Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein

S.-H. Hong1,2, Q. Hao3 and W. Maret1,3,4

1Center for Biochemical and Biophysical Sciences and Medicine, Department of Pathology, Harvard Medical School, One Kendall Square, Cambridge, MA 02139 and 3Departments of Preventive Medicine and Community Health and Anesthesiology, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA 2Present address: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

4 To whom correspondence should be addressed at the Department of Preventive Medicine and Community Health, The University of Texas Medical Branch E-mail: womaret{at}utmb.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Each of the two domains of mammalian metallothioneins contains a zinc–thiolate cluster. Employing site-directed mutagenesis and chemical modification, fluorescent probes were introduced into human metallothionein (isoform 2) with minimal perturbations of the structures of these clusters. The resulting FRET (fluorescence resonance energy transfer) sensors are specific for each domain. The design and construction of a sensor for the {alpha}-domain cluster is based on a FRET pair where a C-terminally added tryptophan serves as the donor for a fluorescence acceptor attached to a free cysteine in the linker region between the two domains. Molecular modeling studies and steady-state fluorescence polarization anisotropy measurements suggest unrestricted motion of the tryptophan donor, but limited motion of the AEDANS ({[(amino)ethyl]amino}naphthalene-1-sulfonic acid) acceptor, putting constraints on the use of the {alpha}-domain sensor with this FRET pair as a spectroscopic ruler. The fluorescent metallothioneins allow distance measurements during binding and removal of metals in the individual domains. The overall dimensions of the apoprotein, thionein, for which no structural information is available, do not seem to be significantly different from those of the holoprotein. The single- and double-labeled fluorescent metallothioneins overcome a longstanding impediment in studies of the function of this protein, namely its lack of intrinsic probe characteristics.

Keywords: fluorescent probes/FRET sensor/metallothionein/site-directed mutagenesis/zinc–thiolate clusters


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Visualizing molecular processes by imaging techniques is at the forefront of approaches that aim to understand the function of biomolecules at the single molecule level and in living cells. We have been interested in introducing fluorescent probes into mammalian metallothioneins (MTs) for this purpose. These relatively small proteins have 60+ amino acids. Among those, 20 are cysteines that are the only ligands for seven divalent transition metal ions in two metal–thiolate clusters. Mammalian MTs are involved in the control of normal cellular metabolism and adaptation to various types of stresses (Karin, 1985Go) and their biological functions have been discussed with regard to homeostasis of essential metals such as zinc, antioxidant defense and the handling of toxic metal ions, in particular cadmium (Kang, 1999Go; Miles et al., 2000Go; Nordberg and Nordberg, 2000Go; Vasák and Hasler, 2000Go). The protein is devoid of aromatic amino acids and, except for some chiroptical features in the far-ultraviolet region, the zinc–thiolate clusters have also virtually no spectroscopic characteristics for structure–function studies. Therefore, it would be of considerable practical importance to have fluorescent MT derivatives that could serve as tracers in the cell and as sensors of its metal load and redox state. The function of these proteins focuses on their zinc–thiolate clusters with regard to cellular zinc homeostasis (Vallee, 1995Go) and cellular translocations to mitochondria (Ye et al., 2001Go) and the nucleus (Takahashi et al., 2005Go). A basis for their mechanism of action is the redox activity of the zinc–thiolate clusters, in which the oxidation and reduction of the sulfur of the cysteine ligands is coupled to zinc release and binding (Maret and Vallee, 1998Go; Maret, 2004Go). Both crystal and solution structures of the holoprotein, i.e. MT, are available (Arseniev et al., 1988Go; Robbins et al., 1991Go). However, it was not possible to determine the orientation of the domains relative to each other in solution (Arseniev et al., 1988Go), nor is there any information about the three-dimensional structure of the oxidized protein (Haase and Maret, 2004Go) or the apoprotein, thionein, which is also present in tissues (Yang et al., 2001Go). FRET (fluorescence resonance energy transfer) seems to be uniquely suited to fill this gap of knowledge by providing spatial and dynamic information on these proteins and their interactions with ligands and/or other proteins. Distance measurements from FRET can be complicated by a variety of factors, however, among which lack of orientational averaging of the fluorophores and breakdown of the point-dipole approximation of the Förster theory are the most critical as confirmed in single molecule fluorescence studies (Schuler et al., 2005Go) when using this ‘spectroscopic ruler’ approach (Stryer and Haugland, 1967Go). We approached minimally invasive modification of MT by tagging the termini and the interdomain (linker) region, because any modification within the two domains would be expected to perturb the zinc–thiolate clusters. For labeling specific residues, we have taken advantage of both the free N-terminus of the expressed protein obtained after cleavage of the intein-fusion protein (Hong et al., 2001Go) and an extra cysteine at position 32 in the linker region of the isoform MT-1b (Heguy et al., 1986Go). We have engineered the S32C mutation into the human MT-2 cDNA, expressed the protein and provided evidence that the additional sulfhydryl group does not participate in the zinc–thiolate clusters (Hong and Maret, 2003Go). In fact, owing to its differential reactivity in cadmium-containing MT (CdMT), this cysteine is the only highly reactive cysteine that can be modified with a fluorescent label, which together with a label at the N-terminus forms a specific FRET pair for the N-terminal ß-domain of MT (Hong and Maret, 2003Go). To generate a second FRET sensor that is specific for the {alpha}-domain, we have employed a different strategy. A tryptophan was added at the C-terminus of the S32C mutant, thereby obtaining an MT with an aromatic amino acid as an intrinsic probe. In another FRET pair, this tryptophan then serves as a fluorescence donor to a fluorescence acceptor attached at the cysteine residue in the linker region. The two sensors allow us to examine the structure and function of each zinc–thiolate cluster separately. When the structures of the protein in its metal-bound and metal-free state were investigated with both domain-specific sensors, neither sensor detects a large change in the dimension of the molecule.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Materials

Restriction enzymes and reaction buffers, Taq DNA polymerase and JM109 cells were purchased from Promega (Madison, WI) and New England Biolabs (Beverly, MA); pKF19 vector, M13 RV-N primer, MV1184 cells and long and accurate (LA) Taq polymerase from Takara (Shuzo, Kasatsu, Japan); pTYB11 vector, intein forward primer, ER2566 cells and chitin resin as part of the IMPACT system from New England Biolabs; ampicillin, isopropyl-ß-D-thiogalactopyranoside (IPTG), CdCl2, ZnSO4, 2,2'-dithiodipyridine, DTT, EDTA, subtilisin and carboxypeptidase A from Sigma (St. Louis, MO); and 5-({[(2-iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) from Molecular Probes (Eugene, OR). Primers were synthesized by Integrated DNA Technologies (Coralville, IA).

Site-directed mutagenesis

The S32C MT mutant of human MT-2 was constructed as described (Hong and Maret, 2003Go). The plasmid pKF19 (2204 bp) contains an amber mutation in the Km resistance gene (kmram2) resulting in loss of the Km resistance phenotype in Km-containing medium. The phenotype can be rescued by a suppressor tRNA produced from the genome of a host cell such as JM109. In a suppressor-free (sup°) host such as MV1184 cells, a site-specific mutagenesis of the amber mutant to wild-type (kmr) is the only way to achieve resistance in Km-containing medium. Plasmids were constructed as follows. First, MT cDNA (361 bp) from pTYB11-MT was inserted into the pKF19 plasmid (Figure 1). The pKF19-MT (2535 bp) plasmid was used for construction of the S32C mutant by the oligonucleotide-directed dual amber-long and accurate (ODA-LA) PCR method [Mutant-Super Express Km kit (Takara)] (Figure 1, mutagenesis 1). Polymerase chain reaction (PCR)-based mutagenesis was performed with a mutant primer (S32C) (5'-TGT ACT AGT TGC AAG AAA TGT TGC TGT TCC TGC-3'(+), mismatched codon underlined) and a selection primer (SP) [5'-CGT CTC GCT cAG GCG cAa TC-3'(–), amber stop codons underlined, mutated sequence in lower case], containing the sequence of Kmr [glutamate (CAG or CAA) at the amber stop codons (TAG)]. The resulting plasmid, pKF19-MT(S32C), contains mutated MT (S32C) and Kmr. After selecting the pKF19-MT(S32C) plasmid by its Km resistance in Km-containing medium, the MT insert (381 bp) from the pKF19(S32C) vector was ligated into pKF19 plasmid to construct pKF19-MT(S32C)-kmram2 (2535 bp). Since this plasmid carries an amber stop mutant, it can be used as a template for the second mutagenesis (Figure 1, mutagenesis 2), which was performed with a second mutant primer (W+62) [5'-AGC TGT TGC GCT TGG TGA CTC TAG AG-3' (+), inserted codon underlined] and the selection primer to construct pKF19-MT(S32C/W+62) (2538 bp). This PCR-constructed plasmid pKF19(S32C/W+62) carries double mutated MT (S32C and an additional amino acid, tryptophan, after the 61st amino acid). The pKF19-MT(S32C/W+62) plasmid was selected by its Km resistance in Km-containing medium, the MT fragment (328 bp) excised and inserted into the expression vector pTYB11 (7412 bp) to construct pTYB11-MT(S32C/W+62) (7593 bp). Primers synthesized for DNA sequencing were an amber primer [(+)886 – (+)906 bp (5'-CCG GTG AGA ATG GCA AGA GC-3'(+)] for sequencing of the amber mutation [(+)996 – (+)1014 bp of pK19(S32C/W+62)] of the Kmr region, an M13 RV-N primer [(–)44 – (–)28 bp of pKF plasmids, Takara] [5'-TGT GGA ATT GTG AGC G-3'(+)) for sequencing of the MT cDNA [(+)186 – (+)374 bp] inserted at the multicloning site of pKF19(S32C/W+62), and an intein forward primer [(+)6326 – (+)6385 bp of pTYB11 plasmids] [5'-CCC GCC GCT GCT TTT GCA CGT GAG-3'(+)] for sequencing of the MT cDNA [(+)6513 – (+)6688 bp] in the multicloning site of pTYB11(S32C/W+62). Sequencing was performed at the molecular biology core facility of the Dana-Farber Cancer Institute (Boston, MA).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Strategy for constructing a double mutant MT (S32C/W+62) employing the oligonucleotide-directed dual amber-long and accurate (ODA LA)-PCR method. SP, selection primer; MCS, multicloning site; ER2566/Amp, ER2566 cells grown in Amp-containing medium; JM109/Km or MV1184/Km, JM109 cells or MV1184 cells grown in Km-containing medium, respectively.

 
Overexpression and purification of MT

The plasmids encoding the wild-type human MT-2 gene and the S32C/W+62 mutant were transformed into E.coli ER2566. ER2566 cells were incubated at 37°C until OD600 reached 2–3. Culture flasks were moved to an incubator kept at room temperature and IPTG (0.5 mM) was added to induce protein production. IPTG induction at room temperature avoids the formation of inclusion bodies of the intein–MT fusion protein. ER2566 cells were harvested when OD600 reached 8–10 (usually after 12–14 h of incubation). MT protein was prepared by using the IMPACT T7 system (New England Biolabs) (Chong et al., 1998Go). In this system, the intein–MT fusion protein is recovered by absorption on an affinity resin, T is cleaved from the absorbed fusion protein by DTT-mediated proteolysis and eluted together with the N-terminally cleaved maltose-binding peptide and both are separated by gel filtration (Sephadex G-25, equilibrated with 10 mM HCl, pH 2) after acidification to pH 1 with HCl. The pooled T fractions were reconstituted with CdCl2 or ZnSO4, adjusted to pH 8.6 with Tris base to stabilize the metal–thiolate clusters, lyophilized and separated from free metal ions by gel filtration (Sephadex G-50, equilibrated with 20 mM Tris–HCl, pH 7.4). MT was quantified by determination of thiols ({varepsilon}343 = 7600 M–1 cm–1) with 2,2'-dithiodipyridine (Pedersen and Jacobsen, 1980Go). Metal analyses were performed by atomic absorption spectrometry (Model 2280 instrument, Perkin-Elmer, Norwalk, CT).

Characterization of the S32C/W+62 mutant

Since the mutant has one aromatic amino acid (tryptophan), determination of the concentration of T, which is typically performed at 220 nm, is affected by the absorbance of tryptophan at this wavelength. The molecular extinction coefficient of T (S32C/W+62) ({varepsilon}220 = 83 000 M–1 cm–1) was calculated from that of tryptophan ({varepsilon}220 = 35 000 M–1 cm–1) (Haschemeyer and Haschemeyer, 1973Go; Fasman, 1976Go) and that of T ({varepsilon}220 = 48 000 M–1 cm–1) (Schäffer, 1991Go). Likewise, quantification of CdMT (S32C/W+62) was based on either {varepsilon}220 = 190 000 M–1 cm–1 [{varepsilon}220 = 155 000 M–1 cm–1 (CdMT)+{varepsilon}220 = 35 000 M–1 cm–1 (tryptophan)] or {varepsilon}250 = 106 900 M–1 cm–1 [{varepsilon}250 = 105 000 M–1 cm–1 (CdMT)+{varepsilon}250 = 1 900 M–1 cm–1 (tryptophan)].

S-labeling of MT

The free cysteine in S32C/W + 62 CdMT was labeled after adjusting the pH to 7.4 instead of 8.6 (see Overexpression and purification of MT) and immediately after the reconstitution of purified T with metal ions. The mixture of the reconstituted CdMT (4 mg, 615 nmol) and free metal ions in 20 mM Tris–HCl, pH 7.4 was incubated with 0.4 mg (920 nmol) of 1,5-IAEDANS dissolved in 500 µl of DMSO in a total volume of 4.3 ml, with stirring for 2 h at room temperature and protection from light. Labeled protein was separated from free reagent and free metal ions by gel filtration as described above. The stoichiometry of labeling was determined from a molar absorptivity of {varepsilon}280 = 5600 M–1 cm–1 for tryptophan (Fasman, 1976Go) and {varepsilon}336 = 5700 M–1 cm–1 for 1,5-IAEDANS (Molecular Probes, www.probes.com). When wild-type CdMT was labeled under identical conditions, 20% labeling was observed.

Fluorescence spectroscopy

AEDANS–CdMT (S32C/W+62), free tryptophan or free 1,5-IAEDANS dissolved in 20 mM Tris–HCl, pH 7.4, was excited at 295 nm for tryptophan or 334 nm for IAEDANS and emission spectra were recorded with a FluoroMax-2 fluorimeter (Instruments SA, Edison, NJ).

Steady-state fluorescence polarization anisotropy was measured using a SPEX FluoroMax fluorimeter with Glan-Thompson polarizers. The acceptor (AEDANS) was excited at 334 nm (5.0 nm bandpass) and emitted light measured in L format at 488 nm (5.0 nm bandpass). Fluorescence emission from Trp in the MT mutant (without the AEDANS label attached) was measured at 358 nm with excitation at 295 nm. The bandpass was 5 nm for both excitation and emission. Fluorescence intensities were collected with excitation polarizers in either the vertical (V) or horizontal (H) position and emission polarizers in either the V or H position (IVV, IVH, IHH and IHV) and anisotropy (r) was calculated by the software module of the fluorimeter. Anisotropy values are the averages of five measurements. Solution viscosity was varied by addition of aliquots of a 2 M sucrose solution in 20 mM Tris–HCl, pH 7.4; background fluorescence from sucrose was negligible. Viscosities at 25°C for each concentration of sucrose were determined using an Internet-based calculator (http://www.univ-reims.fr/Externes/AVH/MementoSugar/001.htm). Data were analyzed using the Perrin equation:

(1)
where r0 is the intrinsic anisotropy in the absence of motion, Vh is the hydrodynamic volume, {tau} is the fluorescence decay time, {eta} is the viscosity and kB is the Boltzmann constant.

Distance calculations from FRET experiments

Distances were calculated from , where E is the efficiency of energy transfer and R0 is the Förster radius corresponding to 50% energy transfer efficiency. The efficiency is expressed as the percentage quenching of the donor emission obtained from the area of the donor emission band.

Effect of proteases and EDTA on the distance between the donor and the acceptor

AEDANS–MT (S32C/W+62) was treated with subtilisin to cleave off the ß-domain (Nielson and Winge, 1984Go). For this purpose, 1 ml of MT (3 µM) was incubated with 10 µl of subtilisin (1 mg/ml in MilliQ water) for 24 h at room temperature. The mutant (3 µM) was also incubated with carboxypeptidase A (1 µM) or with EDTA (5 µM) in 20 mM Tris–HCl, pH 7.4, at 25°C. Emission spectra were recorded at different times by exciting proteins at 295 nm. Protein samples incubated for the same periods of time in the absence of reagents served as controls.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Design and construction of a FRET sensor for the {alpha}-domain

In order to construct a FRET sensor for the {alpha}-domain of MT, fluorescent labels have to be attached at two specific positions. Genetic engineering of the MT molecule achieved this goal (Figure 1; see Materials and methods for further details). One position is the thiol of a cysteine that replaces a serine in the linker region of human MT-2 (hMT2), does not interact with the metal ions and in CdMT has sufficiently high differential reactivity for specific modification when compared with the metal-bound cysteines (Hong and Maret, 2003Go). Hence, if the fluorescence donor and acceptor are attached at the N-terminus and at Cys32, respectively (Figure 2, middle), the resulting FRET sensor monitors the ß-domain of MT. If the donor is attached at the C-terminus instead (Figure 2, bottom), the resulting FRET sensor is expected to monitor the {alpha}-domain. C-terminal attachment of a fluorophore was achieved by adding a tryptophan as the C-terminal amino acid in the S32C MT mutant. The S32C/W+62 double mutant has a total of 21 cysteines instead of 20 in the wild-type human MT-2 and has a total of 62 amino acids instead of 61. 1,5-IAEDANS was chosen for specific labeling of Cys32 as it serves as an acceptor for the fluorescence from tryptophan and because the R0 value of this pair is compatible with the distance between the probes as estimated from molecular modeling and because the R0 value is the average useful distance in distance determination by the FRET method.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 2. Construction of FRET sensors for the {alpha}- or ß-domain of human metallothionein, isoform 2 (hMT2). A FRET sensor for the ß-domain was constructed by attaching fluorescent probes at the N-terminus and at an extra cysteine in the linker region between the domains (Hong and Maret, 2003Go). A FRET sensor for the {alpha}-domain was constructed by adding a tryptophan at the C-terminus of MT by site-specific incorporation and attaching a fluorescent probe at the extra cysteine. A = acceptor; D = donor.

 
Characterization of S32C/W+62 MT

When plasmid pTYB11 (S32C/W+62) was used to express MT in E.coli cells, 11.9 mg of thionein (T) were purified from 1 l of culture medium. Hence attachment of an additional amino acid to MT does not affect the yield of MT (12 mg/l) (Hong et al., 2001Go). The (S32C/W+62) double mutant has the characteristic absorption spectrum of a tryptophan-containing protein (Figure 3A, solid line). The absorption spectrum of the T (S32C/W+62) mutant is significantly different from those of either wild-type T or the T S32C mutant (Figure 3A, dashed line). Tryptophan also absorbs light at 220 nm (Fasman, 1976Go), increasing the absorption of the T (S32C/W+62) mutant at this wavelength ({varepsilon}220 = 83 000 M–1 cm–1) relative to wild-type T. With this correction, the protein concentration determined at 220 nm is identical with that obtained from readings at 280 nm ({varepsilon}280 = 5600 M–1 cm–1).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3. Electronic absorption spectra of the S32C/W+62 mutants of MT and T. (A) T (S32C/W+62) mutant (10 µM, solid line), wild-type T (dashed line). (B) AEDANS-labeled CdMT (S32C/W+62) mutant (10 µM, solid line), wild-type CdMT (dashed line).

 
Quantification of the CdMT (S32C/W+62) mutant with 2,2'-dithiodipyridine confirmed that it has 21 thiols when the protein concentration is based on absorbance readings at 220, 250 or 280 nm. Owing to the significant increase in absorbance at 250 nm from the sulfur ligand-to-cadmium charge-transfer transitions, the peak at 280 nm is less well resolved in this species (Figure 3B, dashed line).

Fluorescence labeling with 1,5-IAEDANS

Thiol-specific fluorescence labeling of the S32C/W+62 mutant was achieved at pH 7.4. Optimizing the pH during labeling is critical, because slight changes of the pH value can cause non-specific labeling at the N-terminal amino group of the protein (Hong and Maret, 2003Go). The labeling ratio between protein and 1,5-IAEDANS was 1:1.

The absorbance spectrum of the AEDANS-labeled CdMT (S32C/W+62) mutant (Figure 3B, solid line) differs from that of wild-type CdMT (Figure 3B, dashed line) by having an absorption band with a maximum at 336 nm. Molecular extinction coefficients at 250 nm of both CdMT (S32C/W+62) and AEDANS-CdMT (S32C/W+62) calculated based on the extinction coefficient of MT, free Trp and free 1,5-IAEDANS (Molecular Probes) reveal that both proteins contain seven cadmium atoms.

Fluorescence spectra

When the CdMT (S32C/W+62) mutant is excited at 295 nm, maximum emission occurs at 360 nm (Figure 4A, line 2). The emission of the free amino acid in the same buffer (20 mM Tris–HCl, pH 7.4) is similar to that in the MT mutant (Figure 4A, line 1 vs line 2). When different concentrations of samples were examined, the fluorescence intensity increased linearly below 40 µM protein as calculated from the equation relative fluorescence intensity=[protein concentration (µM)x8326.7]+509.67 for both the MT (S32C/W+62) mutant and free tryptophan. When AEDANS–MT (S32C/W+62) is excited at 295 nm, the donor emission at 360 nm in the presence of the acceptor (Figure 4B, line 2) decreases by 61% compared with that in the absence of the acceptor (Figure 4B, line 1). The total amount of the acceptor in each sample was determined by exciting the protein at 334 nm (Figure 4B, line 3).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Fluorescence emission spectra of MT (S32C/W+62) and 1,5-IAEDANS-labeled mutant. (A) Fluorescence emission spectra of CdMT (S32C/W+62) mutant (line 1, 33 µM) and free tryptophan at the same concentration (line 2) in 20 mM Tris–HCl, pH 7.4. Both samples were excited at 295 nm. (B) Fluorescence emission spectra of 1,5-IAEDANS-labeled CdMT (S32C/W+62) mutant (18.6 µM). In the presence of acceptor (AEDANS), donor emission (line 2) decreases relative to that of the donor at the same concentration in the absence of acceptor (line 1). Energy transfer between donor and acceptor is calculated based on the decreased donor emission when excited at 295 nm. Emission spectrum of the acceptor when excited at 344 nm (line 3).

 
Distance measurement and 3-D molecular modeling

From the 61% decrease in fluorescence, a distance of 19.7 Å between the Trp donor–AEDANS acceptor pair is calculated with an R0 value of 20 Å for this pair (Sun et al., 2001Go). Based on the crystal structure of MT (Robbins et al., 1991Go), shown here with the S32C mutation (Figure 5A, arrow) and depending on the orientation of the AEDANS group, the theoretical range of distances between the pair is estimated by 3-D molecular modeling to be 12.1–19.8 Å (Figure 5B and C). The experimental distance of 19.7 Å is very close to the maximum theoretical value obtained when the acceptor is in a position where it interacts with Lys20 of the ß-domain via a salt bond (Figure 5C).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5. Structure modeling of AEDANS-MT (S32C/W+62). Structures of the MT (S32C) mutant (A) and the AEDANS-MT (S32C/W+62) mutant (B and C) were generated using ChemDraw and energy-minimized in Chem3D (CambridgeSoft Inc.). The structures are presented with the C-terminal {alpha}-domain on the left side. Ser32 was mutated to Cys and Trp62 was added at the C-terminus using the Biopolymer module in Insight II (Accelrys, Inc.). The 3D structure of AEDANS was imported into Insight II and manually attached at Cys32. The structure of the MT (S32C) mutant (A) shows the exclusive availability of Cys32 (arrow) for labeling among all the other Cys residues (rendered with darker shades of grey at the bonds of their C{alpha}-atoms) that are bound to metal atoms. In order to estimate the range of distances between Trp62 (donor) and AEDANS (acceptor) attached at the side chain of Cys32, flexibility was allowed where torsions are possible within the AEDANS probe. Among all rotamers in the standard library of Insight II, the position of Trp shown is the only viable one without clashes with protein side chains. The modeling reveals estimates of 12.14 (B) and 19.82 Å (C) for the distance between donor and acceptor when allowing for different conformations of the AEDANS probe. In (C), the {varepsilon}-amino group of Lys20 (arrow) is within hydrogen bonding distance (3.2 Å) of the sulfonic acid group of the AEDANS probe.

 
Effect of protease and EDTA treatment on FRET signal

Metallothionein is resistant to proteolysis. However, subtilisin in the presence of EDTA, which removes the metals, digests the N-terminal ß-domain leaving the C-terminal {alpha}-domain intact (Winge and Miklossy, 1982Go). We have found that subtilisin abolishes FRET in the ß-domain FRET sensor by cleaving between the probes, i.e. N-terminal to amino acid 32 (Hong and Maret, 2003Go). The effect of subtilisin on the {alpha}-domain FRET sensor (Figure 6, line 2 vs line 1) is small compared with that on the ß-domain FRET sensor (Hong and Maret, 2003Go), demonstrating that the enzyme does not cleave MT in the {alpha}-domain between the two probes. A comparison of the emission spectrum of MT treated with subtilisin (line 2) and that of Trp at the same concentration (line 3) shows how minimal the effect is. The small effect is probably due to perturbation of the interaction between the two domains (Jiang et al., 2000Go). Since subtilisin did not abolish FRET in the {alpha}-domain, we investigated whether carboxypeptidase A would remove the C-terminal tryptophan from MT and abolish FRET. The acceptor emission of AEDANS–MT (S32C/W+62) (Figure 7) increased slightly (line 2 vs line 3) after overnight incubation with carboxypeptidase A. The immediate large increase in the emission at 360 nm (line 2) compared with that of the untreated sample (line 1) is caused by emission from the seven tryptophan residues in the enzyme (Bradshaw et al., 1969Go). The very slow change of FRET occurring over 24 h during carboxypeptidase-mediated proteolysis (Figure 7) demonstrates that the cadmium-bound {alpha}-domain is a very poor substrate for carboxypeptidase A.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6. Effect of subtilisin on FRET of AEDANS–MT (S32C/W+62). AEDANS–MT (S32C/W+62) mutant (3 µM) was incubated with subtilisin for 24 h at 25°C. Excitation spectrum of untreated AEDANS–MT (S32C/W+62) (line 1) or subtilisin-treated MT (line 2) when excited at 295 nm. A comparison of the emission spectrum of MT treated with subtilisin (line 2) and that of Trp at the same concentration (line 3) shows that proteolysis has a minimal effect on energy transfer between donor and acceptor.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7. C-terminal proteolysis of AEDANS–MT (S32C/W+62) by carboxypeptidase A. Emission spectra of AEDANS–CdMT (S32C/W+62) mutant (3 µM) before (line 1) and after incubation with carboxypeptidase A (1 µM) at room temperature for <1 min (line 2) and 24 h (line 3) (excitation: 295 nm).

 
Metal release from MT by EDTA can be followed by measuring the decrease in the sulfur-to-cadmium charge-transfer absorption bands at 250 nm (Vazquez and Vasák, 1988Go). During this reaction, the FRET signal changes. EDTA has no effect on the donor emission in the unlabeled (S32C/W+62) mutant. The distance between the probes appears to change only slightly, i.e. an increase from 19.7 to 20.1 Å is calculated.

Steady-state fluorescence polarization anisotropy

Uncertainties in the orientation factor in the Förster equation can bias the average distances calculated from FRET measurements. Only if the motions of the donor and acceptor are not restricted during the time of fluorescence emission, a value of 2/3 for the orientation factor is approached and the distances can be calculated with some degree of certainty. Our molecular modeling indicates unrestricted motion of the C-terminally attached Trp, but either immobilization or a variable position of the acceptor AEDANS. Therefore, we performed steady-state fluorescence polarization measurements that provide information about the motion of fluorophores attached to a macromolecule (Dale et al., 1979Go). The linear isothermal Perrin plot for the Trp donor indicates that this fluorophore is freely moving (Figure 8A). The extrapolated limiting anisotropy r0 (0.22) is close to that for Trp in peptides (Eftink et al., 1990Go). The Perrin plot for the acceptor, however, is non-linear (Figure 8B), indicating restricted local motion of the label independent of the motion of the protein as a whole, as indeed suggested by the molecular modeling. A segmental motion of the macromolecule is a less likely interpretation of these data, because the structures of the MT domains are organized by 28 bonds from cysteines in the zinc–thiolate clusters and therefore are relatively compact. The restricted motion of the acceptor puts constraints on our distance determination by steady-state fluorescence measurements. Either time-resolved resonance energy transfer measurements (Wu and Brand, 1992Go; Eis and Lakowicz, 1993Go) or attachment of a different acceptor that is freely moving can obviate the possible ambiguities in distances determined by the FRET technique.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8. Isothermal Perrin plots of anisotropy versus viscosity for donor (Trp) and acceptor (AEDANS) fluorescence. The donor (Trp) fluorescence of S32C/W+62 MT was determined in the absence of the acceptor (A) while the acceptor fluorescence was determined with AEDANS-labeled S32C/W+62 MT (B). Viscosities were changed by either adding the protein (3 µM) in buffer containing no sucrose to a protein solution at the same concentration in 1.7 M sucrose or by the reverse procedure, i.e. adding the protein in 1.7 M sucrose to a solution of the protein without sucrose. Two data points (between 150 and 175) were collected twice to demonstrate the close correspondence of measurements performed by either lowering or increasing the viscosity. Correlation coefficients are 0.936 for the fit of the data in (A) and 0.941 and 0.969 for the data at low and high viscosity, respectively, in (B).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Metallothionein has two domains, each enveloping a zinc–thiolate cluster (Robbins et al., 1991Go). The domains are separate entities that bind and release zinc in cysteine-centered redox reactions that couple the redox-inert zinc ion to cellular redox metabolism (Maret and Vallee, 1998Go; Maret, 2004Go). The molecule has no characteristics that would facilitate studies of either metal binding or the redox state of these clusters. Hence we have designed strategies to label MT fluorescently at three different positions in such a manner that the probes do not interfere with the cluster chemistry. Pairs of probes at two positions can act as FRET sensors for each domain. Construction of a FRET sensor for the ß-domain became possible by specifically labeling the N-terminus and by replacing a serine in the linker region between the domains with an additional cysteine (Hong and Maret, 2003Go). A fluorescent probe attached to this cysteine between the domains can also be employed as an acceptor in a FRET pair where a donor is attached at the C-terminus of MT. Since there is no simple strategy to achieve such labeling by chemical modification of a carboxy group, we added a tryptophan at the C-terminus by genetic engineering. The {alpha}-domain MT FRET sensor that is obtained when a fluorescence acceptor is attached to the free cysteine of this mutant overcomes a longstanding impediment in structure–function studies of this protein, namely its virtual lack of the absorption and emission spectroscopic features of most other proteins.

The experimentally determined distance between the probes is 19.7 Å. The molecular modeling performed as part of these studies provides a basis for comparison with distances calculated from FRET measurements. Modeling indicates that there is very little variation possible in the position of the tryptophan residue, which seems to be freely moving at the C-terminus on the surface of the protein. However, the AEDANS label can adopt multiple conformations. Based on the atomic coordinates of MT (Robbins et al., 1991Go), the theoretical distance between the fluorophores is only 12.1 Å in one conformation. In another, however, the sulfonic acid group of the naphthyl moiety is in ion pairing distance (3.2 Å) with the {varepsilon}-amino group of Lys20 (Figure 5C). When fixed in this position, the distance is almost identical with that observed experimentally (19.8 vs 19.7 Å), suggesting that the label adopts such a conformation in the protein. The excellent agreement between the theoretically predicted and the experimentally determined distances (19.8 vs 19.7 Å) suggests that the error introduced by the restricted rotation of the acceptor is small. The distance of 19.7 Å is very close to the R0 value of 20 Å of this donor–acceptor pair. Hence this pair is a much more sensitive FRET sensor than the Alexa488–546 pair used in the ß-domain sensor of MT (Hong and Maret, 2003Go). However, the fluorescence polarization anisotropy results caution us against the interpretation of distance measurement from the steady-state fluorescence without obtaining additional insights into the dynamics of the system by time-resolved fluorescence studies.

In summary, neither the ß-domain (Hong and Maret, 2003Go) nor the {alpha}-domain FRET sensor detects any significant change in the dimensions of the molecule when its metal ions dissociate. These sensors provide new and highly effective probes to monitor the state of the metal–thiolate clusters and possibly their interactions with ligands such as ATP (Maret et al., 2002Go) and to follow their metal binding and release.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Drs James F.Riordan and Bert L.Vallee (Harvard Medical School) for helpful discussions, Dr Jeremy Jenkins for his help in modeling the structure of F/R-MT and Dr D.Wayne Bolen (UTMB) for the use of his spectrofluorimeter. This work was supported by the Endowment for Research in Human Biology and National Institutes of Health Grant GM 065388 (to W.M.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Arseniev,A., Schultze,P., Wörgötter,E., Braun,W., Wagner,G., Vasák,M., Kägi,J.H.R. and Wüthrich,K. (1988) J. Mol. Biol., 201, 637–657.[CrossRef][ISI][Medline]

Bradshaw,R.A., Ericson,L.H., Walsh,K.A. and Neurath,H. (1969) Proc. Natl Acad. Sci. USA, 63, 1389–1393.[Abstract/Free Full Text]

Chong,S., Montell,G.E., Zhang,A., Cantor,E.J., Liao,W., Xu,M.-Q. and Benner,J. (1998) Nucleic Acids Res., 26, 5109–5115.[Abstract/Free Full Text]

Dale,R.E., Eisinger,J. and Blumberg,W.E. (1979) Biophys. J., 26, 161–194.[Abstract]

Eftink,M.R., Selvidge,L.A., Callies,P.R. and Rehms,A.A. (1990) J. Chem. Phys., 94, 3469–3479.[CrossRef]

Eis,P.S. and Lakowicz,J.R. (1993) Biochemistry, 32, 7981–7993.[CrossRef][ISI][Medline]

Fasman,G.D. (1976) In Fasman,G.D. (ed.), Handbook of Biochemistry and Molecular Biology, 3rd edn. Chemical Rubber Company Press, Cleveland, OH, pp. 183–185.

Haase,H. and Maret,W. (2004) Anal. Biochem., 333, 19–26.[CrossRef][ISI][Medline]

Haschemeyer,R.H. and Haschemeyer,A.E. (1973) Proteins: a Guide to Study by Physical and Chemical Methods. Wiley, New York.

Heguy,A., West,A., Richards,R.I. and Karin,M. (1986) Mol. Cell. Biol., 6, 2149–2157.[ISI][Medline]

Hong, S.-H., Toyama,M., Maret,W. and Murooka,Y. (2001) Protein Expr. Purif., 21, 243–250.[CrossRef][ISI][Medline]

Hong,S.-H. and Maret,W. (2003) Proc. Natl Acad. Sci. USA, 100, 2255–2260.[Abstract/Free Full Text]

Jiang,L.-J., Vasák,M., Vallee,B.L. and Maret,W. (2000) Proc. Natl Acad. Sci. USA, 97, 2503–2508.[Abstract/Free Full Text]

Kang,Y.J. (1999) Proc. Soc. Exp. Biol. Med., 222, 263–273.[Abstract/Free Full Text]

Karin,M. (1985) Cell, 41, 9–10.[CrossRef][ISI][Medline]

Maret,W. and Vallee,B.L. (1998) Proc. Natl Acad. Sci. USA, 95, 3478–3482.[Abstract/Free Full Text]

Maret,W., Heffron,G., Hill,H.A.O., Djuricic,D., Jiang,L.-J. and Vallee,B.L. (2002) Biochemistry, 41, 1689–1694.[CrossRef][ISI][Medline]

Maret,W. (2004) Biochemistry, 43, 3301–3309.[CrossRef][ISI][Medline]

Miles,A.T., Hawksworth,G.M., Beattie,J.H. and Rodilla,V. (2000) Crit. Rev. Biochem. Mol. Biol., 35, 35–70.[Abstract/Free Full Text]

Nielson,K.B. and Winge,D.R. (1984) J. Biol. Chem., 259, 4941–4946.[Abstract/Free Full Text]

Nordberg,M. and Nordberg,G.F. (2000) Cell. Mol. Biol., 46, 451–463.[ISI][Medline]

Pedersen,A.O. and Jacobsen,J. (1980) Eur. J. Biochem., 106, 291–295.[Abstract]

Robbins,A.H., McRee,D.E., Williamson,M., Collett,S.A., Xuong,N.H., Furey,W.F., Wang,B.C. and Stout,C.D. (1991) J. Mol. Biol., 221, 1269–1293.[CrossRef][ISI][Medline]

Schäffer,A. (1991) Methods Enzymol., 205, 529–540.[ISI][Medline]

Schuler,B., Lipman,E.A., Steinbach,P.J., Kumke,M. and Eaton,W.A. (2005) Proc. Natl Acad. Sci. USA, 102, 2754–2759.[Abstract/Free Full Text]

Stryer,L. and Haugland,R.P. (1967) Proc. Natl Acad. Sci. USA, 58, 719–726.[Free Full Text]

Sun,H., Yin,D., Coffeen,L.A., Shea,M.A. and Squier,T.C. (2001) Biochemistry, 40, 9605–9617.[CrossRef][ISI][Medline]

Takahashi,Y., Ogra,Y. and Suzuki,K.T. (2005) J. Cell. Physiol., 202, 563–569.[CrossRef][ISI][Medline]

Vasák,M. and Hasler,D.W. (2000) Curr. Opin. Chem. Biol., 4, 177–183.[CrossRef][ISI][Medline]

Vallee,B.L. (1995) Neurochem. Int., 27, 23–33.[CrossRef][ISI][Medline]

Vazquez,F. and Vasák,M. (1988) Biochem. J., 253, 611–614.[ISI][Medline]

Winge,D.R. and Miklossy,K.-A. (1982) J. Biol. Chem., 257, 3471–3476.[Abstract/Free Full Text]

Wu,P. and Brand,L. (1992) Biochemistry, 31, 7939–7947.[CrossRef][ISI][Medline]

Yang,Y., Maret,W. and Vallee,B.L. (2001) Proc. Natl Acad. Sci. USA, 98, 5556–5559.[Abstract/Free Full Text]

Ye,B., Maret,W. and Vallee,B.L. (2001) Proc. Natl Acad. Sci. USA, 98, 2317–2322.[Abstract/Free Full Text]

Received August 9, 2004; revised March 1, 2005; accepted April 22, 2005.

Edited by Harold Scheraga





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
18/6/255    most recent
gzi031v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Hong, S.-H.
Articles by Maret, W.
PubMed
PubMed Citation
Articles by Hong, S.-H.
Articles by Maret, W.