Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with (Ni(2+):nitrilotriacetic Acid (n)-fluorochrome conjugates.

Structural and mechanistic characterization of proteins by fluorescence resonance energy transfer (FRET)1,2 requires the ability to incorporate fluorescent probes at specific, defined sites.2 For proteins that do not contain cysteine residues, site-specific fluorescent labeling can be accomplished by use of site-directed mutagenesis to introduce a cysteine residue at the site of interest, followed by cysteine-specific chemical modification to incorporate the fluorescent probe.2 However, for proteins that contain cysteine residues (most proteins with MW > 50 kDa), site-specific fluorescent labeling is difficult. Three strategies have been reported: (i) intein-mediated labeling (“expressed protein ligation”),3 (ii) oxidation-mediated labeling,4 and (iii) trivalent-arsenicmediated labeling.5 The first two strategies are limited to labeling of protein termini and do not permit in situ labeling (i.e., direct labeling of proteins in cuvettes, gels, blots, or biological samplesswithout the need for a subsequent purification step); the third strategy currently is limited to a single fluorochrome. Here, we report a strategy that permits labeling of termini or internal sites, that permits in situ labeling, and that is compatible with a range of fluorochromes with different spectroscopic and photophysical properties. Our strategy involves use of the “hexahistidine tag”6,7,8s i.e., the amino acid sequence His6s to target site-specific fluorescent labeling. The hexahistidine tag is known to interact tightly with transition-metal complexes, including Ni2+:nitrilotriacetic acid (Ni2+:NTA).6-8 The hexahistidine tag can be introduced at protein termini or internal sites by using standard molecular-biology procedures6 and is widely used in molecular-biology research for affinity-chromatography-based protein purification [with (Ni2+:NTA)-agarose]6 and protein immobilization [with (Ni2+:NTA)-coated surfaces].6,7 We hypothesized that the hexahistidine tag should interact tightly with (Ni:NTA)n-fluorochrome conjugates and thus should be able to mediate site-specific fluorescent labeling (Figure 1). We further hypothesized, based on molecular modeling, that the hexahistidine tag should be able to interact with up to two Ni2+: NTA moieties without steric hindrance. To test these hypotheses, we prepared and analyzed (Ni:NTA)1-fluorochrome conjugates and (Ni:NTA)2-fluorochrome conjugates. We synthesized derivatives of the widely used cyanine fluorochromes Cy3 and Cy52,9 having one pendant Ni2+:NTAmoiety [(Ni:NTA)1-Cy3 and (Ni:NTA)1-Cy5; 1a and 1b in Figure 1a] or two pendant Ni2+:NTAmoieties [(Ni:NTA)2-Cy3 and (Ni:NTA)2-Cy5; 2a and 2b in Figure 1b] by reaction of monoand bissuccinimidyl-ester derivatives of Cy3 and Cy59 with N-(5-amino1-carboxypentyl)iminodiacetic acid,10 followed by reaction with NiCl2 (Figure 1a,b; Table 1). Fluorescence anisotropy experiments11,12 establish that 1a and 1b exhibit relatively low affinity for the hexahistidine tag (KD g 10 μM; Figure 2).11 * To whom correspondence should be addressed. E-mail: ebright@ mbcl.rutgers.edu. † Current address: Lawrence Berkeley National Laboratory, Berkeley, CA 94720. (1) Clegg, R. Curr. Opin. Biotechnol. 1995, 6, 103-110. (2) Selvin, P. Nat. Struct. Biol. 2000, 7, 730-734. (3) (a) Muir, T.; Sondhi, D.; Cole, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705-6710. (b) Ayers, B.; Blaschke, U.; Camarero, J.; Cotton, G.; Holford, M.; Muir, T. Biopolymers 2000, 51, 343-354. (c) Tolbert, T.; Wong, C.-H. J. Am. Chem. Soc. 2000, 122, 5421-5428. (d) Mukhopadhyay, J.; Kapanidis, A. N.; Mekler, V.; Kortkhonjia, E.; Ebright, Y. W.; Ebright, R. H. Cell 2001, 106, 453-463. (4) Geoghegan, K.; Stroh, J. Bioconjug. Chem. 1992, 3, 138-146. (5) (a) Griffin, B.; Adams, S.; Tsien, R. Science 1998, 281, 269-272. (b) Griffin, B.; Adams, S.; Jones, J.; Tsien, R. Methods Enzymol. 2000, 327, 565578. (6) (a) Hochuli, E.; Bannwarth, W.; Dobeli, H.; Gentz, R.; Stuber, D. BioTechnol. 1988, 6, 1321-1325. (b) Crowe, J.; Dobeli, H.; Gentz, R.; Hochuli, E.; Stuber, D.; Henco, K. Methods Mol. Biol. 1994, 31, 371-387. (7) (a) O’Shannessy, D. J.; O’Donnell, K.; Martin, J.; Brigham-Burke, M. Anal. Biochem. 1995, 229, 119-124. (b) Gershon, P.; Khilko, S. J. Immunol. Methods 1995, 183, 65-76. (c) Nieba, L.; Nieba-Axmann, S.; Persson, A.; Hamalainen, M.; Edebratt, F.; Hansson, A.; Lidholm, J.; Magnusson, K.; Karlsson, A.; Plückthun, A. Anal. Biochem. 1997, 252, 217-228. (8) (a) Kienberger, F.; Kada, G.; Gruber, H.; Pastushenko, V.; Riener, C.; Trieb, M.; Knaus, H.-G.; Schindler, H.; Hinterdorfer, P. Single Mol. 2000, 1, 59-65. (b) Schmitt, L.; Ludwig, M.; Gaub, H.; Tampe, R. Biophys. J. 2000, 78, 3275-3285. (9) Mujumdar, R.; Ernst, L.; Mujumdar, S.; Lewis, C.; Waggoner, A. Bioconjug. Chem. 1993, 4, 105-111. (10) Hochuli, E.; Dobeli, H.; Schacher, A. J. Chromatogr. 1987, 411, 177184. Figure 1. (Ni:NTA)n derivatives of cyanine fluorochromes. (a) Synthesis of (Ni-NTA)1-Cy3 (1a) and (Ni-NTA)1-Cy5 (1b). (b) Synthesis of (Ni-NTA)2-Cy3 (2a) and (Ni-NTA)2-Cy5 (2b). (c) Schematic representation of the mode of interaction of 2a or 2b with the hexahistidine tag.