Photoisomerization of the green fluorescence protein chromophore and the meta- and para-amino analogues

Article abstract of DOI:10.1039/b717714c

The Z → E photoisomerization and fluorescence quantum yields for the wild-type green fluorescence protein (GFP) chromophore (p-HBDI) and its meta- and para-amino analogues (m-ABDI and p-ABDI) in aprotic solvents (hexane, THF, and acetonitrile) and protic

Full text of DOI:10.1039/b717714c

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Photoisomerization of the green fluorescence protein chromophore and
the meta- and para-amino analoguesw
Jye-Shane Yang,* Guan-Jhih Huang, Yi-Hung Liu and Shie-Ming Peng
Received (in Cambridge, UK) 15th November 2007, Accepted 21st December 2007
First published as an Advance Article on the web 18th January 2008
DOI: 10.1039/b717714c
The Z - E photoisomerization and fluorescence quantum yields
for the wild-type green fluorescence protein (GFP) chromophore
(p-HBDI) and its meta- and para-amino analogues (m-ABDI
and p-ABDI) in aprotic solvents (hexane, THF, and acetonitrile)
and protic solvents (methanol and 10–20% H2O in THF) are
reported. The dramatic decrease in the quantum yields on going
from aprotic to protic solvents indicates the important role of
solvent–solute hydrogen bonding in the nonradiative decay path-
ways. The enhanced fluorescence of m-ABDI is also discussed.
torsion,5 not only its disagreement with a volume-conserving
path but also the absence of any explicit conical intersection
near the twisted intermediate is the deficiency.3 Although a
concerted torsion of the C–C and CQC bond2,6 is volume-
conserving, it encounters an unfavorable uphill coordinate.3,4
Another common problem for the TICT and hula-twist me-
chanisms is the lower possibility of these torsions being
effectively inhibited by the protein matrix.10
We report herein a new approach toward understanding the
nonradiative pathways of p-HBDI through quantitative de-
termination of the Z - E photoisomerization quantum yields
(FZE) for p-HBDI and its para- and meta-amino analogues
p-ABDI and m-ABDI in different solvents. Our results have
led to a new excited-decay mechanism proposed for p-HBDI in
fluid solutions.
The green fluorescence protein (GFP) discovered in the Pacific
jellyfish Aequorea victoria has been widely used as a biological
marker.1 The green fluorescence (508 nm) is known to result
from the anionic form of the chromophore, p-hydroxybenzy-
lideneimidazolinone (p-HBI), through excited-state proton
transfer (ESPT) from the phenolic oxygen to the protein
matrix.1 However, the dramatic difference between the fluor-
escence quantum yield (Ff) for p-HBI in the protein b-barrel
(Ff B 0.8) and that in fluid solutions (Ff o 10À3) has puzzled
scientists for many years.1–10 More specifically, a unified
answer for the questions as to: (1) what the ultrafast non-
radiative decay pathway is for both the neutral and anionic
forms of the free chromophore, and (2) how the protein matrix
suppresses such a decay route so efficiently, remained to be
established. In this context, the photodynamics of the dimethyl
derivative of the GFP chromophore (p-HBDI)2–6 and many
other related model systems7,8 and GFP mutants7,9 has been
extensively investigated.
Our design concepts for the investigation of ABDIs are
manifold. First, the excited-state behaviour of the anionic
form of p-HBDI (a phenolate) might be correlated to that of
p-ABDI (an aniline), because the degree of photoinduced
intramolecular charge transfer (ICT) might be similar, as
reflected by the same pKa changes (pKa = 6) on going from
S0 to S1 for 2-naphthol (with a phenolate-like conjugate base)
and the conjugated acid of 2-aminonaphthalene.11 This corre-
lation might resolve the solubility problem upon measuring Ff
and FZE for the anionic form of p-HBDI in nonpolar solvents.
Second, a reliable measurement of FZE requires a stable
E isomer of p-HBDI either in the neutral or anionic forms.
However, it has been reported that p-HBDI undergoes efficient
thermal E - Z isomerization in D2O and CD3OD at room
temperature.12,13 Whereas Falk and coworkers have attributed
the facile thermal isomerization to a weak double-bond char-
acter of the exocyclic CQC bond due to significant delocaliza-
tion of the oxygen lone-pair electrons,13 an alternative
interpretation is the presence of an acidic phenol group (the
pKa value for phenol is 10.0 in H2O and 18.0 in DMSO),14
which self-catalyzes the isomerization reaction. Along this
line, the thermal isomerization would become even more
efficient upon irradiation because of the enhanced acidity of
p-HBDI in S1.8c Since the ABDIs are free of acidic groups (the
The currently discussed nonradiative decay mechanisms for
p-HBDI in fluid solutions are all associated with its torsional
motions, including (a) the torsion of the exocyclic C–C bond
(j) (see Chart 1) that leads to the formation of a twisted
intramolecular charge transfer (TICT) state, and either (b) the
CQC bond torsion (t) or (c) a concerted torsion of both the
C–C and CQC bonds (i.e., the hula twist) that leads to Z - E
photoisomerization. However, none of these mechanisms can
satisfactorily account for all the key experimental and compu-
tational results. For example, the CQC bond torsion3,4 con-
flicts with the volume-conserving nature of the decay process
and with the fast ground-state recovery of p-HBDI in alcohols
(i.e., little E-isomer formation).2 In the case of the C–C bond
Department of Chemistry, National Taiwan University, Taipei,
Taiwan 10617. E-mail: jsyang@ntu.edu.tw
w Electronic Supplementary Information (ESI) available: Experimen-
tal methods, synthetic details, and spectroscopic data for p-ABDI and
m-ABDI; crystal structure data for m-ABDI (CCDC number 667977).
Chart 1
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sum of Ff + Fd (= Ff + 2FZE) is close to 1.0 (0.90–1.08)
within experimental error for both p-ABDI and m-ABDI. We
can thus conclude that in aprotic solvents the nonradiative
decay channel is mainly the CQC bond torsion. The absence
of solvent-polarity dependence of the Ff + 2FZE values and
the observation of an increase of Ff at the expense of FZE on
going from p-ABDI to m-ABDI support the assumption that
Fd = 2FZE and the conclusion. It should also be noted that
the much larger Ff values for m-ABDI vs. p-ABDI in aprotic
solvents resembles the case of the m- vs. p-aminostilbene.19
Evidently, there exists a close photochemical correlation be-
tween the GFP chromophore and aminostilbenes. To our
knowledge, the fluorescence quantum yield for m-ABDI in
hexane is the largest among the various derivatives of the GFP
chromophore.7,20
In contrast, the CQC bond torsion no longer dominates
the nonradiative decay of ABDIs in protic solvents on the
basis of the fact that the value of Ff + 2FZE is much lower
than 1.0 (Table 1) and solvent-dependent. Although the FZE
values in pure water were unavailable due to the solubility
problem, low values of FZE for both ABDIs in pure water can
be readily predicted on the basis of the dramatic reduction of
Fig. 1 The 1H NMR spectra for (A) p-HBDI and (B) p-ABDI (B2
mM) in DMSO-d6 and CD3OD (a) before and (b) after 20 min of
irradiation with 350 nm light and (c) after 24 h at room temperature in
the dark. The E/Z ratio is the same for the conditions (b) and (c) for all
cases except for p-HBDI in CD3OD, where the Z form completely
isomerized back to the E form.
F
ZE on going from pure THF to that containing only 10–20%
pKa value for aniline is 30.7 in DMSO),15 complication of the
thermal E - Z isomerization for the determined FZE would be
reduced. Indeed, as shown in Fig. 1, the E - Z thermal
isomerization of p-HBDI is negligible in DMSO-d6 but effi-
cient in CD3OD, consistent with the larger pKa value of
phenol in aprotic vs. protic solvents. In addition, the E isomers
of p-ABDI and m-ABDI are rather stable in both DMSO-d6
and CD3OD. Third, comparison of the amino-modified GFP
chromophores and aminostilbenes is inherently interesting,
because the issue of TICT formation vs. photoisomerization
has also been extensively discussed for the latter systems.16
Our recent studies on trans-aminostilbenes have shown that
the single-bond torsion that leads to the TICT state does not
couple with the trans - cis double-bond torsion.16 In other
words, the formation of a TICT state competes with the
trans - cis photoisomerization, leading to low photoisomeri-
zation quantum yields (Fts). Thus, the FZE values might allow
us to elucidate the excited-decay mechanism for ABDIs and
thus for p-HBDI, provided that the C–C and CQC bond
torsions in the latter are also decoupled.
water. The lower FZE values for m-ABDI vs. p-ABDI is
consistent with the slower photoisomerization process and
thus the lower ability to compete with the new nonradiative
decay channel. Regarding the new nonradiative decay channel,
the possibility of TICT state formation through the C–C bond
torsion can be excluded, because the marked difference in
acetonitrile vs. methanol could hardly be interpreted in terms
of the comparable solvent polarity.16 Furthermore, the related
chromophore p-CBMNI (see Chart 1) also displays a dramatic
decrease in Ff on going from aprotic dioxane (0.22) to protic
glycerol (0.023),7 but it cannot be a consequence of TICT
formation due to the lack of an explicit electron donor.
Therefore, hydrogen bonding-induced deactivation of the
excited ABDIs is proposed, since the phenomenon of H-
bond-mediated vibronic coupling to the ground state is known
to be an important radiationless decay channel for both inter-
and intramolecular H-bonded species.21,22 In particular, it has
been well demonstrated by studies of several arylethylenes that
the ultrafast Z - E photoisomerization can be completely
inhibited by intramolecular H-bonding.21
The ABDIs were synthesized using our modification of
Niwa’s procedure described in the ESIw.17 As is the case for
p-HBDI, the Z isomers of the ABDIs were obtained. One piece
of solid evidence is provided by the X-ray crystal structure of
m-ABDIw.
Table 1 Quantum yields for fluorescence (Ff) and Z - E photoi-
somerization (FZE) for p-HBDI, p-ABDI, and m-ABDI in protic and
aprotic solvents
p-HBDI
p-ABDI
m-ABDI
Both the values of Ff and FZE for p-HBDI, p-ABDI, and
m-ABDI in protic (MeOH and H2O–THF) and aprotic
(hexane, THF, and MeCN) solvents are shown in Table 1.
Since the calculated potential energy profiles for p-HBDI6a
resemble those for stilbenes, where the conical intersection
locates at a twisted angle of 901, it is reasonable to assume
that the probability of product formation for the E vs. Z
Ff
FZE
Ff
FZE
Ff
FZE
Solvent
Hex
THF
o10À3 0.53a
o10À3 0.46
o10À3 0.48a
o10À3 B0.1
o10À3 B0.2
o10À3 B0.1
o10À3 0.45a
o10À3 0.49
o10À3 0.50a
o10À3 0.17
o10À3 0.37
o10À3 0.28
0.34
0.28
0.37a
0.35
MeCN
MeOH
H2O–THFb
H2O–THFc
a
0.16 À3 0.45a
o10
0.08
o10À3 0.08
o10À3 0.06
isomer from such
a perpendicularly twisted geometry
is ca. 50%, in analogy to the case of stilbenes.18 As a result,
the quantum yield for the CQC bond torsion (Fd) can be
obtained as 2FZE. In all the investigated aprotic solvents, the
For the purpose of solubility, Hex and MeCN contain 20% THF for
b
c
H2O–THF = 1 : 9 (v/v). H2O–THF =
the measurement of FZE
.
2 : 8 (v/v).
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led to a new photodynamic picture for the GFP chromophore.
Further studies toward the identification of the deactivating
H-bonding modes for p-HBDI in protic solvents are in pro-
gress in our laboratory.
We thank the National Science Council and Ministry of
Education of Taiwan, ROC, for financial support.
Fig. 2 Simplified scheme for the radiationless decay channels for p-
HBDI and its amino analogues p-ABDI and m-ABDI in bulk protic
vs. aprotic solvents.
Notes and references
The photochemical behaviour of p-HBDI essentially paral-
lels that of p-ABDI (Table 1), although attempts to obtain
more accurate FZE values for p-HBDI in protic solvents are
hampered by the unstable E isomer in protic solvents.12,13 The
similar behaviour of p-HBDI and p-ABDI is reminiscent of the
common photodynamic scheme for the neutral and anionic
forms of p-HBDI.1,2 Thus, the correlation between p-ABDI
and the anionic p-HBDI appears to be appropriate.
1 (a) R. Y. Tsien, Annu. Rev. Biochem., 1998, 67, 509; (b) M.
Zimmer, Chem. Rev., 2002, 102, 759; (c) M. Zimmer, Cis–trans
Isomerization in Biochemistry, ed. C. Dugave, Wiley-VCH, Wein-
heim, 2006, pp. 77.
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Accordingly, we might conclude that p-HBDI simulta-
neously possesses two different ultrafast raditionless decay
channels (Fig. 2). Whereas the decay in protic solvents in-
volves both the solute–solvent H-bond-mediated internal con-
version (major) and CQC bond torsion (minor), the CQC
bond torsion alone accounts for the decay in aprotic solvents.
It is important to note that H-bonding-induced deactivation is
consistent with the volume-conserving photodynamic scheme
observed for p-HBDI in alcohols.2 In addition, the deactivat-
ing H-bonded intermediate appears to be different from the
Franck–Condon excited state, and its formation requires the
reorganization of the solvent H-bonding network and vibra-
tional relaxations of the excited p-HBDI in order to account
for the previously proposed two-state two-mode model.2c
Although the exact deactivating H-bonding modes remain to
be determined, the most possible candidates are the H-bonds
between the solvent molecules (H-bond donor) and the imi-
dazolinone group (H-bond acceptor) of p-HBDI. One reason
is that the imidazolinone group becomes a better H-bond
acceptor in the lowest excited state (S1) than in the ground
state (S0) as a result of ICT. Another reason is that the imino
nitrogen of the imidazolinone group is not H-bonded to any
water molecules or neighbouring residues inside the protein b-
barrel.1 It appears that the protein matrix not only suppresses
the CQC bond torsion3,10 but also prevents the formation of
the specific H-bonding modes in the excited state, which
accounts for the high fluorescence quantum yield of GFP. It
should be pointed out that a general H-bonding effect in the
excited-state decay of p-HBDI and its derivatives has been
noted.2,7 However, the argument of the presence of certain
H-bonding modes that dominate the nonradiative decays of
p-HBDI in protic solvents had not been recognized. Meech
et al. had considered solvent–chromophore H-bonding as a
possible nonradiative mechanism for p-HBDI, but it was finally
ruled out in view of the fact that this channel alone cannot
interpret their data in acetonitrile (aprotic) vs. methanol (protic).2c
In summary, we have provided for the first time the Z - E
photoisomerization quantum yields for p-HBDI and its amino
analogues p-ABDI and m-ABDI in both aprotic and protic
solvents. In addition, m-ABDI displays record-high fluores-
cence in aprotic solvents at room temperature. The solvent-
dependent fluorescence and photoisomerization behaviour has
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