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Bioconjugate Chem. 2008, 19, 1124–1126
Strategy for Efficient Site-Specific FRET-Dye Labeling of Ubiquitin Michael Wen-Pin Kao,§,† Li-Ling Yang,§,‡ Jacky Chih-Kai Lin,† Tsong-Shin Lim,| Wunshain Fann,*,‡ and Rita P.-Y. Chen*,† Institute of Biological Chemistry, Academia Sinica, Taipei 115, Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, and Department of Physics, Tunghai University, Taichung 407, Taiwan, R. O. C. Received December 27, 2007; Revised Manuscript Received May 1, 2008
To study conformational changes within a single protein molecule, sp-FRET (single pair fluorescence resonance energy transfer) is an important technique to provide distance information. However, incorporating donor and acceptor dyes into the same protein molecule is not an easy task. Here, we report a strategy for the efficient double-labeling of a protein on a solid support. An ubiquitin mutant with two Cys mutations, one with high solvent accessibility and the other with low solvent accessibility, was constructed. The protein was bound to magnetic beads and reacted with the dyes. The first dye reacted with the side-chain of the Cys with the high solvent accessibility and the second with the other Cys under partially denaturing conditions. Using this method, we can easily label two dyes in a site-specific way on ubiquitin with a satisfied yield. The labeling sites for donor and acceptor dyes can be easily swapped.
As a spectroscopic ruler, the FRET (fluorescence resonance energy transfer) technique can provide invaluable information on structural dynamics and has been widely used in studying protein-protein interactions using donor and acceptor dyes attached to different protein molecules (1, 2). To study conformational changes within a single molecule, two dyes have to be added to the same molecule, which is a challenge in the case of proteins (3, 4). Cysteine (Cys) is the most common residue used for labeling due to its unique functional group. Labeling a short peptide with two dyes is straightforward and can be achieved by synthesizing the peptide chemically and adding the dye during the procedure (5). In contrast, labeling a protein with two different dyes is more difficult. Small labeled proteins can be obtained by chemical ligation of a short labeled segment to the rest of the protein (6). For big proteins, one method is to mix the protein with both dyes and purify the double-labeled protein from unlabeled protein, donor-labeled protein (labeled at either one or two sites), and acceptor-labeled protein (labeled at either one or two sites) (7–9). Another more popular method for protein labeling is to add one dye first, then react the labeled protein with the second dye. (10, 15) This method has been used to double-label the cold shock protein (Csp) with Alexa-488 and Alexa-594 (13, 14, 16) and chymotrypsin inhibitor 2 (CI2) and acyl-CoA binding protein (ACBP) with Alexa-532 and Alexa-647 (15). One disadvantage of this method is that protein labeled at a single site with the first dye must be separated from the unlabeled protein and protein labeled at both sites with the first dye. To avoid the formation of the two sites-labeled protein, a substoichiometric quantity of the first dye is usually used, which makes the coupling yield low. * To whom correspondence could be addressed. Rita P.-Y. Chen, Institute of Biological Chemistry, Academia Sinica, No. 128, Sec. 2, Academia Rd, Nankang, Taipei, 115, Taiwan; Tel. +886-2-2785-5696, Fax +886-2-2788-9759, E-mail:
[email protected]. Wunshain Fann, Institute of Atomic and Molecular Sciences, Academia Sinica, No. 1, Sec. 4, Roosevelt Rd, Taipei, 10617, Taiwan; Tel. +886-223668237, E-mail:
[email protected]. § These authors made an equal contribution. † Institute of Biological Chemistry, Academia Sinica. ‡ Institute of Atomic and Molecular Sciences, Academia Sinica. | Tunghai University.
However, it cannot control the position where the first dye is added. Hong and Maret (17) labeled metallothionein in a more specific way. The donor dye, Alexa-488, was added to the N-terminal amino group of metallothionein and the acceptor dye, Alexa-546, was reacted with the Cys residue in the linker region of the protein. Ha et al. (18) also used this method to label the Cys residue and N-terminus of Staphylococcal nuclease with TMR and Cy5, respectively. Here, we report a strategy for the efficient double-labeling of a protein on a solid support (Scheme 1). The choice of labeling sites is the most critical point in making the labeling more site-specific. Because the yield of the labeling reaction depends on the degree of exposure of the sulfhydryl group of the Cys residue to the environment, we can easily add the first dye to a more exposed Cys, then add the second to a less exposed Cys with the aid of denaturant. Haas and co-workers (11) measured the reactivity of six Cys in adenylate kinase by determining the rate constants for the reaction of the six single-Cys mutants with 5,5′-dithiobis(2nitrobenzoic acid), then chose one highly reactive cysteine and one less reactive cysteine as the labeling sites. Based on the measured reactivity, they showed that the reactivity of reaction sites could be predicted using a computer program (19). Here, we chose the 76 amino acid protein, ubiquitin, which has no cysteine residues, as our target and used commercial software to predict the side-chain solvent accessibility of different sites after mutation to Cys. We aimed to add one Cys in the N-terminal region and one in the C-terminal region. One ubiquitin mutant with a single Cys insertion between Met1 and Gln2, named m[C]q, was predicted, using DiscoVery Studio software (version 1.7, Accelrys, USA), to have a higher solvent accessibility. Its side-chain solvent accessible surface was predicted as 56.1 ( 8.7 Å and the percentage of residue solvent accessibility as 46.5 ( 7.3% based on 20 energy-minimized structures. A Ser65 f Cys mutant, named S65C, had a predicted side-chain solvent accessible surface of 28.0 ( 7.0 Å and a percentage of residue solvent accessibility of 21.0 ( 5.4%. Another important feature of our strategy is the use of magnetic beads, which can tolerate high concentrations of denaturants, such as 6 M GdnHCl and 8 M urea. The advantages of carrying out the reaction on a solid support include the
10.1021/bc700480j CCC: $40.75 2008 American Chemical Society Published on Web 05/29/2008
Communications
Bioconjugate Chem., Vol. 19, No. 6, 2008 1125
Scheme 1. Design of Labeling Strategy
Figure 1. HPLC profiles of the dye coupling reactions of the proteins m[C]q (A) and S65C (B). The samples were analyzed by HPLC on a Vydac C18 column and the proteins detected by their absorbance at 220 nm. The proteins were resolved using a 30 min gradient of acetonitrile (15% for 5 min; 15-27% for 5 min; 27-35% for 20 min) in 0.1% trifluoroacetic acid.
following: (1) avoiding disulfide bond formation in the protein; (2) easy removal of free dye after reaction for reuse, thus saving on cost; and (3) avoiding protein aggregation under partially denaturing conditions. A 6-His tail was added to the end of ubiquitin for bead binding. For the dye pair, we selected Alexa Fluor 488 and Alexa Fluor 594 as our donor and acceptor dyes, respectively. The mutant proteins m[C]q and S65C were expressed separately. As predicted, m[C]q was easily labeled with dye. Met1 in m[C]q was found to be removed by cell proteases, but its removal did not affect the reactivity of Cys2 with reagent. The dye coupling reaction was almost complete within two hours (Figure 1A). In contrast, S65C could not be labeled without the aid of denaturant (Figure 1B). When 4 M GdnHCl was added to the reaction mixture, the coupling yield of S65C was g76%. A mutant for double-labeling needs one Cys with high solvent accessibility and one with low solvent accessibility. These two sites with different reactivities were therefore selected as our labeling sites. The double mutant protein, named m[C]q/S65C, was constructed and expressed and showed the same structure and structural stability as wild-type ubiquitin using circular dichroism spectroscopy and nuclear magnetic resonance (see Supporting Information). Importantly, our method can also be used to easily swap the dye labeling positions. When Alexa-488 was added first and Alexa-594 second to m[C]q/S65C, a double-labeled protein with the donor dye at the N-terminal end and the acceptor dye at the C-terminal end, denoted A488-m[C]q/S65C-A594, was obtained. The emission spectra of A488-m[C]q/S65C-A594 showed acceptor emission due to energy transfer when the donor was excited (Figure 2A). When the labels were swapped and Alexa594 added to m[C]q/S65C before Alexa-488, the protein A594m[C]q/S65C-A488 was obtained. The excitation spectra of
Figure 2. (A) Fluorescence emission scan of A488-m[C]q/S65C-A594 and A488-m[C]q/S65C. The excitation wavelength is 493 nm. (B) Fluorescence-excitation scan of A488-m[C]q/S65C-A594 and A594m[C]q/S65C-A488. The emission wavelength is 620 nm. Samples were dissolved in distilled water with 0.1% Tween-20 and 5 mM DABCO (1,4-diazabicyclo[2.2.2]octane).
A488-m[C]q/S65C-A594 and A594-m[C]q/S65C-A488 were compared in Figure 2B. From the comparison of the spectra, different contributions of the donor dye in the acceptor
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fluorescence suggested that the dye-labeling position can affect the efficiency of energy transfer. In protein-labeling studies, heterogeneity of the donor and acceptor labeling sites can only be ignored if each dye does not interact with the surface of the protein and is able to rotate freely or if the interaction does not interfere with the photophysics of the fluorophores and the stability of the protein. Using the normal Cys-labeling strategy, site-specific labeling is not easy to achieve, but our strategy makes this possible and can also be used to make the reversely labeled protein for comparison.
ACKNOWLEDGMENT This work was supported by an Academia Sinica program project (AS-94-TP-A01). We are grateful for the technical support of Dr. Huei-Chun Chang of the Biophysical Instrumentation Laboratory in the Institute of Biological Chemistry. We thank Dr. Kay-Hooi Khoo and Ms. Chi-Chi Chou for the mass analysis. Proteomic mass spectrometry analyses were performed by the Core Facilities for Proteomics Research located at the Institute of Biological Chemistry, Academia Sinica, supported by a National Science Council grant (NSC 95-3112-B-001-014) and the Academia Sinica. The NMR spectra were obtained at the High-Field Nuclear Magnetic Resonance Center (HFNMRC) supported by the National Research Program for Genomic Medicine. Supporting Information Available: Methods for protein expression, dye labeling, and computer calculation; circular dichroism spectra of ubiquitin, m[C]q/S65C, A488-m[C]q/S65C and A488-m[C]q/S65C-A594 (Figure S1); denaturation curves of ubiquitin, m[C]q/S65C, A488-m[C]q/S65C and A488-m[C]q/ S65C-A594 (Figure S2); TOCSY spectra of ubiquitin and m[C]q/S65C (Figure S3); kinetic trace of A488-m[C]q/S65CA594 during refolding (Figure S4); mass results of m[C]q/S65C, A488-m[C]q/S65C, and A488-m[C]q/S65C-A594 after trypsin digestion (Figure S5); electrophoresis images of ubiquitin and its mutants with and without dye-labeling (Figure S6). This material is available free of charge via the Internet at http:// pubs.acs.org.
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