Lanthanide-Binding Tags with Unnatural Amino Acids: Sensitizing

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Lanthanide-Binding Tags with Unnatural Amino Acids: Sensitizing Tb3+ and Eu3+ Luminescence at Longer Wavelengths Anne M. Reynolds, Bianca R. Sculimbrene, and Barbara Imperiali* Departments of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Received November 19, 2007; Revised Manuscript Received February 1, 2008

Lanthanide-binding tags (LBTs) are small, genetically encoded, versatile protein fusion partners that selectively bind lanthanide ions with high affinity. The LBT motif features a strategically positioned tryptophan residue that sensitizes Tb3+ luminescence upon excitation at 280 nm. Herein, we describe the preparation of new LBT peptides that incorporate unnatural amino acids in place of tryptophan, and which sensitize both Tb3+ and Eu3+ luminescence at lower energies. We also report the semisynthesis of proteins tagged with these new LBTs using native chemical ligation. This expands the scope of LBTs and will enable their wider use in luminescence applications.

Understanding the localization and fate of proteins in ViVo is a key research objective in cell biology. Toward this end, fluorescence spectroscopy has become a widely used technique, with much effort devoted toward the development and incorporation of suitable fluorophores into biological molecules. Lanthanide ions represent attractive complements to the more commonly used organic fluorophores, as these species exhibit long-lived (millisecond) luminescence lifetimes, large Stokes shifts, unpolarized luminescence, and sharp emission peaks. Due to these advantages, the use of lanthanide-based probes is becoming more common in applications such as luminescence spectroscopy (1, 2) and luminescence resonance energy transfer (LRET) (3, 4). To overcome the inherently low absorption of lanthanide ions, researchers have developed sensitizing fluorophores that, upon excitation, transfer energy to the lanthanide (5, 6). However, few general methods to incorporate lanthanide-based probes into proteins exist, and these are generally limited to substitution of lanthanides at native calcium-binding sites (7–9), incorporation of unnatural amino acids with side-chain chelators (10), and covalent labeling of reactive side chains using lanthanide chelates (11, 12). Recent work in our laboratory has been focused on the development of lanthanide-binding tags (LBTs), which are short peptides (17–20 amino acids) that selectively and avidly bind lanthanide ions (13–16). Originally based on EF-hand Ca2+binding loops, LBTs have been optimized for Tb3+ binding through multiple rounds of split-and-pool peptide library synthesis (14, 16). In these motifs, the indole ring of a strategically positioned tryptophan residue sensitizes Tb3+ luminescence (17). These multitasking tags have been appended to various proteins and used to detect peptide–protein interactions using LRET (18). The paramagnetic lanthanides have also been used to aid in the determination of protein structure by NMR spectroscopy (19, 20), and the heavy atoms provide scattering power for phasing X-ray crystallographic data (21). While clearly advantageous for these in Vitro studies, the UV light necessary for tryptophan excitation (280 nm) is not ideal in cellular systems (22), and the ability to sensitize only Tb3+ limits their more general use in luminescence applications. Herein, we report the development of new LBTs that incorporate * To whom correspondence should be addressed. Tel: 1-617-2531838. Fax: 1-617-452-2419 . E-mail: [email protected].

Figure 1. Sequences of peptides LBT(Trp), LBT(cs124), and LBT(Acd), where the side chain of the amino acid denoted X is illustrated.

non-natural amino acids as sensitizers, which feature lowerenergy excitation and sensitize Eu3+ as well as Tb3+ luminescence. In these studies, we targeted two unnatural amino acids as potential lanthanide sensitizers in LBTs. The first is based on carbostyril 124 (cs124), which has been used as a sensitizer of both Tb3+ and Eu3+ luminescence in the context of polyaminocarboxylate chelates (23, 24). The second is based on the acridone (Acd) fluorophore, which has been previously used to sensitize Eu3+ luminescence in small molecules (25–28). These fluorophores were selected because they are known to sensitize lanthanide luminescence, making them attractive initial targets in the context of LBTs, and they are excited at lower energies (337 and 390 nm, respectively). Finally, the acridone fluorophore is highly photostable, making it particularly attractive for potential in ViVo applications. For our studies, both of the sensitizers were incorporated into amino acid building blocks for use in solid-phase peptide synthesis (SPPS). The cs124 moiety was coupled to the side chain of glutamic acid via the aniline nitrogen (29), while the acridone-containing amino acid was prepared via a literature procedure (30). Both of the amino acids were NR-Fmoc-protected and incorporated into LBT peptides at the optimal position for sensitizing lanthanide luminescence Via standard SPPS (Figure 1) (17). The parent LBT containing tryptophan was also prepared for comparison (16). Luminescence spectra of all three LBT peptides in the presence of lanthanide ions are illustrated in Figure 2. LBT(cs124) sensitizes Tb3+ luminescence upon excitation at 337 nm, and the intensity of the emission at 544 nm is higher than that of LBT(Trp) (Figure 2a). The presence of the new sensitizer does not significantly alter the Tb3+ affinity, as the dissociation constant (KD), determined by direct titration, is 34 nM (Table 1) compared to 19 nM for LBT(Trp) (16). LBT(cs124) also

10.1021/bc700426c CCC: $40.75  2008 American Chemical Society Published on Web 02/15/2008

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Figure 2. (a) Emission spectra of LBTs (50 nM) in the presence of Tb3+ (200 nM) with excitation at 280 nm for LBT(Trp) (---) and 337 nm for LBT(cs124) (—). (b) Emission spectra of LBTs (50 nM) in the presence of Eu3+ (200 nM) with excitation at 337 nm for LBT(cs124) (—) and 390 nm for LBT(Acd) ( · · · · ). (c) Titration of LBT(Acd) (50 nM) with Eu3+ (•), and fit derived from SPECFIT/32 (—). Table 1. Comparison of Ln3+ Binding Affinities of LBT Peptides and Tagged Proteinsa construct LBT(Trp) LBT(cs124) LBT(Acd) LBT(Trp)-Crk(SH2) LBT(cs124)-Crk(SH2) LBT(Acd)-Crk(SH2) b

KD(Tb3+)

KD(Eu3+)

b

19 nM 34 ( 3 nM 35 ( 5 nM 46 ( 12 nM

10 ( 2 nM 52 ( 2 nM 69 ( 4 nM 54 ( 17 nM

a Titrations performed at a peptide/protein concentration of 50 nM. From ref (16).

effectively sensitizes Eu3+ luminescence, and titration with Eu3+provides a dissociation constant of 10 nM. This slightly tighter binding of Eu3+ versus Tb3+was unexpected based on our previous observations that a similar LBT sequence binds lanthanides other than Tb3+ with lower affinity (15), though the difference is not large between these two lanthanide ions. The cs124-containing amino acid thus allows for the synthesis of new LBTs that sensitize both Tb3+ and Eu3+ luminescence. The only disadvantage of the cs124 sensitizer in this context is the sensitivity of the fluorophore to photobleaching. Under constant illumination, the luminescence intensities decrease 10–15% over 60 s, but remain nearly constant when O2 has been removed (Figure S1). Additionally, the initial luminescence intensities nearly double when deoxygenated buffers are used. We therefore used deoxygenated buffers in all experiments with this sensitizer. LBT(Acd) also sensitizes Eu3+ luminescence upon excitation at 390 nm, with a higher overall luminescence intensity than LBT(cs124) (Figure 2b). The spectrum was collected using a 570 nm long-pass filter to reduce interference from acridone fluorescence. The dissociation constant, determined by direct titration of LBT(Acd) with Eu3+, is 52 nM (Figure 2c), which is only slightly weaker than the other LBTs (Table 1). No luminescence was observed in the presence of other lanthanides including Nd3+, Tb3+, Er3+, Tm3+, and Yb3+. Finally, because the acridone moiety is extremely photostable (Figure S1), LBTs featuring this sensitizer such as LBT(Acd) will be extremely useful in applications where continuous irradiation is required; this characteristic was also reported in previous studies in which the acridone amino acid was used as a fluorophore (31). As is the case for other lanthanide-based probes, the luminescence intensities of these new LBTs are lower than typical organic fluorophores, with detection limits in the lownanomolar range in the case of the peptides sensitizing Eu3+ and the picomolar range for peptides sensitizing Tb3+. However, the long-lived lanthanide luminescence provides the ability to perform time-gated experiments that minimize background autofluorescence (1, 2), increasing the overall sensitivity.

With these promising results, we next targeted the assembly of full-length proteins tagged with the new LBTs. We chose the SH2 domain of the Crk adaptor protein as the test case, as this is a protein that has been previously tagged with LBTs for LRET experiments (18). While several methods exist for incorporating unnatural amino acids into proteins (32, 33), we chose to prepare the LBT-tagged proteins Via semisynthesis using native chemical ligation (NCL) (34). In this approach, one fragment bearing a C-terminal thioester is reacted with a second fragment containing an N-terminal cysteine residue. Reversible thioester exchange leads to an S-to-N acyl rearrangement to form a native amide bond. Using the NCL strategy, the desired LBT can be appended to the N-terminus of Crk(SH2), which requires the peptide to be prepared with a C-terminal thioester and the protein to be expressed with an N-terminal cysteine. Toward this end we synthesized LBTs containing either tryptophan or the unnatural amino acids at the sensitizing position on highly acid-labile TGT resin. This allows the completed peptides to be released from the resin using 0.5% TFA in CH2Cl2, while leaving the side-chain protecting groups intact (Figure S2). The free carboxyl termini were then coupled to benzyl mercaptan to generate the protected thioester peptides (35, 36). The side-chain protecting groups were removed under standard cleavage conditions to provide the LBTthioesters, which were purified by reverse-phase HPLC and confirmed by ESI-MS. Crk(SH2) was overexpressed in E. coli as a GST fusion protein construct with a tobacco etch virus (TEV) protease recognition site (ENLYFQC) (37), such that proteolytic cleavage affords the desired protein with an N-terminal Cys residue (Figure 3a). After purification on Glutathione Sepharose and subsequent TEV cleavage, the appropriate peptide thioester was added directly to the crude reaction mixture along with sodium 2-mercaptoethanesulfonate (MESNA, 150 mM) to facilitate rapid thioester exchange. From gel electrophoresis analysis, the ligations proceeded to 80–90% completion, and did not progress further after 16 h (Figure 3b). The excess thiol and peptide were removed by dialysis, while the GST and His6-tagged TEV were removed with Glutathione Sepharose and Ni-NTA agarose, respectively. The LBT-tagged proteins were isolated with >90% purity, with a small amount of unligated protein as the major byproduct. An advantage of the new sensitizers, particularly in the case of Acd, is that they can be used to visualize the tagged proteins. For example, visualization of an SDS-PAGE gel on a shortwave UV transilluminator (300 nm) reveals a very weak fluorescence signal from the tryptophan residues in LBT(Trp)-Crk(SH2), a stronger signal from the cs124 moiety in LBT(cs124)-Crk(SH2), and very intense fluorescence from the acridone moiety in

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Figure 3. (a) Semisynthesis of LBT-tagged Crk(SH2) proteins. (b) Coomassie-stained SDS-PAGE gel showing GST-TEV-Crk(SH2) (lane 1), products of TEV cleavage (lane 2), and products of NCL with peptide 3a (lane 3). Similar results were observed in the semisynthesis of 4b and 4c.

Figure 4. SDS-PAGE gel of semisynthetic LBT-tagged Crk(SH2) proteins, visualized on a transilluminator (300 nm, left), then stained with Coomassie Brilliant Blue (right). Lane 1 ) LBT(Trp)-Crk(SH2); Lane 2 ) LBT(cs124)-Crk(SH2); Lane 3 ) LBT(Acd)-Crk(SH2).

LBT(Acd)-Crk(SH2) (Figure 4). Staining the same gel with Coomassie Brilliant Blue reveals that all proteins are present in approximately the same quantities. The emission of the acridone amino acid was detected with as little as 4.5 µg of protein and is comparable to detection of protein with the Coomassie stain. This provides a simple method for monitoring the ligations using SDS-PAGE using standard laboratory equipment; the sensitivity could likely be significantly improved by using a high-tech imaging instrument. The luminescence properties of the new LBTs were quite similar in the context of the full proteins, where LBT(Trp)Crk(SH2) sensitizes Tb3+ luminescence, LBT(cs124)-Crk(SH2) sensitizes both Tb3+ and Eu3+ luminescence, and LBT(Acd)Crk(SH2) sensitizes only Eu3+ luminescence. The trends in overall luminescence intensities are the same, with cs124 being brighter than tryptophan for Tb3+ and acridone being brighter than cs124 for Eu3+ (Figure S3). Titrations revealed that the binding constants for the respective lanthanides are all in the low nanomolar range and are similar to those of the peptides (Table 1). An important feature of LBTs is that water molecules are completely excluded from the inner coordination sphere of the bound lanthanide (15). This is important for the luminescence properties, as bound water molecules lead to luminescence quenching via energy transfer to the O-H bonds. To ensure that the change of sensitizer and lanthanide has not affected this coordination, luminescence decay experiments were performed in varying ratios of H2O and D2O. The luminescence lifetimes obtained from these decay experiments were used to calculate the number of water molecules, q, bound to Eu3+ in the new LBT-proteins using a literature procedure (38, 39). The results provided q values of 0.006 for LBT(Acd)-Crk(SH2) and -0.06 for LBT(cs124)-Crk(SH2). (Uncertainties in q values are estimated to be (0.5, owing to errors in the measured luminescence decay rates and the calculated proportionality

constants (39).) These results provide further evidence that the coordination environment around Eu3+ is similar to that for Tb3+ in the optimized LBTs studied to date. In summary, we have developed LBTs featuring two new sensitizers that are excited at lower energy and sensitize luminescence of Tb3+ and Eu3+. Other advantageous photophysical properties of LBTs have not been affected, as these LBTs bind lanthanides tightly and exclude water molecules from their inner coordination spheres. We have established a general method for appending LBTs with lower-energy excitation to the N-terminus of a target protein using native chemical ligation and semisynthesis. This expands the utility of this class of lanthanide-based probes and current work is aimed at using them in ViVo.

ACKNOWLEDGMENT This research was supported by the NSF-CRC program (CHE0304832). A.M.R. and B.R.S. acknowledge the NIH for Ruth L. Kirschstein National Research Service Awards. We also thank Prof. Martin Schwarz (University of Virginia) for a generous gift of the clone expressing the Crk SH2 domain, and Emi Sei for initial synthetic contributions. Supporting Information Available: Figures S1-S3, plus detailed experimental procedures and characterization data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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