The effects of antibodies on stilbene excited-state energetics

Article abstract of DOI:10.1002/anie.200602720

(Figure Presented) You light up my life! Antibodies, when bound to a donor-acceptor-substituted stilbene, strongly fluoresce with colors ranging from blue to green. Photophysical and structural analyses indicate that antibody binding alters the excited-st

Full text of DOI:10.1002/anie.200602720

Angewandte
Chemie
Antibody–Stilbene Complexes
DOI: 10.1002/anie.200602720
The Effects of Antibodies on Stilbene Excited-
State Energetics**
Feng Tian, Erik W. Debler, David P. Millar,
Ashok A. Deniz, Ian A. Wilson,* and Peter G. Schultz*
Antibodies can be used to control the energetics and reactions
of molecules in their ground states as well as in their
electronically excited states.[1] For example, antibodies have
been generated that catalyze the light-dependent cleavage of
thymine dimers, and that modulate the excited-state behavior
of trans-stilbene.[2,3] In particular, Lerner and co-workers
generated a blue-fluorescent antibody, 19G2, which binds
trans-stilbene to give a strongly fluorescent complex. Detailed
structural and photophysical studies revealed that the anti-
body stabilizes the planar excited-state configuration through
the formation of an exciplex between TrpH103 of the antibody
and stilbene. To further explore the effects of the well-defined
environments afforded by antibody binding sites on excited-
state energy surfaces, we have generated antibodies to the
donor–acceptor-substituted stilbene trans-4-N,N-dimethyl-
amino-4’-cyanostilbene (DCS).
=
about the C C bond and undergoes internal conversion to
give a mixture of trans- and cis-stilbene[4]), the photophysics
of donor–acceptor-substituted stilbenes are more compli-
cated.[5] A number of experimental and theoretical results
have been interpreted in terms of an additional excited state,
1a*, which corresponds to a relatively low-lying, twisted
intramolecular charge-transfer (TICT) state in which the
[6–8]
À
styryl-anilino C C bond is rotated (Scheme 1).
Others
have questioned the necessity of a TICT state and have
suggested that the photophysics of substituted DCS analogues
can be explained with a two-state model.[9–11]
Monoclonal antibodies (Mabs) provide a unique oppor-
tunity to study the photophysics of DCS in distinct steric and
electronic enviroments without introducing any substituents.
To this end, antibodies were generated against the DCS
derivative 2, as well as trans-4-N,N-dimethylamino-4’-nitro-
stilbene (DNS) derivative 1, an analogue with a larger dipole
moment (7.42D) [13] which might more closely approximate
Unlike the widely accepted two-state model of photo-
physical unsubstituted trans-stilbene (Scheme 1), which con-
sists of an initial emissive 1t* state and a subsequently
populated nonemissive 1p* state (which is twisted by 908
[*] E. W. Debler,[+] Prof. I. A. Wilson
Department of Molecular Biology
and The Skaggs Institute for ChemicalBiool gy
The Scripps Research Institute
10550 North Torrey Pines Road, BCC206
La Jolla, CA 92037 (USA)
Fax: (+1)858-784-2980
E-mail: wilson@scripps.edu
Dr. F. Tian,[+] Prof. P. G. Schultz
Department of Chemistry
and The Skaggs Institute for ChemicalBiool gy
The Scripps Research Institute
10550 North Torrey Pines Road, SR202
La Jolla, CA 92037 (USA)
Fax: (+1)858-784-9440
E-mail: Schultz@scripps.edu
Prof. D. P. Millar, Prof. A. A. Deniz
Department of Molecular Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Scheme 1. Excited-state photochemistry of free 2 (black) and hypothe-
sized primary pathway of the antibody–2 complexes (green). Dashed
lines: nonradiative transition; solid lines: radiative transitions.
FC=Franck–Condon excited state.
[+] These authors contributed equally to this work.
[**] We thank Sharon Ferguson for expert assistance in Fab preparation
and Dr. M. L. Tsao for his help in manuscript preparation. We
acknowledge support from the NIH (grant GM56528 to P.G.S. and
grant GM38273 to I.A.W.), The Skaggs Institute (predoctoral
fellowship for E.W.D.), and the TSRI graduate program (Jairo H.
ArØvalo fellowship for E.W.D.).
the polar excited state of DCS. These antibody–stilbene
complexes are highly fluorescent and emit blue to green light.
Mabs 16E3, 17E3, and 17A4 elicited against hapten 2 and
Mab 11G10 raised against the more polar hapten 1 were
spectroscopically characterized (Table 1). The absorption
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Table 1: Steady-state spectraldata and affinities for haptens 1 and 2,
antibody–2 complexes, and the 11G10–1 complex.
antibody–2 complexes are two to four times higher than that
of free 2; the rate constants for nonradiative decay, knr,
obtained from time-resolved measurements decrease corre-
spondingly (Table 2). All of the antibody–2 complexes have
large blue-shifts (59–99 nm) with respect to free 2 (Figure 2)
and emit light of distinct colors ranging from blue for the
17E3–2 complex (475 nm) to green for the 11G10–2 complex
(515 nm).
Hapten and labs
complex
lem
emax ”10À3 Stokes shift Ff
Kd
[nm]
[nm] [nm] [mÀ1 cmÀ1
]
[cmÀ1
]
1
430
532
373
424
410
412
396
[a]
7.30
9.45
14.1
23.0
16.4
14.4
14.6
[a]
0
[a]
11G10–1
2
11G10–2
16E3–2
17A4–2
17E3–2
684
574
515
490
505
475
4177
9388
4168
3982
4470
4200
0.002 13
0.16
0.32
0.60
0.64
0.57
[a]
23
30
50
75
Table 2: Results of time-resolved fluorescence spectroscopy of 2 and
antibody–2 complexes.
[a] Not applicable.
Hapten and
complex
tf
tr =tf/Ff
[ns]
kr =1/tr
knr =1/tfÀkr
[ns]
[nsÀ1
]
[nsÀ1
]
spectra of free 2 and antibody–2 complexes display no
substructure (Figure 1). While free 2 absorbs with a lmax of
373 nm, the absorption maxima of antibodies 16E3, 17E3, and
2
1.01
2.68
2.78
2.88
2.53
6.3
8.4
4.6
4.5
4.4
0.16
0.12
0.22
0.22
0.23
0.83
0.25
0.14
0.13
0.17
11G10–2
16E3–2
17A4–2
17E3–2
Figure 1. Steady-state absorption spectra of 2 (10 mm) and antibody–2
complexes (10 mm) in phosphate-buffered saline (PBS) containing 5%
DMF at room temperature.
Figure 2. Steady-state fluorescence spectra of 2 (10 nm) and antibody–
2 complexes (10 nm) in PBS containing 5% DMF at room temper-
ature. The labs values listed in Table 1 were used as lex for each
corresponding spectrum.
17A4 complexed with 2 are red-shifted by 23–40 nm. When 2
is bound to antibody 11G10, the absorption maximum is red-
shifted by 51 nm. This shift is consistent with the notion that
11G10, which was generated to the more polar DNS analogue
1, is more complementary to the highly polar Franck–Condon
(FC) excited state of DCS. The significant red-shifts of all
antibody–stilbene complexes suggest that preorganized anti-
body combining sites are more effective in stabilizing the FC
excited states of 1 and 2 than aqueous bulk solvent, which
cannot reorganize to solvate the more polar excited state on
the timescale of light absorption.
The steady-state fluorescence spectra of 2 and antibody–2
complexes are also broad and devoid of any substructure. In
contrast to the situation in bulk solution, in which the band
widths at half-maximum increase with solvent polarity,[9] band
widths at half-maximum for the antibody–2 complexes are
narrower than those of free 2, suggesting that some rotatory
conformations of 2 are constrained in the antibody combining
sites. As expected for a stilbene molecule with an increased
The overall blue-shift in fluorescence of the antibody–2
complexes relative to 2 in water can be ascribed to the
preorganized nature of antibody combining sites. Bulk
solvent has sufficient time to reorganize to preferentially
1
stabilize the polar excited t* state of 2 relative to the less
polar ground state prior to radiative decay. On the other hand,
although the antibody binding site is more complementary to
the polar excited state of DCS than to the ground state, it
cannot reorganize to the same degree as bulk water, resulting
in the large blue-shifts in fluorescence of the antibody–2
complexes. Alternatively, if we assume that free 2 relaxes to
an emissive low-lying TICT state (1a*), destabilization of the
transition state by the antibody would lead to a switch to 1t* as
the emissive state of the antibody–2 complexes, which would
equally well explain the blue-shift in fluorescence. The
relative ordering of the antibodies with respect to the
increasing emission maxima is the same ordering as for
increasing absorption maxima (Table 1). This finding is
consistent with a similar underlying mechanism for the two
=
activation energy of rotation around the C C bond in the
excited state, the fluorescence quantum yields (Ff) of the
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events, that is, stabilization of the FC excited state (absorp-
tion) and the emitting state (fluorescence) by the antibodies
through electrostatic and van der Waals interactions.
To characterize the nature of an antibody–stilbene com-
plex and its effect on the photophysics of DCS in atomic
detail, we have determined the crystal structure of Fab 11G10
in complex with hapten 1 at 2.75-Š resolution (Figure 3, for
details see the Supporting Information). The stilbene moiety
is bound in a planar conformation. Rotation around the
green fluorescent complexes. Spectroscopic and structural
data suggest a model in which the relatively rigid protein
environment sterically increases the activation barriers con-
1
1
necting the planar t* to the twisted excited states p* (and
possibly a*) and modulates the energetics of the fluorescent
1
1t* state (Scheme 1). The antibodies appear to be more
effective in stabilizing the polar FC excited state of 2 relative
to the ground state, but less effective than bulk water in
selectively stabilizing the light-emitting state. In practical
terms, the relatively long excitation and emission wavelengths
of the antibody–2 complexes may make them useful for in
vitro and in vivo applications as fluorescent biosensors.
=
À
excited-state C C bond as well as the styryl-anilino C C bond
are likely to be restricted by interactions between the ligand
and antibody residues PheL94, LeuL89, LeuL36, TyrH33, TyrH95
,
and ValH93 (Figure 3c). The binding pocket possesses a
relatively high percentage of polar residues in immediate
vicinity to the ligand. The bottom of the cavity exhibits a
positive electrostatic potential (Figure 3b) due to the pos-
itively charged guanidinium group of ArgL46 (Figure 3a,c).
Towards the mouth of the cavity, the electrostatic potential
becomes partially negative. Importantly, the charge distribu-
tion in the combining site together with the aromatic ring
systems of TyrH33, TyrH95, and PheL94 are well suited to stabilize
charge separation in the excited state of DCS derivative 2.
In summary, we have generated antibodies that bind
donor–acceptor-substituted stilbenes to afford strong blue to
Received: July 8, 2006
Revised: September 2, 2006
Published online: November 7, 2006
Keywords: antibodies · fluorescent probes · stilbene
.
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Figure 3. Antibody combining site of 11G10 in complex with hapten 1
with electron density contoured at 1.5s. a) A slice through the center
of the antibody combining site is shown. The only hydrogen bond
between 1 and 11G10 is illustrated as a red dashed line. b) Represen-
tation of the electrostatic surface; similar view to that in (a). c) View of
the antibody binding pocket rotated horizontally by 908 to the view in
(a).
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