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Deeply Inverted Electron-Hole Recombination in a Luminescent Antibody-Stilbene Complex

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Science  29 Feb 2008:
Vol. 319, Issue 5867, pp. 1232-1235
DOI: 10.1126/science.1153445

Abstract

The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedented ability to produce bright luminescence and has been used as a biosensor in various applications. We show that the prolonged luminescence is not stilbene fluorescence. Instead, the emissive species is a charge-transfer excited complex of an anionic stilbene and a cationic, parallel π-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody.

An excited-state complex (exciplex) formed by the interaction of an electronically excited molecule with a ground-state partner features charge transfer to a various extent and typically exhibits structureless emission that is red-shifted from the emissive features of its individual components (1, 2). Among the rare examples of exciplex-like behavior in proteins (3) is the conjugate of monoclonal antibody EP2-19G2 with the trans-stilbene hapten 1 (Scheme 1), which emits intense blue light upon ultraviolet (UV) excitation (movie S1) (4).

Fig. 1.

(A) Steady-state absorption spectra of 1 (10 μM) and antibody-1 complexes (10 μM) in PBS containing 3% DMF at room temperature. (B) Steady-state emission spectra of 1 (20 nM) and antibody-1 complexes (20 nM) in PBS containing 3% DMF at room temperature. Antibody was used in large excess to ensure that the dye was completely bound by protein. (C) Time-resolved emission decay profiles of antibody-1 complexes (3 μM) obtained with picosecond excitation at 303 nm. Decays were measured by time-correlated single-photon counting. Decays were recorded in 4096 channels with a time increment of 22 ps/channel and were normalized relative to the number of counts recorded in the peak channel.

Fig. 2.

Electrostatic and shape complementarity of the hapten 1 in (A) the EP2-25C10 and (B) the EP2-19G2 antibody-combining site. Slices through the center of the binding sites are shown. The heavy and light chains are colored in blue and green, respectively. The electrostatic potential was calculated in APBS (30) and mapped onto the surface with the color code ranging from –30 kT/e (bright red) to +30 kT/e (dark blue). Both binding pockets are highly apolar, but strongly differ in their depth and penetration of the variable antibody domain. (C) Crystal structure of purple-fluorescent antibody EP2-25C10 in complex with 1 (yellow). The 2FoFc electron density map around hapten 1 is contoured at 1.5σ. (D) Crystal structure of the blue-emissive antibody EP2-19G2 in complex with 1 (yellow) (4). TrpH103 undergoes parallel π-stacking with 1 and forms a charge-transfer complex in the excited state. In contrast, stilbene 1 in EP2-25C10 does not engage in any π-stacking interactions with tryptophan.

Fig. 3.

(A) Superimposition of the EP2-19G2 variable chains (VH + VL domains) (red) onto other 20 catalytic antibodies (gray) based on framework regions. Only the VH domains are shown. TrpH103 of EP2-19G2 is represented as a red stick. The β strands of one β sheet are labeled according to convention and clearly deviate from the corresponding strands in other antibodies. (B) The highly conserved TrpH103 features an unusual rotamer in antibody EP2-19G2 with respect to the indole side chains in the other antibodies. (C) Key residues at the VH/VL interface of EP2-19G2 (blue and green) and of a representative canonical antibody (in this case, Diels-Alder catalytic antibody 13G5, PDB ID code 1A3L, gray). Considerable structural displacements and reorientations occur over the entire interface. For clarity, some regions of EP2-19G2, as well as the backbone of 13G5, were omitted and not all conserved contacts, such as the bidentate hydrogen bond between GlnH39 and GlnL38, are displayed. The CDRs H3 and L3 are labeled.

Fig. 4.

Ground- and excited-state potential surfaces for EP2-19G2-1. After UV excitation of 1 (step 1), the stilbene excited singlet oxidizes TrpH103 to produce the charge-transfer complex (step 2); the interplanar distance Q between the cation radical TrpH103 and the stilbene anion 1 is shorter than in the ground state, owing to strong electrostatic binding in the hydrophobic protein cavity. In step 3, the excited charge-transfer complex returns to the ground state via electron-hole recombination that generates bright blue light in the rigid site. Radiative decay in the purple-fluorescent antibody EP2-25C10 occurs from the stilbene singlet excited state.

Scheme 1.

In striking contrast to this highly luminescent complex, electronically excited trans-stilbene is only weakly fluorescent in solution, owing to efficient nonradiative decay via cis-trans isomerization (5). Unlike other antibody-stilbene complexes (4, 6), the radiative lifetime of EP2-19G2-1 is increased by more than two orders of magnitude with respect to free 1, which substantially exceeds those of stilbene exciplexes formed with small organic molecules (7). From extensive examination of the structures and photophysical properties of several antibody-1 conjugates, we have concluded that the bright blue-emissive species is a tryptophan:stilbene charge-transfer excited complex that undergoes deeply inverted electron-hole recombination in a rigid protein matrix.

Guided by the crystal structure of EP2-19G2-1 (4), we identified seven antibody residues in van der Waals' contact with the stilbene aromatic system. These seven residues were then conservatively mutated and the corresponding proteins were expressed as single-chain variable antibody fragments (scFv) (8, 9). Spectroscopic measurements indicated that mutation of TrpH103 to Phe (TrpH103Phe) and TyrL34 to Phe (TyrL34Phe) markedly reduced antibody-1 emissions when compared to scFv wild-type (wt)-1 [Fig. 1B and fig. S1 (9)]. As observed in the crystal structure of the EP2-19G2-1 complex, the indole ring of TrpH103 is π-stacked parallel to the deeply buried phenyl ring of the stilbene ligand at an interplanar distance of 3.5 Å, whereas the TyrL34 phenoxyl group is roughly perpendicular to the stilbene molecule, pointing toward its central double bond. Thus, we focused on these two residues, with the wild-type and TyrL36Phe mutant scFv fragments acting as controls. To exclude the possibility that the much weaker emission is the result of vastly diminished binding, we confirmed that mutant scFv constructs still bind tightly to hapten 1 (Table 1) (9).

Table 1.

Spectral data and affinities of antibody-1 complexes and hapten 1. All measurements were made in phosphate-buffered saline (PBS) [10 mM sodium phosphate, 150 mM NaCl (pH 7.4)] and 3% dimethyl formamide (DMF) cosolvent, at 20°C (9). Quinine bisulfate in 0.5 M H2SO4 was used as a quantum yield reference with Φf = 0.546 (29). The anisotropy r is defined as r = (I-I)/(I + 2I), where I and I are the emission intensities measured parallel and perpendicular to the vertical excitation polarization plane. The total emission intensity after pulsed excitation was fit by the multi-exponential function I(t) = SΣi αiexp(-ti), convoluted with the instrument response function, where S, αi, and τi are the overall scaling factor, decay amplitude, and decay time of component i (9). The long component 1 of the emission decay (>20 ns) is essentially abolished (amplitude α1 of less than 0.1%) in the TrpH103Phe mutant, and the relatively high anisotropy of free 1 reflects the limited depolarization that can occur during the very short lifetime of the excited state. λabs and λem, wavelength of absorption and emission maxima, respectively; Kd, dissociation constant; NA, not applicable.

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The absorption spectra of TrpH103Phe-1 and TyrL36Phe-1 closely resemble that of wt-1, featuring well-resolved, vibronic features (Fig. 1A). By contrast, the corresponding absorption system of TyrL34Phe-1 is less structured and similar to that of free 1 in solution. The highly structured absorption system suggests that stilbene is in a constrained environment within the binding pockets of the TrpH103Phe and TyrL36Phe mutants, whereas it appears to be less restricted in the TyrL34Phe protein, consistent with its decreased affinity and fluorescence anisotropy (Table 1).

The TyrL34Phe-1 complex exhibits less intense, red-shifted emission compared with that of wt-1 (Fig. 1B). By contrast, the emission spectrum of TrpH103Phe-1 differs markedly from that of wt-1, with a large blue-shift (λmax at 366 nm) and even lower intensity (0.12 quantum yield) than observed for the TyrL34Phe-1 (0.23), with a vibronically structured shoulder on the low-energy side. For comparison, both wt-1 and TyrL36Phe-1 emit much more intensely, with quantum yields of 0.57 and 0.55, respectively (Table 1). Notably, the scFv wt-1 emission quantum yield is lower than that of Fab (fragment antigen binding) wt-1 (0.71) and is most likely attributable to a less rigid binding pocket in the single-chain construct, an interpretation that also is consistent with its lower affinity for the hapten (Table 1).

Our steady-state emission measurements show clearly that the TrpH103 mutation greatly enhances nonradiative decay of electronically excited EP2-19G2-1. By using picosecond, time-resolved emission spectroscopy, we found that the long component (>20 ns) of the multi-exponential luminescence decay is essentially abolished (amplitude <0.1%) in TrpH103Phe-1 (Fig. 1C and table S1); this component, which is a major feature in the radiative decay of both wt-1 (17%) and TyrL36Phe-1 (27%), is also reduced substantially in TyrL34Phe-1 (2%) (Table 1). Because of the presence of a long decay time, greatly exceeding the radiative lifetime of 1 itself, we suggest that a Trp:stilbene charge-transfer excited complex is responsible for the slow emissive decay component.

TrpH103 is a highly conserved framework residue of the immunoglobulin fold and, owing to its deep burial within the variable domain, rarely interacts with antigens. However, the TrpH103 indole closely associates with the “distal” phenyl ring of 1 in the EP2-19G2-1 complex. If this Trp:stilbene constellation were, indeed, unique to the blue-luminescent antibody EP2-19G2-1, we would expect a different ligand interaction with TrpH103 in purple- and blue-purple–fluorescent antibodies obtained from the same immunizations with hapten 1 (4).

To test this hypothesis, we determined the crystal structure of the purple-fluorescent complex of Fab EP2-25C10 with 1 to 2.5 Å resolution (table S2). EP2-25C10-1 has an emission maximum at 380 nm with a quantum yield of 0.27 and an average lifetime of 4.9 ns (4). The high quantum yield with respect to free stilbene most likely means that excited-state cis-trans isomerization is disfavored in the complex because the crystal structure shows that the stilbene molecule is bound in a cavity of high shape complementarity (Fig. 2A).

The binding pocket of purple-fluorescent EP2-25C10 (Fig. 2A) is as highly hydrophobic as that of blue-luminescent EP2-19G2 (Fig. 2B), suggesting that polarity effects do not account for the 30-nm blue shift in its emission maximum. However, the overall position of the stilbene ligand within the variable part of the antibody is appreciably different in these two antibodies; in EP2-25C10, the stilbene is translated by 6 Å toward the protein surface and does not penetrate the antibody-combining site as deeply as in the EP2-19G2 complex (Fig. 2, A and B).

Consequently, the deep-seated TrpH103 interacts minimally with stilbene in the purple-fluorescent antibody, consistent with the relatively high emission energy. Furthermore, no EP2-25C10 tryptophan residue is involved in face-to-face or face-to-edge π-stacking interactions with stilbene (Fig. 2C). The crystal structure of the green-fluorescent antibody 11G10 in complex with a donor-acceptor–substituted stilbene reveals a binding mode similar to that in EP2-25C10 (6), providing strong evidence that the parallel π-stacking interaction of 1 with TrpH103 is attributable to the unusually deep burial of stilbene in the EP2-19G2 binding cavity.

Further structural analysis revealed that the side-chain conformation of TrpH103 is unusual in EP2-19G2, whereas TrpH103 of EP2-25C10 corresponds to the canonical rotamer that is prevalent in most antibody structures at this position (Fig. 3B). However, superimposition of the variable domains of EP2-19G2, based on the conserved framework regions, with more than 20 other antibodies, including EP2-25C10 and 11G10, yielded large root-mean-square deviations (greater than 2.0 Å) (Fig. 3A) and revealed a relatively rare disposition of heavy- and light-chain variable (VH and VL) domains with respect to each other in this antibody (Fig. 3C). In contrast, the individual EP2-19G2 VH and VL domains superimpose well onto their counterparts in other antibodies.

The geometry of the VH/VL interface in antibodies is generally highly conserved through invariance of ∼15 side chains (10, 11). Examination of the VH/VL interface of EP2-19G2 uncovered several notable deviations from the canonical interface and revealed factors that may synergistically contribute to the unusual relative configuration of VH and VL. Large deviations in the backbone conformations of the complementarity determining regions (CDRs) H3 and L3 are introduced by ProH101 and ProL96, respectively (Fig. 3C). ProL96 leads to a considerable displacement of conserved framework residue TrpH47 which, in turn, propagates this perturbation to neighboring residues and strands in VH (Fig. 3C). The base of VH and VL also undergoes substantial rearrangements that include SerL43 and GlnH105. The latter residue now points toward VL, unlike that in all other superimposed antibody structures. Bulky side chains at the base of CDR L1 and at the beginning of the subsequent framework region (e.g., TyrL34 and TyrL36) also rearrange considerably. Thus, substantial variations at the conserved VH/VL interface provide a structural framework for the unusual mode of ligand recognition in EP2-19G2 that is responsible for the Trp:stilbene photophysics in the antibody-1 complex.

Why, then, is the luminescence of EP2-19G2-1 so intense? Consider steps 1 to 3 in the following scheme (see also Fig. 4), where TrpH represents the TrpH103 indole side chain carrying the N-H ring proton and hv is the photon energy:

  1. [1/TrpH] + hν → [1*/TrpH]

  2. [1*/TrpH] → [1⚫–/TrpH⚫+]*

  3. [1⚫–/TrpH⚫+]* → [1/TrpH] + hν′

In the first step, 1 absorbs a photon upon ultraviolet (UV) illumination. Because the singlet excited state of stilbene is a strong electron acceptor (1214), an electron is transferred very rapidly from TrpH103 to 1* to form a charge-transfer excited complex (step 2). Conversion of singlet-excited stilbene to the charge-transfer state does not occur at low temperatures, as only stilbene fluorescence is seen below 240 K (4). Owing to their close parallel interaction, the two aromatic rings in this charge-transfer state are tightly bound, which greatly disfavors nonradiative decay, because coupling of this deep and narrow excited well to the ground-state potential energy surface would be very weak (Fig. 4). As a result, radiationless deactivation would be expected to be much slower than radiative decay, hence giving rise to the exceptionally bright blue emission (step 3). Because the driving force for charge recombination is much greater than a reasonable estimate of the reorganization energy, and its rate is orders of magnitude lower than predicted for coupling-limited electron tunneling over a short distance in a folded polypeptide, we conclude that the intense luminescence is attributable to deeply inverted electron transfer (15, 16). In view of this light-generating mechanism, the “blue-fluorescent antibody” EP2-19G2 should really be called a “blue-emissive” or “blue-luminescent” antibody.

Because roughly 3 eV of photon energy is stored in the charge-transfer excited state, it is predicted to be both a powerful reductant and oxidant. We examined the redox activity of the charge-transfer state in experiments in which irradiation of EP2-19G2-1 was followed by flash-freezing, yielding a weak electron paramagnetic resonance signal that is attributable to a neutral tyrosyl radical having a small dihedral angle [fig. S2 (9)] (17). We suggest that a relatively small population of charge-transfer states decays by electron transfer from a tyrosine to the tryptophan radical cation, a proposal that is supported by our finding that the addition of an electron acceptor, namely [Co(NH3)5Cl]2+, greatly enhances the radical signal (17). It is likely that the stilbene anion radical in the charge-transfer state would be oxidized rapidly by Co(III), leaving the Trp cation radical without its electron-transfer partner. The flash-quench–generated [1/TrpH⚫+] cation would then have time to oxidize any nearby protein residue, and our experiments show that tyrosine is the main electron donor.

Charge separation and recombination between a chromophore and tryptophan or tyrosine have been investigated previously in other systems (1821). Very efficient fluorescence quenching is observed in most cases. Notably, the loss of fluorescence is due to very rapid charge recombination following femtosecond electron transfer between riboflavin and a parallel, π-stacked tryptophan after electronic excitation of the riboflavin-binding protein (18). Similarly, the strong fluorescence of fluorescein is quenched upon binding to antibody 4-4-20 via electron transfer from a parallel, π-stacked tyrosine in the antibody-combining site (19, 20); further, the fluorescence of an anticalin-fluorescein complex is efficiently quenched by rapid electron transfer from either a coplanar tryptophan or tyrosine to singlet excited fluorescein (21). We conclude that the very bright blue luminescence of EP2-19G2-1 is attributable to electron-hole recombination of the Trp:stilbene charge-transfer excited state held in the rigid EP2-19G2 matrix that disfavors nonradiative decay.

Protein luminescence (22) only rarely (if ever) occurs by electron-hole recombination in a charge-transfer excited state embedded in a polypeptide matrix. The distinctive photophysical properties of the antibody-stilbene complex have already been exploited in chiral sensing for high-throughput screening for the evaluation of catalysts in asymmetric synthesis (23, 24), sensing mercury (25), DNA hybridization assays (26, 27), and for analysis of accessible cysteine residues on viral surfaces (28). The programmed generation of antibodies against other chromophores may yield novel protein-ligand systems with similar charge recombination-induced luminescence phenomena and further biosensor applications.

Supporting Online Material

www.sciencemag.org/cgi/content/full/319/5867/1232/DC1

Materials and Methods

Figs. S1 to S3

Tables S1 and S2

References

Movie S1

References and Notes

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