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Probing Protein Conformations by in Situ Non-Covalent Fluorescence Labeling Jennifer Julia Strunk, Ingo Gregor, Yvonne Becker, Peter Lamken, Suman Lata, Annett Reichel, Jörg Enderlein, and Jacob Piehler* Institute of Biochemistry and Cluster of Excellence Macromolecular Complexes (CEF), Johann Wolfgang Goethe-University, Frankfurt/Main, Germany, Department of Molecular Neurosensorics, Caesar Research Centre, Bonn, Germany, and Institute of Physical and Theoretical Chemistry, Eberhard-Karls-University, Tu¨bingen, Germany. Received May 20, 2008; Revised Manuscript Received November 11, 2008
The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for sitespecific incorporation of FRET acceptors into proteins, which were covalently labeled with a donor fluorophore. We demonstrate versatile application of this approach for exploring the conformation of the type I interferon receptor ectodomains ifnar1-EC and ifnar2-EC. Substantial ligand-induced conformational changes of ifnar1EC, but not ifnar2-EC, were observed by monitoring the fluorescence intensity and the fluorescence lifetime of the FRET donor. Time-resolved fluorescence correlation spectroscopy revealed a substantial conformational flexibility of ifnar1-EC and a ligand-induced tightening. Our results demonstrate that protein labeling with tris-NTA/fluorophores enables for efficient quantitative intramolecular FRET analysis.
INTRODUCTION Protein-protein recognition frequently imposes conformational changes on the interaction partners. These conformational changes can modulate protein function, which plays a key role in many biological processes, in particular, in biological signaling cascades. Dissecting the conformational dynamics on the level of individual proteins proved particular promising for understanding the molecular basis of bindinginduced structural changes (1-4). Single molecule fluorescence techniques are particularly capable of monitoring conformational dynamics in real time (5-9). One of the main challenges in this approach, however, is the need to incorporate appropriate fluorescence probes as reporter for conformational changes into the protein. For monitoring distance changes within macromolecules, typically fluorescence resonance energy transfer (FRET) is employed, which requires site-specific dual-color labeling. Covalent coupling of thiolreactive fluorescence dyes to free cysteines is the most common approach toward site-specific labeling of proteins. For dual-color labeling, however, this approach is not suitable, requiring the separation of different species (10). Thus, alternative strategies for site-specific incorporation of a second fluorescence dye into proteins are required. Fusion with fluorescent proteins (11, 12) or with other proteins or peptide tags, which can be post-translationally labeled in a selective manner based on biomolecular recognition, is a powerful tool for labeling proteins (13-17). The size of the proteins involved in labeling and their flexible attachment to the target proteins, however, heavily reduce the distance resolution, which can be probed by intramolecular FRET. Thus, approaches for site-specific fluorescence with small * Corresponding author. Institute of Biochemistry, Biocenter N210, Max-von-Laue-Straβe 9, 60438 Frankfurt am Main, Phone +49 (0)69 79829468, Fax +49 (0)69 79829495. E-mail: j.piehler@ em.uni-frankfurt.de.
recognition units are particularly promising for studying protein conformations (17-21). Recently, we have described multivalent chelator heads (MCH) that recognize oligohistidines with subnanomolar affinity and high specificity (22). These chelators have been used for site-specific, quantitative labeling of His-tagged proteins in solution with several different fluorescence probes (23). Here, we have employed MCH conjugates (Figure 1a-c) for studying the conformational organization of the extracellular domain of a cytokine receptor and its changes upon ligand binding. The principle is depicted in Figure 1d,e: the proteins were site-specifically labeled with a fluorescence donor through covalent attachment through a free cysteine residue. Using MCH conjugates, a fluorescence acceptor is site-specifically incorporated in situ through a C-terminal decahistidine tag and the conformations can be probed by FRET (Figure 1d). The extracellular domains of the type I interferon receptor (24) subunits carrying a C-terminal decahistidine tag (ifnar1-H10 and ifnar2-H10) were employed as model systems for conformational analysis. While the highaffinity subunit ifnar2-H10 does not change its structure upon interferon binding (25), we have recently observed complex ligand-induced conformational changes of ifnar1-H10 (26). Ifnar1-H10 and ifnar2-H10 were site-specifically labeled through additional cysteines. For ifnar1-H10, the mutation N23C on the N-terminal Ig-like domain was incorporated by site-directed mutagenesis. The protein was labeled with the maleimide-functionalized green fluorescent dyes Oregon Green 488 (OG488) or Alexa Fluor 488 (AF488). In order to ensure quantitative ligand binding to ifnar1-H10, we used the interferon triple mutant H57A, E58A, and Q61A (IFNR2HEQ), which binds ifnar1-H10 with ∼40-fold higher affinity (∼150 nM) than the wild-type (27). Ifnar2-EC, which binds both IFNR2 wt and HEQ with high affinity (∼5 nM), was employed as a reference protein, and three mutants with single cysteines in different positions were prepared for
10.1021/bc8002088 CCC: $40.75 2009 American Chemical Society Published on Web 12/22/2008
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Figure 1. Principle of the fluorescence quenching assay. (a-c) MCH-based reporter probes used for monitoring conformational changes: (a) FEW646 tris-NTA, (b)AT565tris-NTA, and (c) BTtris-NTA. (d) By site-specific incorporation of the quencher dye through the His-tag, fluorophores attached to the protein are quenched in a distance-dependent manner. (e) NMR-based model of the ternary complex formed by ifnar2-EC (blue) with IFNR2-HEQ (red) (34). The position of residues mutated into cysteines for site-specific labeling (S35C, S131C, and T169C) are colored orange ifnar2.
experimentally calibrating the FRET efficiencies (indicated in Figure 1e).
EXPERIMENTAL PROCEDURES Synthesis, Protein Expression, and Labeling. The trisNTA/fluorophore conjugates with ATTO 565 (AT565tris-NTA) and FEW Chemicals S0387 (FEW646tris-NTA) were synthesized and loaded with Ni(II) ions as described previously (23, 28). Ifnar1-EC with a C-terminal decahistidine-tag (ifnar1-H10) was expressed and purified as described before (29). Ifnar1 without His-tag (ifnar1-tl) was prepared from an ifnar1-H10 containing a factor Xa cleavage site upstream of the His-tag by proteolytic cleavage. Ifnar2-H10 was cloned into the same expression vector as ifnar1-H10 (pAcGP67B) and expressed in Sf9 insect cells as described for ifnar1-H10 (29). Single cysteine mutants of ifnar1-H10 (N23C) and ifnar2-H10 (S35C, S131C, and T169C, respectively) were generated by ligase chain reaction. The proteins were expressed and purified as wild-type ifnar1-H10. Thiol-specific labeling of these cysteine mutants required reduction of these additional cysteine residues without reducing the disulfide bonds in the proteins. Different conditions were found to be suitable for the different proteins: ifnar1 N23C was reduced by incubation with 3 mM tricarboxyethylphospine and 1 mM EDTA for 1 h; ifnar2-H10 mutants S35C, S131C, and T169C were reduced using 300 µM dithiothreitol for 1 h. After removing the reducing agent by SEC (Superdex 200 in 20 mM Tris pH 7.5, 50 mM sodium chloride), a 3-fold molar excess of
the maleimide-functionalized fluorescence dye (OG488 and AF488 maleimide from Molecular Probes) was added and incubated overnight. The labeled proteins were further purified by ion exchange chromatography to separate unlabeled and multiply labeled species, followed by a final polish by SEC in 20 mM Hepes pH 7.5 and 150 mM NaCl (HBS). Cuvette Fluorescence Assays. Fluorescence quenching experiments were carried out in HBS complemented with 1 mg/mL BSA. Fluorescence spectra were recorded with a Cary Eclipse (Varian, Germany) and fluorescence lifetime measurements were carried out in a FluoTime 200 instrument (Picoquant GmbH, Germany) using a 120 µL cuvette. A solution of 100 nM covalently labeled protein was filled into the cuvette, and the fluorescence measurement was carried out. Subsequently, 250 nM of FEW646tris-NTA, AT565tris-NTA, or BTtris-NTA was added to the protein solution in the cuvette, incubated for 15 min, and then the fluorescence spectra or fluorescence lifetime was recorded. Subsequently, 1 µM IFNR2-HEQ was added, and fluorescence spectra or fluorescence lifetime was recorded after 5 min. The stock solutions of the tris-NTA conjugates and IFNR2-HEQ were so concentrated that the change of volume was negligible. Fluorescence spectra were acquired with an excitation of OG488 and AF488 at 470 nm (5 nm slit-width). For ensemble fluorescence lifetime measurements, OG488 was excited at 493 nm by a pulsed LED (PLS500, Picoquant GmbH, Germany) with 500 ps pulse width and 20 MHz repetition rate. Fluorescence lifetimes were fitted by a double-
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exponential decay. For evaluation of the FRET efficiency and the donor-acceptor distance, we treated each component independently. Time-Resolved Fluorescence Correlation Spectroscopy. Time-resolved fluorescence correlation spectroscopy was carried out in a home-built confocal microscope that was described previously (30). The excitation laser used was a pulsed diode laser at 470 nm wavelength (PDL 470, PicoQuant GmbH, Germany) generating pulses with ca. 50 ps pulse width and 40 MHz repetition rate. Fast electronics (PicoHarp 300, PicoQuant GmbH, Germany) was used for recording the detected photons in time-correlated time-tagged recording mode. From these raw data, the autocorrelation curves G(τ) were calculated by crosscorrelating photons from two different single-photon avalanche photodiodes. Binding experiments were carried out with 1 nM OG488ifnar1-H10 or AF488ifnar1-H10 mixed with 100 nM unlabeled ifnar1-H10 in HBS complemented with 1 mg/mL BSA. The mixture was incubated with 250 nM AT565tris-NTA for 15 min prior to the measurements, and with 1 µM IFNR2HEQ for 5 min. For measurements with no addition of the trisNTA conjugates, 1 mM EDTA was added in order to remove contaminating transition metal ions. Data were acquired over 30 min. The fitting of the normalized autocorrelation function data was done using the standard model introduced by Rigler et al. (31). To account for the complex photophysical processes, this model was extended using three exponential decay terms. Hence, the following model function was used to describe the data: G(τ) ) GD(τ)GF(τ) + 1 with GD(τ) )
1 1 N (1 + τ ⁄ τ ) 1 + τ ⁄ (γτ ) diff √ diff
and GF(τ) )
1
1-
∑
Aj
[
3
1-
]
∑ A [1 - exp( ⁄ )] τ
j
j)1
τj
3
where τ is the lag time, τdiff the diffusion time, τj the time constants of photophysical processes, and γ the aspect ratio of the confocal volume.
RESULTS AND DISCUSSION Fluorescence quenching in solution was explored by using either AT565tris-NTA or FEW646tris-NTA binding to the decahistidine-tag of OG488ifnar1-H10. Typical assays using spectral fluorescence detection are shown in Figure 2. Upon addition of AT565tris-NTA, the fluorescence of OG488ifnar1-H10 was quenched by more than 50% (Figure 2a). The same fluorescence quenching was observed in the presence of a 10-fold excess of unlabeled, tagless ifnar1-EC (ifnar1-tl), confirming specific and exclusive binding of AT565tris-NTA to the His-tag of ifnar1-H10 (data not shown). Moreover, no change in fluorescence was observed after scavenging excess AT565tris-NTA by adding 200 nM His-tagged maltose binding protein (MBP-H10) to the AT565 tris-NTA/OG488ifnar1-H10 complex, excluding nonspecific interaction of excess AT565tris-NTA with other regions of ifnar1H10. On the basis of the calculated Fo¨rster radius for this pair of fluorescence dyes (∼58 Å), this extent of fluorescence quenching suggests a distance of less than 6 nm between dye attached to N23 and the C-terminal His-tag. Strikingly, a substantial increase in fluorescence was observed upon binding of the ligand IFNR2-HEQ (∼70% of the signal before adding the quencher), suggesting a substantial spatial rearrangement of the Ig-like domains. Fluorescence dequenching by the ligand
binding was fully reversed upon adding unlabeled, tagless ifnar1EC (ifnar1-tl), corroborating that indeed ligand binding was responsible for this effect. No change in fluorescence intensity was observed upon ligand binding to OG488ifnar1-H10 without AT565 tris-NTA (Figure 2c), confirming that fluorescence dequenching was not caused by interaction of the ligand with the fluorophore. Also, changes in the signal of the acceptor fluorescence were observed upon addition of the ligand. However, owing to the rather high excess of AT565tris-NTA (250 nM) compared to the fluorescence donor (100 nM), the background signal from direct excitation of AT565 was already rather high. Furthermore, the quantum yield of AT565tris-NTA is rather low due to strong quenching (by ∼80%) of the fluorophore by the chelated Ni(II) ions (23). Similar experiments were carried out with FEW646tris-NTA as a quencher (Figure 2b). As expected, substantially lower decrease of the fluorescence signal was observed upon binding of FEW646tris-NTA to OG488ifnar1-H10 (∼25%, Figure 2b). Taking the theoretical Fo¨rster radius for this pair of dyes (∼47 Å) into account, this effect confirmed an effective distance of less than 6 nm between N23 and the N-terminus. Again, the fluorescence recovered substantially upon addition of IFNR2-HEQ (∼90% of the signal before adding the quencher), corroborating the spatial arrangement and the distance between the N-terminal Ig-like domain and the C-terminus. Dissociation of the FEW646trisNTA from the His-tag by addition of 50 mM imidazole resulted into a full recovery of the fluorescence signal, confirming that the changes in fluorescence were due to interactions between FEW646 tris-NTA and the His-tag. The role of Ni(II) ions for the quenching of the covalently attached dye was explored by complex formation with BTtrisNTA instead of the fluorescent tris-NTA conjugates. Strikingly, no change in fluorescence of OG488ifnar1-H10 was observed upon complex formation (Figure 2d). Stable binding of BTtris-NTA and thus incorporation of Ni(II) ions was confirmed by subsequent addition of 250 nM AT565tris-NTA, which did not quench the fluorescence under these conditions, because the decahistidine tag was already blocked by BTtris-NTA. These experiments furthermore corroborated the high specificity of the fluorescence quenching by site-specific incorporation through the His-tag. Ifnar1-H10 labeled with AF488 instead of OG488 as a fluorescence donor yielded similar results (Supporting Information), in agreement with the highly similar photophysical properties of AF488 and OG488. As a further control, as well as for calibrating the quenching signals, similar experiments were carried out with several single cysteine mutants of ifnar2H10, which were used for site-specific labeling at different positions within the molecule (cf. Figure 1e). Upon addition of FEW646 tris-NTA, the fluorescence decreased by ∼20% (Figure 2). Importantly, however, no recovery of the fluorescence was observed after ligand binding. This is in line with the rigid structure of the protein observed in NMR, which remained essentially unchanged upon ligand binding (32). These results corroborate that the fluorescence changes observed for ifnar1H10 were indeed due to conformational changes. Again, stronger quenching by ∼35% was observed for binding of AT565tris-NTA (spectra not shown). On the basis of the distances between the C-terminus of ifnar2-EC and the mutated residue as determined from the NMR structure of ifnar2-EC, the quenching amplitudes obtained for different mutants were used for a distance calibration. The relative quenching amplitudes obtained for FEW646trisNTA and AT565tris-NTA for different distances are compared in Figure 2f and fitted by the Fo¨rster function. From these calibration curves, Fo¨rster radii of 48 Å and 59 Å were estimated for FEW646tris-NTA and AT565tris-NTA, respectively, which were in excellent agreement with the Fo¨rster radii calculated from
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Figure 2. FRET experiments in solution. (a,b) Fluorescence spectrum of 100 nM OG488ifnar1-H10 before and after adding 250 nM AT565trisNTA (a) or FEW646tris-NTA (b), and upon addition of the ligand IFNR2-HEQ (1 µM). In (a), IFNR2 HEQ was competed out of the complex by addition of 5 µM ifnar1-tl; in (b), FEW646tris-NTA was competed out of the complex by addition of 50 mM imidazole. (c) Fluorescence of 100 nM OG488ifnar1-H10 without tris-NTA conjugate before and after addition of 1 µM IFNR2-HEQ. (d) Fluorescence of 100 nM OG488ifnar1H10 before and after addition of 250 nM BTtris-NTA, followed by the addition of 250 nM AT565tris-NTA. (e) Fluorescence spectrum of 100 nM OG488ifnar2-H10 S35C before and after adding 250 nM FEW646tris-NTA, and upon addition of the ligand (1 µM). (f) Comparison of relative quenching with FEW646tris-NTA and AT565tris-NTA for ifnar2-H10 labeled at three different positions and fit of the Fo¨rster equation. Table 1. Relative Quenching and Estimated Mean Intramolecular Distances in OG488ifnar1-H10 OG488
ifnar1-H10 a
AT565
tris-NTA tris-NTA
FEW646
OG488
ifnar1-H10/IFNR2-HEQ
Q [%]
r [Å]
b
Q [%]a
r [Å]b
55 ( 2 25 ( 2
56 ( 1 56 ( 1
30 ( 2 10 ( 2
67 ( 1 68 ( 1
a Relative loss in fluorescence compared to OG488ifnar1-H10. b Distance between donor and quencher as estimated from the Fo¨rster radii (47 Å for FEW646 tris-NTA and 58 Å for AT565tris-NTA).
absorbance and emission spectra assuming a quantum yield of 50% for the protein-bound OG488 (47 Å and 58 Å, respectively). On the basis of these Fo¨rster radii, the distances between N23 from the C-terminus before and after ligand binding were estimated (Table 1). Distances of ∼56 Å before and ∼67 Å (AT565tris-NTA) as well as ∼68 Å (FEW646tris-NTA) after ligand binding were obtained, highly consistent for both quencher. Thus, the N-terminal domain moves away from the C-terminal domain by ∼11-12 Å. The multidomain architecture and the ligand-induced conformational change suggested that a flexible organization of the Ig-like domains of ifnar1 may contribute to the intramolecular FRET. We, therefore, further explored fluorescence quenching and ligand-induced dequenching by fluorescence lifetime measurements (Figure 3a,b). An overall decrease of the fluorescence lifetime was observed upon binding of AT565tris-NTA (Figure 3a) as well as FEW646tris-NTA (Figure 3b), which was followed by a recovery upon ligand binding. The differences in the decrease and recovery of the fluorescence lifetimes qualitatively matched well with the spectral data. The fluorescence lifetime curves obtained with AT565tris-NTA yielded substantially stronger changes in the fluorescence lifetime compared to FEW646tris-NTA and were therefore analyzed in more detail. All samples showed a biexponential decay indicating a rather complex scenario. We interpreted this as a two-state environment of the attached label,
which might be either due to linker flexibility that allows the label to find two possible positions on the protein surface or due to conformational dynamics of the protein (see below). For OG488 ifnar1-H10 alone, we observed two lifetimes of ∼2.5 ns and ∼4.4 ns (cf. Supporting Information). After binding of AT565 tris-NTA, both lifetimes decreased (1.5 and 3.9 ns, respectively), but the effect was much more prominent in the case of the short-lifetime component. Upon addition of the ligand, both lifetimes increased (2.0 and 4.0 ns, respectively), and again the change in the short lifetime component was more pronounced. Using the previously estimated Fo¨rster radii, we could conclude that upon ligand binding the C-terminal His-tag moves by ∼12-14 Å away from the labeled position near the N-terminus (detailed in the Supporting Information), consistent with the conformational changes estimated from intensity changes. We further explored the conformational dynamics of ifnar1H10 by means of fluorescence correlation spectroscopy. FRET introduces an additional channel that de-excites the fluorescence of the fluorophore. For fluctuations of FRET efficiencies due to conformational dynamics, one expects to observe an additional decay term in the autocorrelation function. For this purpose, 1 nM AF488ifnar1-H10 mixed with 100 nM unlabeled ifnar1-H10 was incubated with 250 nM AT565tris-NTA, and fluorescence fluctuations in the confocal volume were followed by time-correlated single photon counting (TCSPC). Under these conditions, a similar excess of AT565tris-NTA was maintained as in the cuvette experiments, thus ensuring the same specificity and complete complex formation. Typical autocorrelation functions obtained for AF488ifnar1-H10 in different states are compared in Figure 3c and d. Normalization of the autocorrelation curves to individual fluorophores (data not shown) confirmed the intramolecular FRET signals obtained by ensemble measurements. A clear change in the shape of the correlation curve was observed upon binding of AT565tris-NTA (Figure 3c). Upon ligand binding, the autocorrelation curves
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Figure 3. Probing conformational dynamics by time-resolved fluorescence experiments. (a) Fluorescence lifetime of OG488ifnar1-H10 before and after adding AT565tris-NTA, and upon addition of the ligand IFNR2-HEQ. (b) Fluorescence lifetime of OG488ifnar1-H10 before and after adding FEW646 tris-NTA, after addition of the ligand IFNR2-HEQ, and after treatment with imidazole. (c) Autocorrelation curves for AF488ifnar1-H10, after binding of AT565tris-NTA, and after ligand binding (IFNR2-HEQ) to the AF488ifnar1-H10/AT565tris-NTA complex. The fits of the curves are shown as dotted lines. (d) Autocorrelation curves for AF488ifnar1-H10 without AT565tris-NTA before and after ligand binding (IFNR2-HEQ). The fit of the curves are shown as dotted lines.
recovered to a very similar behavior as observed prior to the binding of AT565tris-NTA. These observations suggest that an additional mode of dynamic quenching takes place in the AF488 ifnar1-H10/AT565tris-NTA complex, which is lost upon complex formation with the ligand. In contrast, no changes in the shape of the autocorrelation curves were observed without AT565 tris-NTA (Figure 3d). Fitting of the autocorrelation curves revealed a transition time of ∼200 µs, which we ascribe to conformational dynamics of ifnar1-H10 in the unliganded state. The amplitude of this photophysical decay term was 25% of the diffusional autocorrelation amplitude (Supporting Information Table 2). Very similar autocorrelation curves were obtained when the same experiments were carried out with OG488ifnar1H10 (data not shown). These results clearly corroborate a conformational flexibility of ifnar1-H10, which is largely lost upon ligand binding. This observation is in line with the fact that the three N-terminal domains simultaneously interact with the ligand, thus reducing the potential flexibility of the molecule in the linker between SD2 and SD3.
CONCLUSIONS These results demonstrate that noncovalent labeling by multivalent chelator heads efficiently complements cysteinespecific labeling for probing protein conformations. The key advantage of this approach is that an acceptor dye can be rapidly incorporated into the protein in situ in a quantitative
fashion without the addition of other reagents being required. Thus, the fluorescence of the covalently attached fluorescence donor can be readily quantified before and after attachment of the acceptor dye, and the FRET efficiencies can be quantified reliably from the donor quenching even if the donor dye is coupled at a low degree of labeling. Compared to other noncovalent labeling techniques, the localization of the fluorophore is very well defined, though not as well as for a dye chemically coupled to a cysteine residue. The method is therefore particularly suited to explore the domain organization of multidomain proteins, which was applied to the ectodomain of ifnar1 (26). Binding of tris-NTA/fluorophore conjugates to the His-tag is fast, quantitative, and reversible under mild conditions, and suitable for labeling in live cells (23, 33). Our studies demonstrate the elegance and versatility of labeling through tris-NTA/fluorophore conjugates and its compatibility with different fluorescence detection techniques including TCSPC-based FCS.
ACKNOWLEDGMENT We thank Thomas Gensch for help with the fluorescence lifetime instrument. This project was supported by grants to J.P. from the DFG (Emmy-Noether Program PI 405/1 and SFB 628), and from the Human Frontier Science Program (RGP60/2002). J.P. is a Heisenberg Professor funded by the DFG (PI 405/3) and was supported by the Cluster of Excellence “Macromo-
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lecular Complexes” at the Goethe University Frankfurt (DFG Project EXC 115). Supporting Information Available: Spectroscopic FRET experiments with AF488ifnar1-H10 and AT565tris-NTA and tabled fitting results for time-resolved fluorescence and FCS measurements. This material is available free of charge via the Internet at http://pubs.acs.org.
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