1
R2
1
2
3
4
5
6
7
8
9
H
Me
Me
Me
Me
Me
Me
Et
10
11
12
13
14
15
16
17
2-Me
2-Me
3-Me
2-Et
4-Me
4-Et
n-Bu
n-Pent
n-Pr
n-Pr
Et
n-Pr
n-Pr
i-Pr
2-Me
3-Me
4-Et
2,5-Me
4-Me
2-Et
4-Me
2-Me
2-Me
2-Me
Et
n-Pr
a Lengths of the molecules provided in SI.
Scheme 1. Torsional Modes in Excited States of BMIs
Figure 1. Partial 1H NMR (500 MHz, 10 mM NaB4O5(OH)4 in D2O)
spectra for the capsular assemblies of (i) cis-9@OA2 and (ii) trans-9@OA2.
‘*’ represents residual cis-9@OA2.
Using previously described methods,5 we synthesized a number
of model FP chromophores, i.e., benzylidene-3-methylimidazoli-
dinones (“BMIs”), with alkyl groups on both the phenyl and
imidazolidinone rings (see R1 and R2 in Table 1) and exposed them
to buffered solutions of OA in D2O. Complexation of the chro-
mophores6 is associated with upfield NMR chemical shifts com-
pared to CD3CN solvent, corresponding to placement of protons
within the shielding region of the hydrophobic (aromatic) cavity.
As we anticipated from earlier studies,7 the para-alkyl derivatives
exhibited strong shielding of both para- and N-alkyl groups within
the cavity (see Figure 1 and Supporting Information, SI). In contrast,
meta- and ortho-alkyl groups were shielded less effectively,
suggesting that the para groups anchor the aryl group at one end
of the capsule.
proxy for the aromatic OA interior,8 that were at variance with
expectations based upon anchoring of the aryl groups. Thus neither
the para-alkyl group nor the meta-alkyl group was effective in
ameliorating internal conversion, as evidenced by little change in
emission quantum yield. In contrast, the ortho-alkyl groups
increased the emission quantum yield by ca. 1 order of magnitude
in the case of 9 (See Figure 2 and Table 2).
Free FP chromophores, as those in some FPs, undergo relatively
efficient cis/trans isomerization.9 Thus we were intrigued by the
possible effect of encapsulation on the photochemistry as well.
Indeed, all of the chromophores, which were synthesized in their
initial cis configuration, readily underwent isomerization in CH3CN
to reach a photostationary state (PSS) with cis/trans ranging from
ca. 40:60 to 60:40 (see Table 2). In contrast, irradiation in the OA
capsule produced, in most cases, PSS favoring the less stable trans
isomer, reaching g94% trans in the case of 1, 9, or 11.
Examination of the effect of encapsulation on fluorescence led
to quantum yields, relative to those in benzene, which served as a
† Georgia Institute of Technology.
‡ University of Miami.
9
1498 J. AM. CHEM. SOC. 2010, 132, 1498–1499
10.1021/ja908870k  2010 American Chemical Society

C O M M U N I C A T I O N S
the photostationary state toward the trans isomer, reflecting the more
favorable equilibrium for encapsulation of the trans. Similarly, the
slow ground-state isomerization of the chromophore also drives
formation of the trans isomer, which becomes the stable form in
the presence of the octaacid.
Curiously, the propensity for cis/trans isomerization does not
correlate readily with internal conversion efficiency. Isomerization
of stilbene-like molecules, for instance, is generally associated with
formation of a twisted state which quenches fluorescence.10 In our
case, the presence of an ortho substituent inhibits internal conversion
without necessarily inhibiting isomerization. We are forced to
conclude that, within the OA capsule, the ortho substituent prevents
ready twisting around the single bond (motion φ in Scheme 1).
Thus when single-bond rotation is prevented, less efficient internal
conversion allows either fluorescence or double-bond isomerization
to take place. In solution, however, the combination of a one-bond
flip with efficient single-bond twisting allows both ready isomer-
ization and efficient internal conversion. As we have observed, in
the protein a low frequency motion is largely responsible for the
loss of fine structure and contributes heavily to internal conver-
sion.11
Figure 2. Fluorescence of cis-9 in aerated solution (red ) benzene, blue
) OA).
b c
,
Table 2. Isomerization Ratio,a TPSS
,
and Emission Quantum
Yields within OA and in Organic Solvents
OA (cavitand)
After
CD3CN
Emission
After
irrad.
(cis:trans)
Φf
(in OA)
×10-3
Φf
(in PhH)
irrad.
(cis:trans)
TPSS
min
,
No.
×10-3
1
2
8
9
10
11
5:95
22:78
22:78
6:94
24:76
2:98
6
8
8
2
10
10
50:50
43:57
58:42
39:61
39:61
39:61
3.27
1.35
3.20
10.0
4.90
2.16
1.40
0.98
1.30
1.47
1.09
1.19
MD/MM calculations (see SI) allowed location of the most
efficient fluorophore 9 inside the capsule (see Figure 3), consistent
with the NOESY spectra (see SI). These were not conclusive as to
the barrier to isomerization in the 2-methyl case or to the preference
for the trans isomer. Further studies on this issue are in progress.
a
1
Monitored from H NMR spectra. Irradiations were done by using a
310 nm cutoff filter. b Time required to reach PSS. c In CD3CN TPSS was
invariably 10 min.
Acknowledgment. We thank the National Science Foundation
(CHE-0809179 and CHE-0848017) for support and Rajib Choudhury
for help with Figure 3. A.B. acknowledges a fellowship from the
Center for Organic Photonics and Electronics (COPE) at Georgia
Tech.
Supporting Information Available: Experimental information,
synthetic details, characterization, and additional spectroscopic informa-
tion is provided. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Figure 3. Fluorophore cis-9 within OA dimer cavity.
The PSS favoring trans is rationalized by the more efficient
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