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
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Abstract |
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Keywords: fluorescent probes/FRET sensor/metallothionein/site-directed mutagenesis/zincthiolate clusters
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Introduction |
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Materials and methods |
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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, 2003). 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).
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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 23. 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 inteinMT fusion protein. ER2566 cells were harvested when OD600 reached 810 (usually after 1214 h of incubation). MT protein was prepared by using the IMPACT T7 system (New England Biolabs) (Chong et al., 1998). In this system, the inteinMT 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 metalthiolate clusters, lyophilized and separated from free metal ions by gel filtration (Sephadex G-50, equilibrated with 20 mM TrisHCl, pH 7.4). MT was quantified by determination of thiols (
343 = 7600 M1 cm1) with 2,2'-dithiodipyridine (Pedersen and Jacobsen, 1980
). 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) (220 = 83 000 M1 cm1) was calculated from that of tryptophan (
220 = 35 000 M1 cm1) (Haschemeyer and Haschemeyer, 1973
; Fasman, 1976
) and that of T (
220 = 48 000 M1 cm1) (Schäffer, 1991
). Likewise, quantification of CdMT (S32C/W+62) was based on either
220 = 190 000 M1 cm1 [
220 = 155 000 M1 cm1 (CdMT)+
220 = 35 000 M1 cm1 (tryptophan)] or
250 = 106 900 M1 cm1 [
250 = 105 000 M1 cm1 (CdMT)+
250 = 1 900 M1 cm1 (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 TrisHCl, 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 280 = 5600 M1 cm1 for tryptophan (Fasman, 1976
) and
336 = 5700 M1 cm1 for 1,5-IAEDANS (Molecular Probes, www.probes.com). When wild-type CdMT was labeled under identical conditions, 20% labeling was observed.
Fluorescence spectroscopy
AEDANSCdMT (S32C/W+62), free tryptophan or free 1,5-IAEDANS dissolved in 20 mM TrisHCl, 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 TrisHCl, 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:
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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
AEDANSMT (S32C/W+62) was treated with subtilisin to cleave off the ß-domain (Nielson and Winge, 1984). 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 TrisHCl, 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.
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Results |
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In order to construct a FRET sensor for the -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, 2003
). 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
-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.
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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., 2001). 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, 1976
), increasing the absorption of the T (S32C/W+62) mutant at this wavelength (
220 = 83 000 M1 cm1) 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 (
280 = 5600 M1 cm1).
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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, 2003). 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 TrisHCl, 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 AEDANSMT (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).
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From the 61% decrease in fluorescence, a distance of 19.7 Å between the Trp donorAEDANS acceptor pair is calculated with an R0 value of 20 Å for this pair (Sun et al., 2001). Based on the crystal structure of MT (Robbins et al., 1991
), 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 12.119.8 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).
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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 -domain intact (Winge and Miklossy, 1982
). 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, 2003
). The effect of subtilisin on the
-domain FRET sensor (Figure 6, line 2 vs line 1) is small compared with that on the ß-domain FRET sensor (Hong and Maret, 2003
), demonstrating that the enzyme does not cleave MT in the
-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., 2000
). Since subtilisin did not abolish FRET in the
-domain, we investigated whether carboxypeptidase A would remove the C-terminal tryptophan from MT and abolish FRET. The acceptor emission of AEDANSMT (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., 1969
). The very slow change of FRET occurring over 24 h during carboxypeptidase-mediated proteolysis (Figure 7) demonstrates that the cadmium-bound
-domain is a very poor substrate for carboxypeptidase A.
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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., 1979). 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., 1990
). 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 zincthiolate 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, 1992
; Eis and Lakowicz, 1993
) or attachment of a different acceptor that is freely moving can obviate the possible ambiguities in distances determined by the FRET technique.
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Discussion |
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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., 1991), 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
-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 donoracceptor pair. Hence this pair is a much more sensitive FRET sensor than the Alexa488546 pair used in the ß-domain sensor of MT (Hong and Maret, 2003
). 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, 2003) nor the
-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 metalthiolate clusters and possibly their interactions with ligands such as ATP (Maret et al., 2002
) and to follow their metal binding and release.
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Acknowledgements |
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Received August 9, 2004; revised March 1, 2005; accepted April 22, 2005.
Edited by Harold Scheraga
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