CHEMBIOCHEM COMMUNICATIONS DOI: 10.1002/cbic.201402204
Site-Specific Dual Labeling of Proteins by Using Small Orthogonal Tags at Neutral pH Jan Grnewald,[a] David H. Jones,[a] Ansgar Brock,[a] Hsien-Po Chiu,[a] Badry Bursulaya,[a] Kenneth Ng,[a] Todd Vo,[a] Paula Patterson,[a] Tetsuo Uno,[a] James Hunt,[b] Glen Spraggon,*[a] and Bernhard H. Geierstanger*[a] To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor FceRI, which is a key event in allergic disease.
Labeling of proteins with multiple fluorescent dyes has become a universal tool for monitoring structural changes within biopolymers by Fçrster resonance energy transfer (FRET).[1] This quantum phenomenon is based on the distancedependent energy transfer between donor and acceptor molecules. An important requirement for these types of observation is the site-specific introduction of a donor–acceptor pair into the protein of interest. One approach is based on fusing the target protein with a donor–acceptor pair of intrinsically fluorescent proteins.[2] However, genetic fusion typically restricts labeling to N- and C termini and can potentially impair protein folding, structure, and function. Furthermore, the relatively large size of such fusions (for example, 26.9 kDa for green fluorescent protein) negatively impacts the resolution of measurable distances within the target protein. Alternatively, labeling with spectrally diverse small-molecule fluorophores can be accomplished chemically or enzymatically. For instance, two-color fluorescence labeling has been accomplished by using sortases with distinct substrate specificities,[3] solid-phase expressed protein ligation,[4] and chemoselective modification of a cleaved intein fusion protein.[5]Although [a] Dr. J. Grnewald, Dr. D. H. Jones, Dr. A. Brock, Dr. H.-P. Chiu, Dr. B. Bursulaya, K. Ng, T. Vo, P. Patterson, Dr. T. Uno, Dr. G. Spraggon, Dr. B. H. Geierstanger Genomics Institute of the Novartis Research Foundation 10675 John-Jay-Hopkins Drive, San Diego, CA 92121 (USA) E-mail: [email protected] [email protected] [b] Dr. J. Hunt Novartis Institutes for Biomedical Research Wimblehurst Road, Horsham, West Sussex, RH12 5AB (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402204.
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these strategies minimize the potential impact of labeling on protein folding, the installation of a FRET pair involves multistep protocols and is still restricted to terminal protein regions. These limitations can be overcome by using thiol-reactive fluorescent probes for the modification of engineered cysteine residues.[6] However, site-specific labeling might require the deletion of surface-exposed native cysteine residues, thereby potentially interfering with the folding and stability of the target protein. Furthermore, the introduction of unpaired cysteine residues can potentially trigger protein aggregation, and the heterogeneity imposed by random labeling of two engineered cysteine residues with donor and acceptor dyes makes meaningful combinations of FRET pairs difficult. One way to eliminate this lack of selectivity is to use either a Cys mutant combined with a non-canonical amino acid (ncAA)[6b] or two ncAAs with bio-orthogonal reactivities (pazido-phenylalanine and 2-amino-8-oxononanoic acid) to incorporate two probes into the same protein. In the latter case, dual labeling with the appropriate dye molecules afforded homogeneous protein–fluorophore conjugates for FRET studies.[6c, 7] Although elegant, the approach is currently restricted to proteins that can be expressed in Escherichia coli, and the pH-dependence of the oxime ligation reaction of 2-amino-8oxononanoic acid greatly reduces the efficiency of labeling at pH values of 7 or higher. Here we present an alternative approach that enables site-specific dual labeling of both bacterially and mammalian-expressed proteins at neutral pH by utilizing the orthogonality between the chemical labeling of genetically encoded pyrroline-carboxy-lysine (Pcl), a demethylated analogue of pyrrolysine,[8] and the post-translational modification of a 12-mer S6 peptide sequence (GDSLSWLLRLLN).[9] We have previously demonstrated that the Pcl side chain can readily and specifically be conjugated to a wide range of 2-aminobenzaldehyde-activated molecules.[10] Analogously, phosphopantetheinyl transferases (PPTases) have been established as versatile tools for the site-specific labeling of proteins with structurally diverse molecules through the use of 11- or 12-mer peptidic recognition sequences such as the S6 peptide.[9, 11] Here, we show that both reactions can be performed simultaneously in an efficient one-pot process to incorporate a donor–acceptor pair site-specifically into a protein of interest at neutral pH. Notably, this bio-orthogonal dual-tag system is fully compatible with bacterial and mammalian expression systems, due to the ability of the pyrrolysyl-tRNA/tRNA synthetase pair to shuttle between both expression hosts.[12] To this end, we demonstrate the site-specific incorporation of FRET pairs ChemBioChem 2014, 15, 1787 – 1791
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CHEMBIOCHEM COMMUNICATIONS into E. coli-expressed Z domain of staphylococcal protein A and into HEK293-expressed human immunoglobulin E fragment crystallizable (IgE-Fc). In the latter proof-of-concept model, site-specific two-color labeling was used to monitor the binding of IgE-Fc to its high-affinity receptor FceRI. Because of its low molecular weight and ease of expression and purification, Z domain protein has been frequently used to demonstrate the in vivo incorporation of ncAAs in response to a non-sense codon (TAG) in E. coli.[6b, 13] Two Z domain mutants were constructed, containing a Pcl residue in place of a surface-exposed proline at position 23, as well as an S6 peptide sequence appended either to the N terminus (S6-Z-Pcl23) or to the C terminus (Pcl23-Z-S6). The dual-tagged proteins were expressed in E. coli BL21(DE3) cells co-transformed with two plasmids encoding the corresponding Z domain construct with a non-sense codon substitution at position 23, the wild-type
www.chembiochem.org pyrrolysyl-tRNA synthetase (PylS), its cognate tRNA (PylT), and the two biosynthetic enzymes PylC and PylD. The last two catalyze the synthesis of Pcl from the precursor compound d-ornithine,[8] which was added to Terrific Broth growth medium at a final concentration of 5 mm. Culturing in shake flasks and subsequent nickel nitrilotriacetate (Ni-NTA) chromatography afforded purified S6-Z-Pcl23 and Pcl23-Z-S6 in yields of 8 and 18 mg L 1, respectively. Next, we tested whether the orthogonal reactivity of the Pcl amino acid side chain and the S6 peptide allowed concurrent labeling of the proteins with different fluorophores for subsequent FRET experiments. In a one-pot reaction, 5 mm of dualtagged Z domain (S6-Z-Pcl23 or Pcl23-Z-S6) was treated with a mixture of 200 mm of 1 and 20 mm of 2 (Scheme 1) in the presence of 1.4 mm of Sfp PPTase. Incubation for 16 h at 37 8C and pH 7.5 resulted in almost complete formation of the de-
Scheme 1. Fluorogenic compounds used in this study. The fluorescent dyes Alexa Fluor 488 and Alexa Fluor 594 were chemically linked to 2-aminobenzaldehyde to provide compounds 1 and 4, respectively, and to coenzyme A to provide compounds 3 and 2, respectively).
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sired dual-labeled Z domain constructs, S6-2-Z-Pcl23-1 and Pcl23-1-Z-S6-2, as confirmed by ESI-MS analysis of the crude labeling mixtures prior to cyanoborohydride addition (Figure 1 A). No noticeable amount of uncoupled or mono-labeled Z domain was detectable, thus indicating that highly efficient conjugation of the proteins at neutral pH had been achieved. Furthermore, no peaks originating from double labeling with either 1 or 2 were observed, thus confirming selective modification of the two tags with the corresponding fluorophore-
(Figure 1 B), thus indicating that the presence of the co-labeled Alexa Fluor 594 acceptor dye causes substantial quenching of donor emission through FRET. Furthermore, incubation of the dual-labeled Z domain proteins S6-2-Z-Pcl23-1 and Pcl23-1-Z-S62 with guanidinium chloride (Gdm·HCl) resulted in an elevated ratio of donor/acceptor fluorescence (I520 nm/I620 nm), thus suggesting an increased average interfluorophore distance due to disturbance of the protein fold at Gdm·HCl concentrations above 1 m (Figure 1 C, Figures S1, S2). In order to demonstrate that the Pcl and S6 twotag system is not restricted to bacterially expressed proteins, the approach was also applied to the Fc domain of IgE (IgE-Fc). IgE-Fc contains three immunoglobulin domains (Ce2, Ce3, and Ce4) and requires expression in mammalian cells to preserve N-linked glycosylation, which is essential for it to maintain full functionality. Dual labeling of IgE-Fc with a FRET pair has previously provided valuable insight into the conformational changes associated with the binding of IgE to its high-affinity receptor FceRI, a key event in allergic disease because it results in the priming of mast cells with allergen-specific IgE.[14] An engineered IgE-Fc construct (IgE-Fc2–4) containing N- and C-terminally fused fluorescent proteins was used as a conformational FRET probe to monitor the IgE·FceRI protein–protein interaction in solution.[2c] Consistent with crystallographic data,[15] increased FRET suggested a more bent conformational state of IgE when complexed with FceRI. In contrast, binding of the anti-IgE therapeutic IgG antibody omalizumab (Xolair) resulted in a reduced FRET signal, thus suggesting that the antibody-bound IgE-Fc adopts an inhibitory conformation that is unable to interact with FceRI.[2c] Subsequently, a similar construct, this time labeled through a mutated cysteine at position 289 and a BirA tag at the C terminus, enabled the selecFigure 1. One-pot dual labeling with a FRET pair at neutral pH. A) Charge-state-deconvotion and characterization of an antibody that capluted ESI-MS spectra of the crude labeling mixtures confirm labeling of S6-Z-Pcl23 (left) and Pcl23-Z-S6 (middle) with both 1 and 2. MS verification of singly labeled Pcl23-1-Z-S6 is tured IgE in a previously unobserved conformation.[6c] shown on the right. Calculated molecular weights of S6-2-Z-Pcl23-1 (Pcl23-1-Z-S6-2), S6-2Furthermore, mono-labeling of a truncated form of Z-Pcl23 (Pcl23-Z-S6-2), S6-Z-Pcl23-1 (Pcl23-1-Z-S6), and S6-Z-Pcl23 (Pcl23-Z-S6) are 11 326.4 Da, IgE-Fc has been used to develop FRET-based assays 10 520.5 Da, 10 098.1 Da, and 9292.2 Da, respectively. B) Fluorescence emission spectra of for similar purposes,[16] again achieved by the engidual-labeled Pcl23-1-Z-S6-2 (a) and mono-labeled Pcl23-1-Z-S6 (c) reveal significant FRET-mediated donor emission quenching in the presence of covalently attached 2. neering of a free cysteine (Cys367 in this case). ThereMeasurements were carried out in 1.5 m Gdm·HCl with the excitation wavelength set to fore, dual labeling of IgE with the Pcl-S6 dual tag 23 485 nm. C) The ratio of donor and acceptor emission (I520 nm/I620 nm) for S6-2-Z-Pcl system appeared to be an ideal test-case for this new 1 (c) and Pcl23-1-Z-S6-2 (a) increases as a function of Gdm·HCl concentration (excilabeling technology, requiring neither bulky fluorestation at 485 nm). Corresponding fluorescence emission spectra are provided in the Supporting Information. RFU: relative fluorescence units. cent proteins nor the engineering of free cysteine residues. Given the compatibility of both tags with linked reactive moieties. small molecules of diverse structure,[9–11] we selected the freHaving demonstrated the simultaneous and selective introquently used Alexa Fluor 488/594 FRET pair for site-specific duction of two different fluorophores into Z domain as dual labeling of IgE-Fc. To demonstrate the versatility of the a model protein, we next performed FRET experiments with dual-labeling approach, the reactive groups linking the Alexa the single- and dual-labeled constructs Pcl23-1-Z-S6 and Pcl23-1Fluor dyes (compounds 3 and 4; see Scheme 1) were switched relative to those used in the Z domain labeling experiment Z-S6-2 (for procedures and characterization data see the Sup(compounds 1 and 2; see Scheme 1). porting Information). Fluorescence emission spectra were reWe identified suitable incorporation sites for Pcl and S6 tags corded between 505 and 700 nm at a fixed excitation wavewithin IgE-Fc with the aid of the X-ray structures 2WQR and length of 485 nm. Notably, Pcl23-1-Z-S6-2 displayed only 31 % 2YZQ, which reflect the apo and receptor-bound states of the (relative to Pcl23-1-Z-S6) of Alexa Fluor 488 emission at 520 nm 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 2. Schematic model for dual-labeled IgE-Fc in its apo and holo conformations. Structures of unbound IgE-Fc2–4 (left) and IgE-Fc2–4 bound to the extracellular domain of the FceRI a chain (right) are based on the PDB IDs 2WQR and 2Y7Q, respectively. Possible positions of the attached Alexa Fluor 488 donor and Alexa Fluor 594 acceptor dyes are represented by green and purple spheres, respectively. According to this model, receptor binding increases the distances between sphere centers due to the conformational change upon IgE·FceRI complex formation (FceRI in green) and by the steric effects that the receptor produces by binding between the acceptor molecules. Specifically, the average intra- and interchain FRET pair distances (black and red lines, respectively) increase by about 6 and 10 , respectively. Interfluorophore distances were averaged over 50 conformations that were generated by use of the program MacroModel from Schrçdinger, LLC (Supporting Information). The schematic representation was generated by use of PyMol (DeLano Scientific, LLC).
chromatography. According to UV/Vis spectroscopy, the degrees of labeling for 3 and 4 were found to be 90 and 70 %, respectively, resulting in a dual-labeling efficiency of 63 % (Figure S4). In addition, electrophoretic separation and subsequent fluorescence imaging of Pcl367-4-IgE-Fc-S6-3 confirmed the high specificities of both labeling reactions (Figure S5). These results demonstrate that the Pcl and S6 two-tag system enables the efficient and site-specific dual labeling of complex proteins that cannot be produced in bacterial hosts. In order to investigate whether the Pcl and S6 dual-labeled IgE-Fc operated as a FRET biosensor to detect receptor binding, we acquired fluorescence emission spectra of Pcl367-4IgE-Fc-S6-3 (200 nm) in the absence and in the presence of a saturating concentration of an FceRI mutant protein (1 mm; for details regarding the FceRI mutant protein see the Supporting Information). Using a fixed excitation wavelength of 495 nm (5 nm bandwidth), we compared the emission intensity of Pcl367-4-IgE-Fc-S6-3 with that of a mixture of absorbance-matched singly labeled IgE-Fc molecules: Pcl367-4-IgE-FcS6 and Pcl367-IgE-Fc-S6-3. As shown in Figure 3 A, Pcl367-4IgE-Fc-S6-3 exhibits reduced donor emission (518 nm) and enhanced acceptor emission (616 nm) relative to the mixture of singly labeled controls, consistently with intramolecular FRET
molecule. Figure 2 shows a model of the FRET-labeled IgE-Fc, with the S6 tag covalently attached to Alexa Fluor 488 (donor), which is fused to the C terminus of the Ce4 domain, and the Pcl residue carrying Alexa Fluor 594 (acceptor) substituted for Lys367 in a surface-exposed loop region of the Ce3 domain. The possible spatial positions of both fluorescent dyes are illustrated by spheres. As illustrated in Figure 2, FceRI binding to the Ce3 domain of IgE-Fc leads to a displacement of both Pcl367-linked Alexa Fluor 594 dyes, consistently with the dislocation of the corresponding sphere centers. According to molecular modeling with the MacroModel program (Schrçdinger, LLC), this receptor-mediated displacement of the acceptor dyes results in increases in the average intra- and interchain distances of the FRET pair by about 6 and 10 , respectively (for details regarding the molecular modeling of fluorophorelabeled IgE-Fc see the Supporting Information). Dual-tagged IgE-Fc construct Pcl367-IgE-Fc-S6 was expressed in HEK293 cells after transient transfection with four plasmids encoding the mutated human IgE-Fc domain (Ce2–4), the wildtype pyrrolysyl-tRNA synthetase-tRNACUA pair, as well as the two Pyl biosynthetic enzymes PylC and PylD (for detailed procedures regarding protein expression, purification, labeling, and characterization see the Supporting Information). The cell culture medium was supplemented with 5 mm of d-ornithine for Pcl generation, and after affinity purification with immobilized anti-IgE-Fc monoclonal antibody, 3.7 mg of Pcl367-IgE-FcS6 was obtained from 1 L of culture. No truncated IgE-Fc fragment derived from incomplete non-sense codon suppression was observed by SDS-PAGE (Figure S3). To introduce a FRET pair into IgE-Fc site-specifically, the dual-tagged IgE-Fc protein was sequentially labeled with 4 and 3 at pH 7 and 23 8C (Scheme 1). Excess fluorophore was removed by size-exclusion
Figure 3. IgE·FceRI complex formation suggests an increased interfluorophore distance in the FRET biosensor Pcl367-4-IgE-Fc-S6-3. A) The fluorescence emission spectra (fixed excitation wavelength of 495 nm, emission wavelengths from 510 nm to 700 nm) of dual-labeled Pcl367-4-IgE-Fc-S6-3 (200 nm), as well as of a mixture of singly labeled Pcl367-4-IgE-Fc-S6 and Pcl367-IgE-Fc-S6-3, were acquired in the absence (left) and in the presence of FceRI (right). All samples were absorbance-matched at both excitation wavelengths (495 and 590 nm). Saturating levels of FceRI (1 mm) result in decreased quenching of donor fluorescence and diminished acceptor fluorescence, indicative of an increased interfluorophore distance upon receptor binding. Fluorescence emission spectra were normalized to the maximum intensity of the mixture of singly labeled IgE-Fc constructs at 518 nm. B) The proximity ratios of the Pcl367-4-IgE-Fc-S6-3 biosensor (200 nm; left) and the mixture of Pcl367-4-IgE-Fc-S6 and Pcl367-IgE-Fc-S6-3 (right) are plotted as a function of FceRI concentration. The proximity ratio of the mixture of singly labeled IgE-Fc is largely independent of receptor concentration, whereas the proximity ratio of the dual-labeled IgE-Fc decreases with increasing receptor concentration.
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CHEMBIOCHEM COMMUNICATIONS between 3 and 4. Moreover, the extent of FRET in the dual-labeled protein was found to be dependent on the presence or absence of FceRI. Specifically, IgE·FceRI complex formation was accompanied by enhanced donor emission and diminished acceptor emission; this is indicative of a decreased proximity ratio (see definition in the Supporting Information) between the two fluorescent dyes in Pcl367-4-IgE-Fc-S6-3. These observations can be interpreted as an increase in interfluorophore distance because of receptor-mediated displacement of the acceptor fluorophores. This interpretation is consistent with the model in Figure 2, which predicts increases in the average intra- and interchain distances between donor and acceptor dyes of approximately 6 and 10 , respectively, upon receptor binding. A titration experiment further indicated that for Pcl367-4-IgE-Fc-S6-3, the proximity ratio decreases with increasing FceRI concentration until the concentration of this high-affinity receptor approaches saturation. This is suggestive of the formation of a stable 1:1 complex (Figure 3 B). These observations confirm the integrity of the labeled proteins and indicate that the changes in FRET signal are due to a true protein-binding event. In contrast, the same titration experiment with the mixture of singly labeled IgE-Fc molecules (Pcl367-4IgE-Fc-S6 and Pcl367-IgE-Fc-S6-3) did not produce any significant changes in the proximity ratio as a function of FceRI concentration. These results demonstrate the utility of dual-labeled Pcl367-4-IgE-Fc-S6-3 as a sensitive FRET sensor for probing the IgE·FceRI protein–protein interaction in solution. However, further investigation is required to determine the extent to which the molecule is reporting on receptor-mediated conformational changes relative to other effects such as contact quenching of the acceptor, changes in dipole orientation, or a combination of these phenomena as a result of proximity of the acceptor dye to the protein–protein interaction surface.[16a] In conclusion, we have developed an efficient approach for site-specific dual labeling of proteins under near physiological conditions (i.e., pH 7 and 23 8C) by using two small tags: the Pcl amino acid and the 12-mer S6 peptide. The absence of cross-reactivity between these bio-orthogonal tags can be used for dual labeling in a one-pot reaction. Both tags can be used for conjugation with donor or acceptor dyes. On the basis of previous experiments with individually tagged proteins,[9–10] both tags tolerate conjugation reactions with structurally diverse small molecules, thus making this orthogonal tag system suitable for homogenous two-color labeling as required for FRET-based applications. In addition, incorporation of Pcl and S6 tag can be accomplished at many sites in a variety of proteins.[9–11] Because the incorporation of the tags is compatible with mammalian protein expression, site-specific two-color labeling can also be applied to extensively posttranslationally modified proteins. In particular, we have demonstrated the efficient incorporation of a FRET pair into IgE-Fc and have monitored IgE·FceRI complex formation, a key event in the allergic cascade. The ability to perform dual labeling of complex proteins with small molecules selectively and efficiently at neutral pH might find broad utility in applications beyond the development of fluorescence-based probes.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org Acknowledgements We thank Dr. Lukas Leder for providing FceRI expression plasmids. Keywords: biosensors · FRET · IgE · phosphopantetheinyl transferase · pyrroline-carboxy-lysine
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Site-Specific Dual Labeling of Proteins by Using Small Orthogonal Tags at Neutral pH Jan Grnewald,[a] David H. Jones,[a] Ansgar Brock,[a] Hsien-Po Chiu,[a] Badry Bursulaya,[a] Kenneth Ng,[a] Todd Vo,[a] Paula Patterson,[a] Tetsuo Uno,[a] James Hunt,[b] Glen Spraggon,*[a] and Bernhard H. Geierstanger*[a] To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor FceRI, which is a key event in allergic disease.
Labeling of proteins with multiple fluorescent dyes has become a universal tool for monitoring structural changes within biopolymers by Fçrster resonance energy transfer (FRET).[1] This quantum phenomenon is based on the distancedependent energy transfer between donor and acceptor molecules. An important requirement for these types of observation is the site-specific introduction of a donor–acceptor pair into the protein of interest. One approach is based on fusing the target protein with a donor–acceptor pair of intrinsically fluorescent proteins.[2] However, genetic fusion typically restricts labeling to N- and C termini and can potentially impair protein folding, structure, and function. Furthermore, the relatively large size of such fusions (for example, 26.9 kDa for green fluorescent protein) negatively impacts the resolution of measurable distances within the target protein. Alternatively, labeling with spectrally diverse small-molecule fluorophores can be accomplished chemically or enzymatically. For instance, two-color fluorescence labeling has been accomplished by using sortases with distinct substrate specificities,[3] solid-phase expressed protein ligation,[4] and chemoselective modification of a cleaved intein fusion protein.[5]Although [a] Dr. J. Grnewald, Dr. D. H. Jones, Dr. A. Brock, Dr. H.-P. Chiu, Dr. B. Bursulaya, K. Ng, T. Vo, P. Patterson, Dr. T. Uno, Dr. G. Spraggon, Dr. B. H. Geierstanger Genomics Institute of the Novartis Research Foundation 10675 John-Jay-Hopkins Drive, San Diego, CA 92121 (USA) E-mail: [email protected] [email protected] [b] Dr. J. Hunt Novartis Institutes for Biomedical Research Wimblehurst Road, Horsham, West Sussex, RH12 5AB (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402204.
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these strategies minimize the potential impact of labeling on protein folding, the installation of a FRET pair involves multistep protocols and is still restricted to terminal protein regions. These limitations can be overcome by using thiol-reactive fluorescent probes for the modification of engineered cysteine residues.[6] However, site-specific labeling might require the deletion of surface-exposed native cysteine residues, thereby potentially interfering with the folding and stability of the target protein. Furthermore, the introduction of unpaired cysteine residues can potentially trigger protein aggregation, and the heterogeneity imposed by random labeling of two engineered cysteine residues with donor and acceptor dyes makes meaningful combinations of FRET pairs difficult. One way to eliminate this lack of selectivity is to use either a Cys mutant combined with a non-canonical amino acid (ncAA)[6b] or two ncAAs with bio-orthogonal reactivities (pazido-phenylalanine and 2-amino-8-oxononanoic acid) to incorporate two probes into the same protein. In the latter case, dual labeling with the appropriate dye molecules afforded homogeneous protein–fluorophore conjugates for FRET studies.[6c, 7] Although elegant, the approach is currently restricted to proteins that can be expressed in Escherichia coli, and the pH-dependence of the oxime ligation reaction of 2-amino-8oxononanoic acid greatly reduces the efficiency of labeling at pH values of 7 or higher. Here we present an alternative approach that enables site-specific dual labeling of both bacterially and mammalian-expressed proteins at neutral pH by utilizing the orthogonality between the chemical labeling of genetically encoded pyrroline-carboxy-lysine (Pcl), a demethylated analogue of pyrrolysine,[8] and the post-translational modification of a 12-mer S6 peptide sequence (GDSLSWLLRLLN).[9] We have previously demonstrated that the Pcl side chain can readily and specifically be conjugated to a wide range of 2-aminobenzaldehyde-activated molecules.[10] Analogously, phosphopantetheinyl transferases (PPTases) have been established as versatile tools for the site-specific labeling of proteins with structurally diverse molecules through the use of 11- or 12-mer peptidic recognition sequences such as the S6 peptide.[9, 11] Here, we show that both reactions can be performed simultaneously in an efficient one-pot process to incorporate a donor–acceptor pair site-specifically into a protein of interest at neutral pH. Notably, this bio-orthogonal dual-tag system is fully compatible with bacterial and mammalian expression systems, due to the ability of the pyrrolysyl-tRNA/tRNA synthetase pair to shuttle between both expression hosts.[12] To this end, we demonstrate the site-specific incorporation of FRET pairs ChemBioChem 2014, 15, 1787 – 1791
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CHEMBIOCHEM COMMUNICATIONS into E. coli-expressed Z domain of staphylococcal protein A and into HEK293-expressed human immunoglobulin E fragment crystallizable (IgE-Fc). In the latter proof-of-concept model, site-specific two-color labeling was used to monitor the binding of IgE-Fc to its high-affinity receptor FceRI. Because of its low molecular weight and ease of expression and purification, Z domain protein has been frequently used to demonstrate the in vivo incorporation of ncAAs in response to a non-sense codon (TAG) in E. coli.[6b, 13] Two Z domain mutants were constructed, containing a Pcl residue in place of a surface-exposed proline at position 23, as well as an S6 peptide sequence appended either to the N terminus (S6-Z-Pcl23) or to the C terminus (Pcl23-Z-S6). The dual-tagged proteins were expressed in E. coli BL21(DE3) cells co-transformed with two plasmids encoding the corresponding Z domain construct with a non-sense codon substitution at position 23, the wild-type
www.chembiochem.org pyrrolysyl-tRNA synthetase (PylS), its cognate tRNA (PylT), and the two biosynthetic enzymes PylC and PylD. The last two catalyze the synthesis of Pcl from the precursor compound d-ornithine,[8] which was added to Terrific Broth growth medium at a final concentration of 5 mm. Culturing in shake flasks and subsequent nickel nitrilotriacetate (Ni-NTA) chromatography afforded purified S6-Z-Pcl23 and Pcl23-Z-S6 in yields of 8 and 18 mg L 1, respectively. Next, we tested whether the orthogonal reactivity of the Pcl amino acid side chain and the S6 peptide allowed concurrent labeling of the proteins with different fluorophores for subsequent FRET experiments. In a one-pot reaction, 5 mm of dualtagged Z domain (S6-Z-Pcl23 or Pcl23-Z-S6) was treated with a mixture of 200 mm of 1 and 20 mm of 2 (Scheme 1) in the presence of 1.4 mm of Sfp PPTase. Incubation for 16 h at 37 8C and pH 7.5 resulted in almost complete formation of the de-
Scheme 1. Fluorogenic compounds used in this study. The fluorescent dyes Alexa Fluor 488 and Alexa Fluor 594 were chemically linked to 2-aminobenzaldehyde to provide compounds 1 and 4, respectively, and to coenzyme A to provide compounds 3 and 2, respectively).
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sired dual-labeled Z domain constructs, S6-2-Z-Pcl23-1 and Pcl23-1-Z-S6-2, as confirmed by ESI-MS analysis of the crude labeling mixtures prior to cyanoborohydride addition (Figure 1 A). No noticeable amount of uncoupled or mono-labeled Z domain was detectable, thus indicating that highly efficient conjugation of the proteins at neutral pH had been achieved. Furthermore, no peaks originating from double labeling with either 1 or 2 were observed, thus confirming selective modification of the two tags with the corresponding fluorophore-
(Figure 1 B), thus indicating that the presence of the co-labeled Alexa Fluor 594 acceptor dye causes substantial quenching of donor emission through FRET. Furthermore, incubation of the dual-labeled Z domain proteins S6-2-Z-Pcl23-1 and Pcl23-1-Z-S62 with guanidinium chloride (Gdm·HCl) resulted in an elevated ratio of donor/acceptor fluorescence (I520 nm/I620 nm), thus suggesting an increased average interfluorophore distance due to disturbance of the protein fold at Gdm·HCl concentrations above 1 m (Figure 1 C, Figures S1, S2). In order to demonstrate that the Pcl and S6 twotag system is not restricted to bacterially expressed proteins, the approach was also applied to the Fc domain of IgE (IgE-Fc). IgE-Fc contains three immunoglobulin domains (Ce2, Ce3, and Ce4) and requires expression in mammalian cells to preserve N-linked glycosylation, which is essential for it to maintain full functionality. Dual labeling of IgE-Fc with a FRET pair has previously provided valuable insight into the conformational changes associated with the binding of IgE to its high-affinity receptor FceRI, a key event in allergic disease because it results in the priming of mast cells with allergen-specific IgE.[14] An engineered IgE-Fc construct (IgE-Fc2–4) containing N- and C-terminally fused fluorescent proteins was used as a conformational FRET probe to monitor the IgE·FceRI protein–protein interaction in solution.[2c] Consistent with crystallographic data,[15] increased FRET suggested a more bent conformational state of IgE when complexed with FceRI. In contrast, binding of the anti-IgE therapeutic IgG antibody omalizumab (Xolair) resulted in a reduced FRET signal, thus suggesting that the antibody-bound IgE-Fc adopts an inhibitory conformation that is unable to interact with FceRI.[2c] Subsequently, a similar construct, this time labeled through a mutated cysteine at position 289 and a BirA tag at the C terminus, enabled the selecFigure 1. One-pot dual labeling with a FRET pair at neutral pH. A) Charge-state-deconvotion and characterization of an antibody that capluted ESI-MS spectra of the crude labeling mixtures confirm labeling of S6-Z-Pcl23 (left) and Pcl23-Z-S6 (middle) with both 1 and 2. MS verification of singly labeled Pcl23-1-Z-S6 is tured IgE in a previously unobserved conformation.[6c] shown on the right. Calculated molecular weights of S6-2-Z-Pcl23-1 (Pcl23-1-Z-S6-2), S6-2Furthermore, mono-labeling of a truncated form of Z-Pcl23 (Pcl23-Z-S6-2), S6-Z-Pcl23-1 (Pcl23-1-Z-S6), and S6-Z-Pcl23 (Pcl23-Z-S6) are 11 326.4 Da, IgE-Fc has been used to develop FRET-based assays 10 520.5 Da, 10 098.1 Da, and 9292.2 Da, respectively. B) Fluorescence emission spectra of for similar purposes,[16] again achieved by the engidual-labeled Pcl23-1-Z-S6-2 (a) and mono-labeled Pcl23-1-Z-S6 (c) reveal significant FRET-mediated donor emission quenching in the presence of covalently attached 2. neering of a free cysteine (Cys367 in this case). ThereMeasurements were carried out in 1.5 m Gdm·HCl with the excitation wavelength set to fore, dual labeling of IgE with the Pcl-S6 dual tag 23 485 nm. C) The ratio of donor and acceptor emission (I520 nm/I620 nm) for S6-2-Z-Pcl system appeared to be an ideal test-case for this new 1 (c) and Pcl23-1-Z-S6-2 (a) increases as a function of Gdm·HCl concentration (excilabeling technology, requiring neither bulky fluorestation at 485 nm). Corresponding fluorescence emission spectra are provided in the Supporting Information. RFU: relative fluorescence units. cent proteins nor the engineering of free cysteine residues. Given the compatibility of both tags with linked reactive moieties. small molecules of diverse structure,[9–11] we selected the freHaving demonstrated the simultaneous and selective introquently used Alexa Fluor 488/594 FRET pair for site-specific duction of two different fluorophores into Z domain as dual labeling of IgE-Fc. To demonstrate the versatility of the a model protein, we next performed FRET experiments with dual-labeling approach, the reactive groups linking the Alexa the single- and dual-labeled constructs Pcl23-1-Z-S6 and Pcl23-1Fluor dyes (compounds 3 and 4; see Scheme 1) were switched relative to those used in the Z domain labeling experiment Z-S6-2 (for procedures and characterization data see the Sup(compounds 1 and 2; see Scheme 1). porting Information). Fluorescence emission spectra were reWe identified suitable incorporation sites for Pcl and S6 tags corded between 505 and 700 nm at a fixed excitation wavewithin IgE-Fc with the aid of the X-ray structures 2WQR and length of 485 nm. Notably, Pcl23-1-Z-S6-2 displayed only 31 % 2YZQ, which reflect the apo and receptor-bound states of the (relative to Pcl23-1-Z-S6) of Alexa Fluor 488 emission at 520 nm 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 2. Schematic model for dual-labeled IgE-Fc in its apo and holo conformations. Structures of unbound IgE-Fc2–4 (left) and IgE-Fc2–4 bound to the extracellular domain of the FceRI a chain (right) are based on the PDB IDs 2WQR and 2Y7Q, respectively. Possible positions of the attached Alexa Fluor 488 donor and Alexa Fluor 594 acceptor dyes are represented by green and purple spheres, respectively. According to this model, receptor binding increases the distances between sphere centers due to the conformational change upon IgE·FceRI complex formation (FceRI in green) and by the steric effects that the receptor produces by binding between the acceptor molecules. Specifically, the average intra- and interchain FRET pair distances (black and red lines, respectively) increase by about 6 and 10 , respectively. Interfluorophore distances were averaged over 50 conformations that were generated by use of the program MacroModel from Schrçdinger, LLC (Supporting Information). The schematic representation was generated by use of PyMol (DeLano Scientific, LLC).
chromatography. According to UV/Vis spectroscopy, the degrees of labeling for 3 and 4 were found to be 90 and 70 %, respectively, resulting in a dual-labeling efficiency of 63 % (Figure S4). In addition, electrophoretic separation and subsequent fluorescence imaging of Pcl367-4-IgE-Fc-S6-3 confirmed the high specificities of both labeling reactions (Figure S5). These results demonstrate that the Pcl and S6 two-tag system enables the efficient and site-specific dual labeling of complex proteins that cannot be produced in bacterial hosts. In order to investigate whether the Pcl and S6 dual-labeled IgE-Fc operated as a FRET biosensor to detect receptor binding, we acquired fluorescence emission spectra of Pcl367-4IgE-Fc-S6-3 (200 nm) in the absence and in the presence of a saturating concentration of an FceRI mutant protein (1 mm; for details regarding the FceRI mutant protein see the Supporting Information). Using a fixed excitation wavelength of 495 nm (5 nm bandwidth), we compared the emission intensity of Pcl367-4-IgE-Fc-S6-3 with that of a mixture of absorbance-matched singly labeled IgE-Fc molecules: Pcl367-4-IgE-FcS6 and Pcl367-IgE-Fc-S6-3. As shown in Figure 3 A, Pcl367-4IgE-Fc-S6-3 exhibits reduced donor emission (518 nm) and enhanced acceptor emission (616 nm) relative to the mixture of singly labeled controls, consistently with intramolecular FRET
molecule. Figure 2 shows a model of the FRET-labeled IgE-Fc, with the S6 tag covalently attached to Alexa Fluor 488 (donor), which is fused to the C terminus of the Ce4 domain, and the Pcl residue carrying Alexa Fluor 594 (acceptor) substituted for Lys367 in a surface-exposed loop region of the Ce3 domain. The possible spatial positions of both fluorescent dyes are illustrated by spheres. As illustrated in Figure 2, FceRI binding to the Ce3 domain of IgE-Fc leads to a displacement of both Pcl367-linked Alexa Fluor 594 dyes, consistently with the dislocation of the corresponding sphere centers. According to molecular modeling with the MacroModel program (Schrçdinger, LLC), this receptor-mediated displacement of the acceptor dyes results in increases in the average intra- and interchain distances of the FRET pair by about 6 and 10 , respectively (for details regarding the molecular modeling of fluorophorelabeled IgE-Fc see the Supporting Information). Dual-tagged IgE-Fc construct Pcl367-IgE-Fc-S6 was expressed in HEK293 cells after transient transfection with four plasmids encoding the mutated human IgE-Fc domain (Ce2–4), the wildtype pyrrolysyl-tRNA synthetase-tRNACUA pair, as well as the two Pyl biosynthetic enzymes PylC and PylD (for detailed procedures regarding protein expression, purification, labeling, and characterization see the Supporting Information). The cell culture medium was supplemented with 5 mm of d-ornithine for Pcl generation, and after affinity purification with immobilized anti-IgE-Fc monoclonal antibody, 3.7 mg of Pcl367-IgE-FcS6 was obtained from 1 L of culture. No truncated IgE-Fc fragment derived from incomplete non-sense codon suppression was observed by SDS-PAGE (Figure S3). To introduce a FRET pair into IgE-Fc site-specifically, the dual-tagged IgE-Fc protein was sequentially labeled with 4 and 3 at pH 7 and 23 8C (Scheme 1). Excess fluorophore was removed by size-exclusion
Figure 3. IgE·FceRI complex formation suggests an increased interfluorophore distance in the FRET biosensor Pcl367-4-IgE-Fc-S6-3. A) The fluorescence emission spectra (fixed excitation wavelength of 495 nm, emission wavelengths from 510 nm to 700 nm) of dual-labeled Pcl367-4-IgE-Fc-S6-3 (200 nm), as well as of a mixture of singly labeled Pcl367-4-IgE-Fc-S6 and Pcl367-IgE-Fc-S6-3, were acquired in the absence (left) and in the presence of FceRI (right). All samples were absorbance-matched at both excitation wavelengths (495 and 590 nm). Saturating levels of FceRI (1 mm) result in decreased quenching of donor fluorescence and diminished acceptor fluorescence, indicative of an increased interfluorophore distance upon receptor binding. Fluorescence emission spectra were normalized to the maximum intensity of the mixture of singly labeled IgE-Fc constructs at 518 nm. B) The proximity ratios of the Pcl367-4-IgE-Fc-S6-3 biosensor (200 nm; left) and the mixture of Pcl367-4-IgE-Fc-S6 and Pcl367-IgE-Fc-S6-3 (right) are plotted as a function of FceRI concentration. The proximity ratio of the mixture of singly labeled IgE-Fc is largely independent of receptor concentration, whereas the proximity ratio of the dual-labeled IgE-Fc decreases with increasing receptor concentration.
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CHEMBIOCHEM COMMUNICATIONS between 3 and 4. Moreover, the extent of FRET in the dual-labeled protein was found to be dependent on the presence or absence of FceRI. Specifically, IgE·FceRI complex formation was accompanied by enhanced donor emission and diminished acceptor emission; this is indicative of a decreased proximity ratio (see definition in the Supporting Information) between the two fluorescent dyes in Pcl367-4-IgE-Fc-S6-3. These observations can be interpreted as an increase in interfluorophore distance because of receptor-mediated displacement of the acceptor fluorophores. This interpretation is consistent with the model in Figure 2, which predicts increases in the average intra- and interchain distances between donor and acceptor dyes of approximately 6 and 10 , respectively, upon receptor binding. A titration experiment further indicated that for Pcl367-4-IgE-Fc-S6-3, the proximity ratio decreases with increasing FceRI concentration until the concentration of this high-affinity receptor approaches saturation. This is suggestive of the formation of a stable 1:1 complex (Figure 3 B). These observations confirm the integrity of the labeled proteins and indicate that the changes in FRET signal are due to a true protein-binding event. In contrast, the same titration experiment with the mixture of singly labeled IgE-Fc molecules (Pcl367-4IgE-Fc-S6 and Pcl367-IgE-Fc-S6-3) did not produce any significant changes in the proximity ratio as a function of FceRI concentration. These results demonstrate the utility of dual-labeled Pcl367-4-IgE-Fc-S6-3 as a sensitive FRET sensor for probing the IgE·FceRI protein–protein interaction in solution. However, further investigation is required to determine the extent to which the molecule is reporting on receptor-mediated conformational changes relative to other effects such as contact quenching of the acceptor, changes in dipole orientation, or a combination of these phenomena as a result of proximity of the acceptor dye to the protein–protein interaction surface.[16a] In conclusion, we have developed an efficient approach for site-specific dual labeling of proteins under near physiological conditions (i.e., pH 7 and 23 8C) by using two small tags: the Pcl amino acid and the 12-mer S6 peptide. The absence of cross-reactivity between these bio-orthogonal tags can be used for dual labeling in a one-pot reaction. Both tags can be used for conjugation with donor or acceptor dyes. On the basis of previous experiments with individually tagged proteins,[9–10] both tags tolerate conjugation reactions with structurally diverse small molecules, thus making this orthogonal tag system suitable for homogenous two-color labeling as required for FRET-based applications. In addition, incorporation of Pcl and S6 tag can be accomplished at many sites in a variety of proteins.[9–11] Because the incorporation of the tags is compatible with mammalian protein expression, site-specific two-color labeling can also be applied to extensively posttranslationally modified proteins. In particular, we have demonstrated the efficient incorporation of a FRET pair into IgE-Fc and have monitored IgE·FceRI complex formation, a key event in the allergic cascade. The ability to perform dual labeling of complex proteins with small molecules selectively and efficiently at neutral pH might find broad utility in applications beyond the development of fluorescence-based probes.
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www.chembiochem.org Acknowledgements We thank Dr. Lukas Leder for providing FceRI expression plasmids. Keywords: biosensors · FRET · IgE · phosphopantetheinyl transferase · pyrroline-carboxy-lysine
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