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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA
Reprint requests to: Homme W. Hellinga, Duke University Medical Center, Department of Biochemistry, Box 3711, Research Drive, 415 Nanaline Duke Building, Durham, NC 27710, USA; e-mail: hwh{at}biochem.duke.edu; fax: (919) 684-8885.
(RECEIVED July 2, 2004; FINAL REVISION August 30, 2004; ACCEPTED August 30, 2004)
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Keywords: covalent modification; biosensor; fluorescence resonance energy transfer; surface immobilization
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04965405.
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Introduction |
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TOPAbstractIntroductionResultsMaterials and methodsReferences |
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Cysteines that are oxidized in a disulfide bridge, or that coordinate to a metal, are often protected from covalent modification by thiol-reactive reagents. Protection is readily reversed by reduction or metal chelation. It is therefore possible to engineer proteins with multiple, independently addressable, site-specific covalent attachment points by constructing several cysteines that are (1) unprotected, (2) react to form a disulfide, and (3) participate in metal binding. We have developed a strategy that allows two or three sites to be independently modified by fusing a protein with a single, unprotected cysteine with one or two small domains that contain a Zn2+-binding site or a disulfide bridge (Fig. 1
). These fusion domains are based on a consensus zinc-finger domain, ZifQNK (Shi and Berg 1995). This 32-residue domain has a Cys2His2 primary coordination sphere that binds Zn2+ reversibly with 10–9–10–11 M affinity (Michael et al. 1992). In the absence of Zn2+, the two cysteines can form a disulfide under oxidizing conditions (Knapp and Klann 2000). ZifQNK can therefore be used in either metal-dependent or redox-dependent RTP strategies (MRTP, RRTP). Additionally, we used a truncated, 18-residue version of ZifQNK (
ZIF) in which the single 
-helix bearing the two histidines has been deleted, leaving a two-stranded -sheet containing the two cysteines that readily oxidize to form a disulfide, but do not bind Zn2+ in the reduced form. Thus, ZIF can be used in a RRTP strategy.
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ZIF at the N or C termini of MBP, and demonstrate that these can be used to obtain ligand-responsive FRET between donor and acceptor fluorophores site specifically coupled at position 141 within MBP (MBP141) and the fusion domain. We also construct a FRET relay (Watrob et al. 2003) between three fluorophores in a triply labeled, double-fusion protein. The immobilization of proteins on glass, gold, or other nonbiological substrates is an important aspect of constructing hybrid devices such as biosensors (Willner and Katz 2000, 2003; Willner et al. 2002); it is also an increasingly important component for the construction of protein chips used in genome analysis technologies (Figeys and Pinto 2001). Orientation-specific immobilization using defined attachment points on a protein has numerous advantages over random, multipoint chemi- or physisorption (Lu et al. 1996; Rao et al. 1998; Turkova 1999), especially in cases where binding sites need to be presented, or conformational changes are taken advantage of, such as is the case for the proteins presented in this work. Again, site-specific thiol-mediated covalent linkage strategies offer advantages over noncovalent site-specific linkages such as provided by a oligohistidine C- or N-terminal fusions (Gershon and Khilko 1995; Allard et al. 2002). Here we demonstrate that GBP first labeled with a fluorophore at the unprotected cysteine 149 can be patterned on a glass slide by covalent coupling using reversibly protected cysteines in a ZifQNK fusion peptide.
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Results |
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ZIF to protein with a single, unprotected cysteine (Fig. 1
). In the case of ZifQNK either MRTP, or RRTP strategies can be used; for ZIF, only RRTP is possible. Independent triple labeling can be achieved using a fusion with both ZifQNK (MRTP) and ZifQNK (RRTP).
Differential reactivity of engineered thiols
The multiple labeling scheme requires that protected thiols are significantly less reactive than unprotected thiols, and that protection is reversible. To test this, we reacted cysteine-free MBP (MBPwt), MBP141, MBPwt fused at the C terminus with ZifQNK in the Zn2+ form (MBPwt::ZifQNK•Zn), in the Zn2+-free oxidized form (MBPwt::ZifQNKox), and in the Zn2+-free reduced form (MBPwt::ZifQNKred) with 7-diethylamino-3-(4'maleimidylphenyl)-4-methyloumarin (CPM). CPM becomes fluorescent upon covalent conjugation (Parvari et al. 1983). The reactions were carried out in parallel under typical conditions used for labeling proteins, measuring the increase in fluorescence upon formation of the conjugate (Table 1). Cysteine-free MBPwt shows very slight reactivity, presumably due to reaction with surface lysines, since maleimides react slowly with primary amines as well as thiols (Hermanson 1996). The metal- and oxidatively-protected thiols in MBPwt::ZifQNK•Zn and MBPwt::ZifQNKox react with CPM at the same very slow rate as detected for the thiol-free protein. The unprotected thiols in MBP141, and MBPwt::ZifQNKred react 10,000-fold more rapidly than the protected thiols, with the reaction being >95% complete in 10 min or 30 min, respectively. Both metal- and redox-mediated strategies therefore provide excellent protection and are readily reversible.
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After the first reaction, the protein:fluorophore ratio was determined by absorbance spectroscopy, and was found to be approximately 1:1 in all four cases, consistent with complete reaction of the unprotected thiol in MBP141, and full protection of the two thiols in the ZifQNKox or ZifQNK•Zn2+ domain. In the second reaction, the ZifQNK was first deprotected by addition of chelator or reductant, and reacted with the other fluorophore. The stoichiometry of the reaction was determined by absorbance spectroscopy and mass spectrometry (Fig. 2; Table 2). In all four cases, the ratios were 1:1:2 for protein:fluorophore 1:fluorophore 2, consistent with the expected labeling pattern. The masses were also as expected for the appropriately labeled protein (Table 2). We also separated the labeled MBP141 and ZifQNK domains by thrombin cleavage of the linker peptide to determine the degree of mislabeling (first fluorophore on ZifQNK; second fluorophore on MBP141) by the optical absorbance and retention times of the fragments (Fig. 2). In all four cases, no evidence of mislabeling was observed. Taken together, these results are therefore consistent with the intended, site-specific, double-labeling patterns, and show that both redox- and metal-mediated reversible thiol protection strategies work well with ZifQNK.
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). The distances between the attached fluorophores is expected to be less in the ligand-bound closed conformation than in the open conformation of the apo-protein. It is therefore likely that orientation, rather than distant-dependent effects, dominate the FRET mechanism in this system (Lakowicz 1999). Furthermore, the magnitude of the change differs in the two constructs: MBP141(TMR)::tb::ZifQNK(Cy5)2 shows a threefold change in the ratio of the donor:acceptor emission intensities upon addition of maltose, whereas MBP141(Cy5)::tb::ZifQNK(TMR)2 shows only a 0.1-fold change. The maltose affinities of the labeled and unlabeled proteins are similar (Fig. 3), indicating that the two fluorophores did not significantly perturb the conversion between the open and closed conformations.
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Zif fused to the N terminus, and ZifQNK to the C terminus, using a thrombin-cleavable linker peptide in each case (Zif::tb::MBP141::tb::ZifQNK). Zif and ZifQNK form an orthogonally protected pair: Redox-mediated protection has to be used for Zif, mandating the metal-mediated strategy for ZifQNK in this case. The order in which modifications and deprotections are carried out is important: (1) The unreacted thiol is modified; (2) Zifox is deprotected by reduction, and modified; (3) ZifQNK•Zn2+ is deprotected by chelation, and modified. Steps 2 and 3 cannot be inverted, because deprotection of ZifQNK•Zn2+ requires addition of reductant, which would also deprotect Zifox.
Cy5, TMR, and 5-iodoacetamide fluoroscein (IAF) were used as the labels. Two proteins with different labeling patterns were prepared using the appropriate order of modification and deprotection steps: Zif(IAF)2::tb::MPB141(Cy5)::tb::ZifQNK(TMR)2 and Zif(IAF)2::tb::MPB141(TMR)::tb::ZifQNK(Cy5)2. Labeling stoichiometries were determined by absorbance spectroscopy for the single and double modifications, but not for the triply labeled proteins, due to the spectral overlap of TMR and IAF (Fig. 4A). The stoichiometry was also confirmed by measuring the mass of triple modified protein (Table 2). We determined the degree of mislabeling by cleaving both N- and C-terminal fusions with thrombin and separating the labeled products on HPLC (data not shown). The unprotected cysteine and the ZifQNK cysteines were exclusively modified with the correct fluorophores. The Zif cysteines were correctly labeled with at least one IAF. The IAF reaction did not quite reach completion (~90%), however, leaving the second cysteine in some of the Zif fusions free to react with the fluorophore in the third modification.
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). As predicted, Zif(IAF)2::tb::MPB141(Cy5)::tb::ZifQNK(TMR)2 demonstrated a complete FRET relay but Zif(IAF)2::tb::MPB141(TMR):: tb::ZifQNK(Cy5)2 did not, presumably because the separation between IAF and TMR is within the Förster distance in Zif(IAF)2::tb::MPB141(Cy5)::tb::ZifQNK(TMR)2 (42 Å) but exceeds the Förster distance in Zif(IAF)2::tb::MPB141(TMR)::tb::ZifQNK(Cy5)2 (61 Å). FRET between TMR and Cy5 still occurs in Zif(IAF)2::tb::MPB141(TMR)::tb::ZifQNK(Cy5)2 when TMR is excited (50 Å). The FRET relay demonstrated a maltose-dependent decrease (Fig. 4B).
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). The BMOE pattern was generated by protecting thiol silane from photooxidation with a 10-µm beehive mask as described in Materials and Methods. An image of a slide prepared with a Cy5-modified GBP lacking the ZifQNK fusion was also taken (Fig. 5B). As can be seen, the GBP149(Cy5)::ZifQNK gave the expected square grid pattern corresponding to reaction with the maleimide, whereas the pattern produced by the control protein was significantly dimmer, and is consistent with physisorption of the protein in the irradiated squares where there is a preponderance of negatively charged groups resulting from photooxidation (Bhatia et al. 1992).
Conclusions
We have demonstrated that fusions with one or two zinc finger derivatives allow two or three sites to be modified independently by reversible thiol protection schemes that exploit metal coordination or disulfide formation. We demonstrated that both methods produce orthogonal protein modifications with no apparent mislabeling. Both MBP141(TMR)::th::ZifQNK(Cy5)2 and MBP141(Cy5)::th:: ZifQNK(TMR)2 were rapidly produced by simply switching the order of reactants, unlike many competing methods which require additional synthesis steps (Hofmann and Muir 2002; Zhang et al. 2003).
Both labeling combinations resulted in ligand-induced FRET decreases. MBP141(TMR)::th::ZifQNK(Cy5)2, in particular, generated a larger ligand-mediated signal change than any previously reported intramolecular FRET biosensor (Hofmann et al. 2001; Fehr et al. 2002, 2003; Lager et al. 2003). The large FRET change cannot be explained in terms of distance-dependent effects because the distance change is too small and because the separation between fluorophores gets smaller upon ligand binding, which should produce an increase rather than a decrease in FRET. Instead, we propose that the observed FRET change is due to an orientation effect (Lakowicz 1999). The 2:1 ratio of fluorophores did not appear to interfere with FRET or correct immobilization. We have also demonstrated that both protection methods can be combined to triple modify proteins, and in this case, produce an intramolecular protein FRET relay. FRET relays have utility in overcoming large distances (Watrob et al. 2003) and provide large Stokes shifts. Another use for the triple modification strategy may be to immobilize a FRET biosensor to produce a ratiometric device. We have demonstrated that different modifications can be combined to immobilize Cy5 modified proteins in an orientation-specific pattern.
ZifQNK and Zif fusions are a rapid, straightforward way to add functionalities to almost any protein. The protection strategies are not limited to these domains, however. Other suitable domains containing disulfides or stable metal centers can be used. Furthermore, the metal-mediated protection scheme could be extended to any thiol protected by a tightly binding ligand. Finally, the approach can be even further generalized by using design methods to introduce disulfides (Ivens et al. 2002; Nemeth et al. 2002), metal centers (Hellinga 1998), or ligand binding sites (Looger et al. 2003) in suitable locations.
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Materials and methods |
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Zif N-terminal fusions with the thrombin cleavage sites were: GLVPR|GSTGEKPYKCPECGKSFSRSDHLSRHQRTHQNKKG SHHHHHH and MTGEKPYKCPECGKSFSRSLVPR|GSGG, respectively (cysteines indicated in bold; linker peptide underlined; thrombin recognition site italicized; cleavage site indicated with |). The C-terminal zinc finger fusion was generated by PCR using the following oligonucleotides:
5'-GGAGGTTCAACAGGTGAGAAACCGTACAAGTGCC CGGAGTGTGGCAAATCATTCTCTCGATCGGACCAT, 5'-CG GGATCCTATCACTTCTTGTTCTGATGTGTCCGTTGGTGACG GGATAGATGGTCCGATCGAGAGAATG, and 5'-CTCACCTG TTGAACCTCCCTTGGTCAGCTTAGTCTG. The N-terminal Zif was constructed by PCR using the following oligonucleotides: 5'-GGAATTCCATATGACAGGTGAGAAACCGTACAAGTG CCCGGAGTGTGGC and 5'-CCTTCTTCGATTTTGCCCCCGG ATCCTCGAGGGACGAGCGATCGAGAGAATGATTTGCCA CACTCCGGGCA. Wild-type MBP was used as a template to generate the zinc-finger fusions. The MBP A141C mutant was generated by PCR using the following oligonucleotides: 5'-GAAC TGAATGCAAAGGTAAGAGCGCG and 5'-CGCGCTCTTACC TTTGCATTTCAGTTC. All recombinant constructs were cloned into pET21a for expression.
Protein expression and purification
Recombinant proteins were overexpressed in BL21(DE3). One liter of 2xYT was inoculated with 25 mL from a culture freshly grown to stationary phase (9 h), and grown at 37°C to an optical density of A600 = 0.4, induced with 1 mM IPTG, and grown for a further 2 h. The cultures were supplemented with 100 µM ZnCl2 at induction to ensure viability. For MBP fusions, cell pellets were resuspended in IMAC buffer (20 mM MOPS, 500 mM NaCl, 10 mM imidazole [pH 7.5]), lysed by sonication (2 min), and a cleared lysate produced by centrifugation (25 min, 25,000g). The MBP fusions were purified using nickel-charged IMAC resin followed by gel filtration (Superdex 200). Pure protein was quantified by absorbance (
280 = 66,000 M–1cm–1).
Labeling reaction kinetics
Proteins (1 µM in 50 mM MOPS, 100 mM NaCl [pH 6.0]) were reacted with a fivefold molar excess of CPM (concentrated stock solution in DMSO). The labeling reaction was monitored by following the increase in fluorescence at 470 nm (excitation 385 nm) for the CPM-protein conjugate as a function of time using a fluorescence plate reader (SprectraMAX GeminiXS, Molecular Devices). The values for t1/2 were obtained from fits of the data using a commercial software package (TableCurve 2D, SYSTAT Software, Inc.). All experiments were conducted at 25°C.
Metal-mediated reversible thiol protection
Proteins were exchanged from purification buffer into modification buffer (50 mM MOPS, 100 mM NaCl [pH 6.0]) by gel filtration (Superdex 200). For the first modification (unprotected thiol), 25 µM protein was incubated (room temperature, 30 min; agitated with a roller drum) with 125 µM TCEP, 100 µM ZnCl2, and 250 µM tetramethylrhodamine 5-maleimide or Cy5 dye in a total volume of 1 mL. The reaction then was transferred to a desalting column (BioRad PD10) preequilibrated with modification buffer, collecting the first colored band (modified protein). The labeling efficiency of the first modification was determined as described below. The second pair of thiols were deprotected by chelation in the presence of 5 mM EDTA and 2 mM orthophenathroline (4°C; 8 h). Following removal of the chelators by gel filtration (Superdex 200), the protein was labeled with 500 µM TMR or Cy5 dye in the presence of 250 µM TCEP, (1-mL reaction volume; 1 h, room temperature; agitated on a roller drum). Unincorporated label was removed by a desalting column (BioRad PD10), eluting with 50 mM MOPS, 100 mM NaCl (pH 6.8).
Redox-mediated reversible thiol protection
To chelate any free metal, purified protein was first incubated with 5 mM EDTA and 2 mM o-phenanthroline (4°C, 8 h), followed by exchange into 20 mM Tris, 100 mM NaCl (pH 6.0) on a S200 gel filtration column. In these preparations, the disulfide in the ZifQNK peptide was completely oxidized, as determined by DTMB reactivity. For the first modification (unprotected thiol), 25 µM protein was incubated with 250 µM TMR or Cy5 dye (1-mL reaction volume; room temperature for 30 min; agitated on a roller drum). Free fluorophore was removed by desalting column (see above), and the labeling efficiency was determined as described below. Deprotection by reduction and dye modification were carried out in one step by the addition of 250 µM TCEP and 500 µM Cy5 or TMR (1 h at room temperature). Unreacted material was removed by desalting column (see above).
Triple modification
The unprotected thiol was labeled first using 25 µM protein and 250 µM Cy5 (30 min at room temperature; agitated on a roller drum). After removing unreacted fluorophore by gel filtration (see above), the Zif domain was deprotected and labeled (125 µM TCEP and 250 µM 5-IAF; 30 min at room temperature). Excess 5-IAF was removed by gel filtration. The ZifQNK domain was deprotected by chelation with 5 mM EDTA and 2 mM o-phenanthroline (8 h at 4°C), followed by gel filtration and labeling protein with 150 µM TCEP and 250 µM TMR. The triple labeled product was purified from excess fluorophore by gel filtration (see above).
Determination of fluorophore labeling stoichiometry
Dye–protein ratios were determined using:
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where Afluor. is the absorbance at 650 nm for Cy5 and 525 nm for TMR, Aprotein is the absorbance at 280 nm, protein = 66,000 M–1cm–1, fluor. is 250,000 M–1cm–1 for Cy5, 95,000 M–1cm–1 for TMR and 75,000 M–1cm–1 for 5-IAF, and N is 0.05 (Amersham Biosciences) for Cy5 and 0.3 for TMR.
The equation for dye/dye ratios was:
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where Afluor1 is the absorbance for fluorophore 1, Afluor2 is the absorbance for fluorophore 2, fluor1 is the extinction coefficient for fluorophore 1, and fluor2 is the extinction coefficient for fluorophore 2.
Thrombin cleavage and HPLC purification
Protein was cleaved with biotinylated thrombin according to the manufacturer’s protocol (Novagen Thrombin Cleavage Capture Kit). The cleavage products were separated by HPLC (Waters 2795 Alliance HT, PDA detector) using a C4 reversed phase column (Symmetry 300), eluting with a linear gradient from 20% B to 100% B over 80 min at a flow rate of 1 mL/min (A = water with 0.1% TFA; B = acetonitrile with 0.1% TFA). Peaks were identified by absorbance and elution times. Assignments were confirmed by MALDI-TOF mass spectrometry (Applied Biosystems, Voyager DE).
Fluorescence spectroscopy
Fluorescence emission intensities were measured at 25°C in a stirred 1-cm quartz cell using a fluorimeter (AMINCO Bowman Series 2). Protein samples were diluted to 0.2 µM using 20 mM MOPS, 100 mM NaCl (pH 7.0 buffer). Excitation for TMR and IAF was 530 nm and 490 nm, respectively. Fluorescence emission spectra were collected from 550 nm to 700 nm.
Protein immobilization and confocal imaging
A glass slide was silanized with a 20:1 ratio of bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane:3-mercaptopropyltrimethoxysilane. A pattern was then produced by photooxidation of the 3-mercapto-propyltrimethoxysilane with short wavelength ultraviolet irradiation for 5 min in the presence of a copper mask (10-µm square beehive). Thiols that were protected from photooxidation by the mask were reacted with a homobifuctional crosslinker, bis-maleimidoethane (BMOE). The cysteines in ZifQNK were then de-protected with TCEP, and the GBP149(Cy5)::ZifQNK incubated with the slide to react with the maleimide of BMOE. After 1 h, the substrate was washed with buffer to remove uncoupled protein, and imaged using a Zeiss LSM-410 confocal microscope.
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References |
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