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 confor- mational 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 chymot- rypsin 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. 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(2- nitrobenzoic 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 * 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: pyc@gate.sinica.edu.tw. Wunshain Fann, Institute of Atomic and Molecular Sciences, Academia Sinica, No. 1, Sec. 4, Roosevelt Rd, Taipei, 10617, Taiwan; Tel. +886-2- 23668237, E-mail: fann@gate.sinica.edu.tw. § These authors made an equal contribution. † Institute of Biological Chemistry, Academia Sinica. ‡ Institute of Atomic and Molecular Sciences, Academia Sinica. | Tunghai University. Bioconjugate Chem. 2008, 19, 1124–1126 1124 10.1021/bc700480j CCC: $40.75  2008 American Chemical Society Published on Web 05/29/2008