Evolving the Substrate Specificity of O6-Alkylguanine-DNA

Road, Cambridge CB2 2QH, U.K. ‡Current address: Sanofi-Aventis Deutschland; Chemical Sciences; D-65926 Frankfurt am. Main, Germany. Redesigning the ...
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Evolving the Substrate Specificity of O6-Alkylguanine-DNA Alkyltransferase through Loop Insertion for Applications in Molecular Imaging

Christian Heinis†, Simone Schmitt, Maik Kindermann‡, Guillaume Godin, and Kai Johnsson* Ecole Polytechnique Fédérale de Lausanne (EPFL), Institute of Chemical Sciences and Engineering, CH-1015 Lausanne, Switzerland, †Current address: Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K. ‡Current address: Sanofi-Aventis Deutschland; Chemical Sciences; D-65926 Frankfurt am Main, Germany.

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edesigning the substrate specificity of enzymes is one of the major challenges in protein engineering. As well as advancing our understanding of the relationship between structure and function, the generation of proteins with new substrate specificities has important medical and industrial applications. Classical examples are the conversion of lactate dehydrogenase into a malate dehydrogenase, which required the mutation of only one single amino acid to switch substrate specificity (1), or the redesign of trypsin into a protease with chymotrypsin-like specificity, which required much more extensive mutagenesis (2). Unfortunately, it is the latter case that is representative of the amount of effort typically needed to engineer enzyme substrate specificity. The protocols that are commonly employed to alter the substrate specificity and selectivity of catalysts have been iteratively improved over the past decades, to the point where a new activity can be introduced into an existing unrelated protein scaffold, a recent example being the introduction of ␤-lactamase activity into the ␣␤/␤␣ metallohydrolase scaffold of glyoxalase II (3). However, a recurring problem in protein engineering is that the desired change in substrate specificity can rarely be achieved by a small number of point mutations and more often requires extensive probing of the protein-sequence space (4). Frequently, directed evolution of enzymes based on libraries generated by conventional approaches such as error-prone polymerase chain reaction (PCR) or saturation mutagenesis does not yield an enzyme with the desired specificity at all. The difficulty of altering the substrate specific-

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A B S T R A C T We introduce a strategy for evolving protein substrate specificity by the insertion of random amino acid loops into the protein backbone. Application of this strategy to human O6-alkylguanine-DNA alkyltransferase (AGT) led to the isolation of mutants that react with the non-natural substrate O6-propargylguanine. Libraries generated by conventional random or targeted saturation mutagenesis, by contrast, did not yield any mutants with activity towards this new substrate. The strategy of loop insertion to alter enzyme specificity should be general and applicable to other classes of proteins. An important application of the isolated AGT mutant is in molecular imaging, where the mutant and parental AGTs are used to label two different AGT fusion proteins with different fluorophores in the same living cell or in vitro. This allowed the establishment of fluorescence-based assays to detect protein–protein interactions and measure enzymatic activities.

*Corresponding author, kai.johnsson@epfl.ch.

Received for review July 27, 2006 and accepted September 15, 2006. Published online October 6, 2006 10.1021/cb6003146 CCC: $33.50 © 2006 by American Chemical Society

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Figure 1. Mechanism and substrates used for labeling of AGT fusion proteins. a) General mechanism for the labeling of AGT fusion proteins with O6-benzylguanine (BG) derivatives. b) Structure of labeled PG derivatives: PGBT, PGDIG, PGDF, and PGCy3.

ity of enzymes also became apparent in this work as we tried to reprogram the substrate specificity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT). AGT repairs O6-alkylated guanine in DNA by irreversibly transferring the alkyl group to its active cysteine residue. AGT has previously been exploited by our group as a tag that can be covalently labeled with chemical probes in living cells. The labeling is based on the reaction of AGT fusion proteins with O6-benzylguanine (BG) derivatives carrying a probe attached to the 4-position of the benzyl ring, leading to specific transfer of the probe to the fusion protein (Figure 1, panel a) (5, 6). AGT mutants with orthogonal substrate specificities could be used for simultaneous and specific labeling of different AGT fusion proteins with different synthetic probes, which would be particularly attractive for applications in molecular imaging. We show in this work that attempts to alter the substrate specificity of AGT by directed evolution using protein repertoires generated by traditional mutagenesis techniques (saturation and random mutagenesis) failed. As an alternative to these mutagenesis strategies, we tested here a new approach in which additional amino acid sequences were inserted into the polypeptide chain of the protein to provide new substrate binding elements. AGT mutants with completely random amino acid loops on the surface were generated, and those that catalyzed the desired reaction 576

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were identified through selection and screening techniques. The best AGT mutant isolated can be used in conjunction with previously generated AGT mutants to achieve selective labeling of two different fusion proteins with two different fluorophores in vitro and in living cells. Such protein pairs can be used to explore protein–protein interactions or enzymatic activities using FRET measurements. RESULTS Substrate Design. We sought a substrate that does not react with the AGT mutants in current use, but that might be able to react with an appropriately engineered AGT. Numerous O6-alkylguanine derivatives have been previously synthesized for the inhibition of AGT in tumor therapy (7–10). We focused on O6-propargylguanine derivatives (PG; Figure 1, panel b) as potential substrates for an appropriately mutated AGT because PG has been reported to be a very poor inhibitor of human wild-type AGT (7). Furthermore, its reactivity in SN2 reactions should be comparable to that of benzylguanine. The low reactivity of PG with AGT most likely arises from the poor binding of the substrate to the active site of AGT, which we reasoned could be improved by redesigning the substrate binding site. The difference in size between PG and BG also has the advantage that a mutant that reacts with PG might not react with the more www.acschemicalbiology.org

ARTICLE bulky substrate BG, thus leading to two orthogonal AGT–substrate pairs based on steric complementation (11). PG derivatives were synthesized in which PG was coupled to biotin (PGBT), digoxigenin (PGDIG), diacetylfluorescein (PGDF), and Cy3 (PGCy3) (Figure 1, panel b; see Supporting Information for syntheses of these molecules). Immunoassay of wild-type AGT and previously evolved improved AGT mutants confirmed that PGBT and PGDIG are indeed very poor substrates. PGBT did not compete with BG substrates for reaction with AGT even when used at a 300-fold higher concentration (results not shown). A precise rate for the reaction of AGT with PGBT could not be determined under these conditions because very little product was detected, even after long incubations at high substrate concentrations. We estimated an upper limit of 0.5 s⫺1 M⫺1 for the apparent second-order rate constant of the reaction of the mutant MAGT (4) with PGBT. This value is ⬃1000fold lower than the rate constant of MAGT for BG. MAGT is an engineered mutant with improved expression properties, reduced DNA binding, and increased activity towards BG substrates. Selection of Activities from Random and Saturation Mutagenesis Libraries. Numerous directed evolution strategies have been applied in search of enzyme variants that exhibit activities towards new substrates. In the majority of attempts, one or several amino acid residues were exchanged, either through random or saturation mutagenesis, to obtain mutants that display activities towards the desired substrates. Such classical enzyme engineering strategies have been particularly successful in the improvement of the robustness of catalysts (e.g., thermal stability, solubility, activity in organic solvents) (12) to increase the catalytic turnover of enzymes or for the creation of enantioselective catalysts (13). Following these examples, two repertoires of mutated AGT were generated by random and saturation mutagenesis of the AGT mutant MAGT (4). Members of the randomly mutated library (4 ⫻ 107 transformants) contained an average of 2–3 amino acid mutations per gene, as determined by sequencing 10 clones chosen at random. For the second library, amino acids that are in close proximity to the benzyl group (residues 138–143, 157, 159, and 160) were conservatively mutated using degenerate primers to increase the chance of encoding the wild-type amino acid in each position (5.5 ⫻ 107 transformants). The AGT mutants were displayed on filawww.acschemicalbiology.org

mentous phage as a fusion of the minor coat protein pIII. AGT-phage reacting with PGDIG were isolated on a solid support coated with anti-digoxigenin antibody. In earlier work we have successfully used this strategy to evolve AGT mutants with increased activity towards BG (14, 15) and mutants resistant to an inhibitor of wild-type AGT (4). Four consecutive phage panning rounds were performed with the two libraries in parallel using either 2 or 20 ␮M PGDIG substrate. The number of phage isolated after each round of selection was similar to the level of phage isolated in reactions without the substrate (105–106 transducing units (t.u.)), indicating that the activities of the selected proteins were not improved. Activity measurements of 100 mutants of each library isolated in the fourth selection round confirmed that none of the mutants reacted with the PGDIG substrate with a rate constant significantly ⬎0.5 s⫺1 M⫺1. Insertion of Amino Acid Loops into the Polypeptide Chain of AGT. Because mutation of the existing amino acids in AGT failed to generate activity towards the PG substrate, we envisioned adding new sequence space to AGT by inserting additional amino acid loops into the protein. We reasoned that libraries of AGT with random loops inserted into selected regions of the polypeptide chain could offer a rich source of potential catalysts, as the catalytic center of the enzyme is expected to remain functional. In fact, it has been demonstrated in simulations that DNA insertion is a key step in searching protein space in natural evolution (16). Furthermore, a number of studies have shown that amino acid stretches inserted into selected surface exposed regions of a protein (e.g., loop regions) often leave the structure and the function of a protein unperturbed (17). Insertion of random sequences of 120 amino acids into a surface loop of RNase H, for example, left the activity of 10% of the mutants intact (18), whereas TEM-1 ␤-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity (19, 20). We chose three positions in AGT that are in close proximity to the substrate binding site but are surface exposed. Three libraries were generated by inserting a stretch of five random amino acids into the polypeptide chain of MAGT at each of the three positions (Figure 2). The length of the inserted loop was chosen to be long enough to form a turn and hence to link the opened ends of the enzymes polypeptide chain without impairing protein folding. For library 1, peptide loops were inserted into a turn between the short ␤ strand 7 VOL.1 NO.9 • 575–584 • 2006

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indicating that phage enzymes with improved activities against the PGDIG substrate had become enriched. No enrichment was observed in the selections with libraries 2 and 3. After the fourth round of selection, phage DNA was isolated from each of the libraries, and the genes coding for the enzyme mutants were cloned into the plasmid pGEX-2T for cytoplasmic expression of glutathione S-transferase (GST)-AGT fusion proteins. About 100 clones from each selected library were expressed on a Figure 2. Structure of AGT with BG modeled into the active small scale, and the activity was measured by immunosite (14). For the creation of the loop libraries, stretches of assay as follows: cell lysate was incubated with PGDIG five random amino acids were inserted between amino acids for time periods ranging from 0 to 30 min and blotted in the highlighted regions. One amino acid on each side of onto a hydrophobic membrane. Mutants with activity the insert was also randomized in each library. towards PGDIG became labeled with DIG and were quantified with an anti-digoxigenin antibodyand ␣ helix 8 (amino acid residues 159/160); for library horseradish peroxidase (HRP) conjugate (Figure 3, 2, amino acids were inserted at the terminus of helix 6, panel a). Of 100 mutants selected from library 1, eight close to the asparagine hinge (amino acid residues 135/ reacted efficiently with the PGDIG substrate, whereas no 136); and for library 3, the additional amino acids were active mutants were found among the clones selected from libraries 2 and 3. Library 1 also contained a signifiinserted at the terminus of the third ␤ strand of the N-terminal domain of AGT (amino acid residues 32/33). cantly higher percentage of clones with activity against BG compared with libraries 2 and 3, underscoring the One amino acid on either side of the inserted pentaimportance of the choice of the position for the loop peptide was also randomized, on the basis of the insertion. assumption that these junctions might require a differCharacterization of AGT Mutants. The sequences of ent conformation to accommodate the inserted loop. Transformation of the three libraries into bacterial cells the eight mutants with improved PGDIG reactivity revealed high homology in the added loop (Figure 3, resulted in library sizes of 5.5 ⫻ 107 (library 1), 9.2 ⫻ 107 (library 2), and 2.5 ⫻ 107 (library 3) with ⬎95% of panel b). Amino acid 159, which flanks the inserted pentapeptide at the N-terminus, and amino acid 159a, the clones containing the gene of an AGT mutant. The first amino acid of the inserted loop, were found to be number of AGT mutants that retained their activity glycine and proline in all sequences. Positions 159c, against the BG substrate (and hence their structural integrity) was estimated by phage capture experiments 159d, and 160 were frequently occupied by the amino acids glycine, tryptophan, and glycine, respectively. At with the substrate BG digoxigenin (BGDIG) and phage positions 159b and 159e, the amino acid side chains mixtures of each library. Phages displaying the mutavaried. One of the active mutants (clone 24) contained genized AGT were enriched 150-fold (library 1) and 10-fold (libraries 2 and 3) above background levels at a an insertion of only three amino acids, probably as a result of an impurity in the wobble DNA primer used in BGDIG concentration of 2 ␮M. This indicates that, in library construction. This clone also contained the concontrast to mutant libraries created by saturation mutagenesis of active site residues, a very large portion served motif Gly-Pro-Xaa-Gly seen in the other seven active mutants, which indicates that AGT with different of the library remained catalytically active. loop sizes can also react with PGDIG. Four consecutive phage panning rounds were perThe mutant that showed the highest activity in the formed with all three libraries in parallel using either 2 or immunoassay with the PGDIG substrate (clone 9; 20 ␮M PGDIG substrate. Phage titers were measured after each round of selection, and an increase in the titer 45 s⫺1 M⫺1) was tested for its activity towards PGBT from library 1 was noticed after round 3 (20- and 200(Figure 1, panel b), a substrate in which digoxigenin is fold enrichment at 2 and 20 ␮M PGDIG) and round 4 replaced with biotin. The mutant reacted with PGBT with (30- and 600-fold enrichment at 2 and 20 ␮M PGDIG), the same kinetics as with PGDIG (45 s⫺1 M⫺1), indicat578

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Figure 3. Characterization of selected AGT mutants. a) Example of a typical dot-blot activity screen. Cell lysates were incubated with PGDIG for 0, 3, 10, or 30 min and blotted onto a hydrophobic membrane. The lysates of 24 mutants (including MAGT; line 1, column A) were arrayed on eight lines (1– 8) and three columns (A–C). Product formation was detected using an anti-digoxigenin-antibody-HRP conjugate and a chemiluminescence-based assay. As an example of an activity assay of one mutant, the dots of the most active clone in this screen (6B) are highlighted in a rectangular box. b) Alignment of DNA and amino acid sequences of LAGT and AGT mutants that reacted with PGDIG in the activity screen. Numbers were newly assigned to the mutants and do not relate to the ones in panel a. c, d) Time course of the reactions of AGT54 or LAGT with 1 ␮M BGCy3 in the presence of varying concentrations of PGBT. The assay is based on an increase of the fluorescence intensity (F.I.) of Cy3 upon reaction of AGT with BGCy3 (4).

ing that the evolved activity is independent of the label attached to the poly(ethylene glycol) linker. In contrast to our expectations, clone 9 retained its activity towards BG derivatives; the activity of clone 9 with BG coupled to Cy3 was measured as 480 s⫺1 M⫺1 compared to 520 s⫺1 M⫺1 for the parental clone MAGT (Table 1). The thermal stability of the evolved mutants was measured by incubation of purified enzyme for 15 min at temperatures ranging from 4 to 60 °C and measurement of the residual activity. Despite the insertion of a 5-amino acid loop, the selected mutants were as stable under these www.acschemicalbiology.org

conditions as their progenitor MAGT (TM ⬃55 °C; see Supplementary Figure 1). The high sequence similarity in the loop of the active mutants isolated from library 1 indicates a unique mode of interaction between the mutants and the PG substrate. The higher activity towards the novel substrate may derive from a better positioning of PG in the active site or a higher affinity of the binding site for PG. The fact that the selected mutants retain their activity with BG argues against drastic changes to the structure of the binding site, but a detailed analysis of the mutant will require structural information. VOL.1 NO.9 • 575–584 • 2006

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TABLE 1. Properties of AGT mutants used in this work Activity versus BG kobs (sⴚ1 Mⴚ1)

Mutant

Description

W160

Human wild-type AGT with the single mutation G160W Previously evolved mutant with increased activity towards BG and optimized properties for applications in protein labeling (4) Mutant selected from library 1 with loop inserted at position Glu159/Gly160 of MAGT Previously evolved mutant with increased activity towards BG (4) Mutant with loop from clone 9 inserted at position Glu159/Gly160 of AGT54

AGT AGT

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clone 9 AGT54 L AGT

To validate that the new activity originates from the inserted loop and to generate a clone with even higher activity towards PG substrates, the five additional amino acids 159a–159e, plus the two neighboring amino acids 159 and 160, were grafted into another AGT mutant, termed AGT54, which has an activity 2.5-fold higher than that of MAGT with BG substrates. The superior activity of AGT54 derives from mutations in the residues that interact with the purine heterocycle of BG (115, 116, 150, 151, and 152), which is identical in PG. The mutant resulting from the loop grafting, termed L AGT, has a second-order rate constant of 115 s⫺1 M⫺1 with PGDIG, which is elevated 2.6-fold compared with that of clone 9 (Table 1). This confirms that the inserted loop is responsible for the activity towards PG derivatives and shows that, in this case, the changes generated through loop insertion and site-directed mutagenesis are additive. The latter point should be important for future protein engineering experiments with AGT. To assess the substrate specificity of LAGT and AGT54, we measured the reaction of each protein with BGCy3, a BG derivative labeled with the fluorophore Cy3, and then measured the inhibition of the reaction by competition with the alternative substrate PGBT. The reaction between AGT54 and BGCy3 was unaffected even by a 100-fold excess of PGBT over BGCy3, reflecting the lack of activity of AGT54 against PG derivatives (Figure 3, panel c). In contrast, the reaction of LAGT with BGCy3 was quenched by 50% by a 30-fold excess of PGBT over BGCy3 (Figure 3, panel d). This result is in agreement with the higher activity of LAGT towards BG derivatives compared to that towards PG derivatives. This strategy of inserting additional amino acid sequences into surface regions of a protein to enhance activity towards a new substrate should, in principle, be applicable to other proteins. Surface loops play an important role in the substrate recognition of many enzymes. For example, in many kinases, the difference in specificity originates from variations in the loops connecting the secondary structure elements (21). The insertion of amino acid sequences into surface regions of enzymes may thus also be applied to multi-turnover catalysts. Loop insertion at random positions might be an important alternative to insertion at rationally chosen 580

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Activity versus PG kobs (sⴚ1 Mⴚ1)

400 520

ⱕ0.5 ⱕ0.5

480

45

1300 1250

ⱕ0.5 115

sites. An innovative methodology to insert or delete amino acids in random positions has recently been presented (22). Specific Double-Labeling of AGT Fusion Proteins in Vitro and in Living Cells. To achieve specific labeling of different proteins with different chemical probes, we first needed to determine whether the mutant LAGT could be used as a tag to label fusion proteins in living cells. LAGT was fused either to three copies of the simian virus 40 large T-antigen nuclear localization signal (NLS) or to ␤-galactosidase and transiently expressed in a Chinese hamster ovary (CHO) cell line. Cells were incubated with PGDF (Figure 1, panel b) for 30 min. Confocal microscopy showed specific labeling of LAGT-NLS in the nucleus and LAGT-␤-galactosidase in the cytoplasm (Figure 4, left panels). As a control, we chose W160AGT, human wild-type AGT with the single mutation G160W, whose activity towards BG derivates is comparable to that of LAGT. Cells overexpressing W160AGT did not become labeled, even when incubated with PGDF for extended periods of time (Figure 4, right panels). Next we transiently co-expressed LAGT-␤-galactosidase and W160 AGT-NLS to assess whether the evolved LAGT

Figure 4. Selective fluorescence labeling of LAGT fusion proteins in living CHO cells with PGDF. Left panels: CHO cells transiently expressing LAGT-NLS3 or LAGT-␤galactosidase were incubated with PGDF (20 ␮M) and analyzed by fluorescence microscopy. Right panels: CHO cells transiently expressing W160AGT-NLS3 or W160AGT-␤galactosidase were incubated with PGDF (20 ␮M) and analyzed by fluorescence microscopy. Fluorescence and differential interference contrast (DIC) images of confocal micrographs are overlaid. www.acschemicalbiology.org

ARTICLE mutants could be selectively labeled in the presence of an AGT with wild-type specificity. Incubation of the cells with PGDF led to selective labeling of LAGT in the cytoplasm, while labeling of W160AGT-NLS in the nucleus could not be detected (Supplementary Figure 3). Because LAGT can react with both PG and BG derivatives, selective labeling of LAGT and AGT fusion proteins relies on sequential addition of the substrates. First, the L AGT fusion protein must be labeled to completion with a PG derivative, and then a BG derivative can be added to label the wild-type specific AGT fusion protein. To test the feasibility of this sequential labeling strategy, an equimolar mix of pure LAGT-GST and W160AGT was incubated first with PGDF and then with BG linked to tetramethylrhodamine (BGTMR). Aliquots of the reaction mixture were taken at different time points, subjected to SDS-PAGE, and analyzed for fluorescein and TMR labeling using a laser-based fluorescence scanner with appropriate filters (Figure 5, panel a). Under these conditions LAGT-GST reacts with PGDF to completion within 90 min, and BGTMR added after 90 min reacts exclusively with W160AGT (Figure 5, panel a). Next we attempted a similar sequential labeling in CHO cells transiently co-expressing LAGT-␤-galactosidase and W160AGT-NLS. Sequential labeling with PGDF followed by a red-emitting SNARF-1 derivative of BG (BGSF) (6) led to specific labeling of the corresponding proteins in their different cellular compartments (Figure 5, panel b). The successful reprogramming of the substrate specificity of AGT through loop insertion broadens the scope of application of the AGT labeling approach, as it allows selective labeling of two fusion proteins within the same cell. The use of spectroscopic distinguishable fluorophores is especially useful for studying protein localization and function in living cells. Specific Double-Labeling of AGT Fusion Proteins for FRET Measurements. The ability to perform selective labeling of different AGT proteins with different spectroscopic probes may be of particular use for FRET. This powerful technique is used to investigate protein–protein interactions or to construct sensors for enzyme activities conformational changes of proteins and concentration changes of secondary messengers. So far, FRET measurements in living cells have predominantly used autofluorescent proteins (23, 24). Cyan and yellow fluorescent proteins (CFP and YFP, respectively) are currently the FRET system of choice, and the two proteins have been optimized by various groups with respect to www.acschemicalbiology.org

spectral overlap, dynamic range, and various other properties (25–27). Despite all these efforts, the optimized CFP–YFP pairs still have a number of drawbacks. For example, CFP and YFP in their current form are still reported to be weak dimers, which might affect the interaction of the corresponding fusion proteins (27). Also, it has been reported that the YFP mutant optimized for FRET applications, CyPet, folds poorly at 37 °C (27). Furthermore, efficient FRET pairs of autofluorescent proteins that are orthogonal to the CFP–YFP pairs are not yet available (27). The latter point would be important for the simultaneous monitoring of multiple biochemical processes in living cells. We wanted, therefore, to evaluate whether the new AGT mutant can be used together with parental AGT for the creation of efficient FRET pairs. The proteins FK506 binding protein (FKBP) and

Figure 5. Specific labeling of LAGT and W160AGT fusion proteins with different fluorophores in vitro and in living cells. a) Sequential incubation of an equimolar mixture of purified LAGT-GST and W160AGT (0.5 ␮M each) with PGDF (10 ␮M) and BGTMR (1 ␮M) and subsequent analysis using SDS-PAGE and a laser-based fluorescence scanner. Green color represents fluorescence resulting from fluorescein and red color represents fluorescence resulting from TMR. b) Sequential incubation of human embryonic kidney (HEK) and CHO cells transiently co-expressing L AGT-␤-galactosidase and W160AGT-NLS with PGDF (20 ␮M) and BGSF (5 ␮M) and subsequent analysis using laser scanning confocal microscopy. Green color represents fluorescence resulting from fluorescein, and red color represents fluorescence resulting from SNARF-1. VOL.1 NO.9 • 575–584 • 2006

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rapamycin-binding domain of mTOR (FRB) as fusions with LAGT and MAGT were chosen as a first example for measurements in vitro. The FKBP/ FRB pair was particularly well suited for these experiments as their interaction depends on the presence of the natural product rapamycin, which facilitates control Figure 6. FRET between fluorescence labeled AGT fusion experiments. A soluproteins. a) Emission spectra of PGCy3/BGCy5 labeled tion containing FKBP-LAGT and FRB-MAGT in the presence and absence of equimolar concenrapamycin. b) Emission spectra of PGFL/BGCy5 labeled L M trations of FKBPAGT-DEVD- AGT before and after treatment with L caspase 3 or proteinase K. AGT and FRB-MAGT was sequentially incubated with equimolar concentrations of PGCy3 (Figure 1, panel b) followed by BG linked to Cy5 (BGCy5) and subsequently used without further purification. The selective labeling of FKBP-LAGT with Cy3 and FRB-MAGT with Cy5 was confirmed by SDS-PAGE, visualized using a fluorescence scanner (Supplementary Figure 4, panel a). Excitation of the mixture of the two labeled proteins at 520 nm and measurement of the Cy5 emission at 674 nm showed that addition of rapamycin induced a 250% increase of the Cy5 emission and a change of the emission ratio of Cy3 and Cy5 of 2.9-fold (Figure 6, panel a). When an alternative substrate pair was used, PGFL (Figure 1, panel b) and BGCy5, with excitation at 485 nm, the increase in FRET through addition of rapa-

METHODS Standard chemicals were purchased from Sigma-Aldrich. Enzymes for recombinant DNA work were purchased from MBI Fermentas or New England Biolabs. Sequences of PCR primers used in this work are listed in Supporting Information. Phage Library Creation. The phagemid vector pAK100 (29) was used for the creation of filamentous phage displaying variants of AGT. Mutated MAGT genes were ligated into the two SfiI restriction sites that are located between the gene of the minor coat protein pIII and its leader sequence. The creation of the random mutagenesis library is described in Gronemeyer et al. (15). In

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mycin was even more pronounced (350% increase of Cy5 emission and a 3.8-fold change in the emission ratio of Cy3 and Cy5), emphasizing the need for welloptimized pairs of fluorophores; see Supplementary Figure 4, panel b. We then tested whether the two AGT variants concatenated in a single polypeptide chain could be specifically labeled with two different fluorophores. Such constructs would be valuable for the construction of FRETbased sensors to measure concentration changes of important metabolites or enzymatic activities (23, 24). Towards this end, MAGT and LAGT were linked by the DEVD substrate motif of the endopeptidase caspase 3. The resulting protein LAGT-DEVD-MAGT could be efficiently labeled with the substrates PGFL and BGCy5, as confirmed by SDS-PAGE and subsequent analysis using a fluorescence scanner; see Supplementary Figure 5. Excitation of the doubly labeled fusion protein at 485 nm led to intramolecular FRET and emission at 640 nm (Figure 6, panel b). When the construct was subjected to proteolysis by caspase 3, under conditions where ⬃75% of the protein was cleaved, the FRET-dependent Cy5 emission decreased by ⬃60% and the emission ratio at 540 and 640 nm changed by a factor of 2.7. Complete digestion by proteinase K reduced the Cy5 emission by 77% (Figure 6, panel b and Supplementary Figure 5). Together these experiments demonstrate that doubly labeled AGT fusion proteins are attractive candidates for the construction of FRET-based sensors. Furthermore, different AGT fusion proteins can now be tagged with a large variety of different synthetic fluorophores, which is important for the construction of efficient FRET pairs orthogonal to those of autofluorescent proteins. The emission wavelengths of fluorophores available for labeling of AGT fusion proteins in living cell ranges from blue- to far-red (28). Future experiments will focus on establishing such FRET pairs in living cells.

this library, the gene of MAGT is randomly mutated with an average of 2–3 base mutations per gene. For the creation of the saturation mutagenesis library, the amino acid positions 138 –143, 157, 159, and 160 of MAGT were mutated using partially degenerate primers in a PCR-based method using pAK100M AGT as a template (4). Two overlapping PCR products were formed with the primer pairs p1/p2 and p3/p4. The gel-purified products were assembled in a PCR reaction with the primer pair p1/p4. The three libraries containing an additional stretch of five random residues (loop libraries 1–3) were created by an overlap-

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ARTICLE extension PCR of two PCR products. The overlapping PCR products were created with the primer pairs p5/p6 and p7/p8 for the library with the insertion at the amino acid position 159/160 (loop library 1), with the primer pairs p1/p9 and p10/p4 for the library with the insertion at the amino acid position 135/136 (loop library 2), and with the primer pairs p1/p11 and p12/p4 for the library with the insertion at the amino acid position 32/33 (loop library 3). The degenerated primers were designed in a such way that the two amino acid positions flanking the five residue insert were also randomized. The gel-purified PCR products were assembled in a reaction using the primer pair p13/p14. For all libraries, ligated plasmids were purified and electroporated into Escherichia coli JM101 cells. Phage Selection. Phage were produced in 50-mL cultures essentially as described in Juillerat et al. (14). Phage were separated from cells by centrifugation at 4000 rpm for 20 min at 4 °C. Then, 0.5 mL of the supernatant containing the phage particles (typically 1011 t.u./mL) was blocked with 0.5 mL of phosphate buffered saline (PBS)/milk (4% (w/v) skimmed milk in PBS pH 7.4) and incubated with PGDIG (2 or 20 ␮M) for 10 min. As a positive and negative control of the phage panning procedure, phage of the same preparation were incubated in parallel either with or without the substrate BGDIG. Nonreacted substrate was removed by two rounds of PEG precipitation as follows: Phage were incubated with 250 ␮L of 20% (w/v) PEG 8000, 2.5 M NaCl on ice for 10 min and spun at 13,000 rpm for 10 min at 4 °C. Phage were resuspended in 0.5 mL of PBS/milk, added to preblocked anti-digoxigenin magnetic beads (50 ␮L of beads in 0.5 mL of PBS/milk; Roche), and incubated on a rotating wheel at RT for 30 min. The magnetic beads were then washed once with PBS/milk, six times with PBS/Tween 20 (0.01% (v/v)), and twice with PBS. The reaction tube was replaced at least once during the washing procedure. The phage were eluted with 100 ␮L of 50 mM glycine buffer, pH 2.5. The pH of the eluate was neutralized by addition of 50 ␮L of 1 M Tris-HCl, pH 8. Eluted phage (100 ␮L) were incubated with 10 mL of exponentially growing JM101 for 30 min at 37 °C and plated on a large (20 cm diameter) agar plate containing kanamycin (70 ␮g/mL). For phage titer determination, 20 ␮L of the eluted phage (and eight parallel 10-fold dilutions) were incubated with 180 ␮L of exponentially growing JM101 cells for 30 min at 37 °C. Then, 20 ␮L of the infected cells was spotted on kanamycincontaining agar plates. The infected cells grown on large agar plates were collected and subjected to 2–3 further rounds of phage selection. Activity Screening. Cells harboring the DNA of selected phage were pooled by resuspension of bacterial colonies resulting from phage infection with PBS. Plasmid DNA was extracted from cells, and the genes of the AGT mutants were PCR amplified with the primer pair p15/p16 to attach the sequences of the BamHI and EcoRI restriction sites and a C-terminal polyhistidine tag. The PCR product was ligated into the pGEX-2T vector for expression of AGT mutants as fusion proteins with GST. The plasmids were electroporated into E. coli XL1-Blue. Individual colonies were picked and grown overnight in 3 mL of 2YT/ampicillin media. Cells of these cultures were diluted in 20 mL of Sabouraud dextrose broth (SB) media containing ampicillin to an OD600 of 0.1 and grown at 37 °C. Protein expression was induced when the OD600 reached 0.6 by addition of 1 mM IPTG, and cells were incubated at 24 °C for 4 h. The cells were then pelleted, resuspended in 1 mL of PBS pH 7.4 containing 1 mg/mL lysozyme and 10 ␮g/mL DNase I, and sonicated for 10 s. The cell debris was removed by centrifugation for 30 min at 13,000 rpm at 4 °C, and 50 ␮L of supernatant was incubated with either 20 ␮M PGDIG or 2 ␮M BGDIG for 0, 1, 3, 10, 30, or 90 min. The reactions were quenched by addition of 100 ␮M BG. The reaction mixtures were spotted on a hydrophobic mem-

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brane with a 96-well dot-blot system (Minifild I, Schleicher & Schuell Biosciences). The membrane was blocked in PBS/milk, washed with PBS, blocked in PBS/bovine serum albumin (BSA) (1 mg/mL), and incubated with preblocked anti-digoxigeninHRP antibody conjugate (dilution, 1:1000). The membrane was washed, and the HRP was detected with a chemiluminescencebased assay. Characterization of Active AGT Mutants. Active mutants were expressed in cultures of 100 mL of 2YT containing ampicillin (100 ␮g/mL) at 24 °C for 4 h and purified by either nickel or glutathione affinity chromatography. The eluted protein was dialyzed overnight at 4 °C in 50 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer, pH 7, containing 1 mM DTT. The activity of the purified protein was measured with the dot-blot-based assay described above using either the substrates PGDIG (20 ␮M) or BGDIG (2 ␮M) in combination with the anti-digoxigenin-HRP antibody conjugate or the substrates PGBT or BGBT in combination with streptavidin-HRP. The same assay was used to measure residual activity of the AGT mutants after heat incubation for 15 min at temperatures ranging from 4 to 60 °C. The selectivity of AGT mutants towards different substrates was measured by incubating the protein simultaneously with the two substrates BGCy3 (1 ␮M) and PGBT (1–200 ␮M) at various molar ratios in 50 mM HEPES buffer and 1 mM DTT. The kinetics of the reaction of AGT mutants with BGCy3 was measured as described previously (4). In Vitro FRET Assays. Construction and purification of the fusion proteins FKBP-LAGT, FRB-MAGT, and LAGT-DEVD-MAGT is described in Supporting Information. A solution containing FKBP-LAGT and FRB-MAGT (5 ␮M each) in 50 mM HEPES, pH 7.2, containing 1 mM DTT was incubated sequentially with either PGCy3 or PGFL (4 ␮M, 3 h at RT), then PG (40 ␮M, 1 h, RT, to quench non-reacted LAGT), and finally BGCy5 (4 ␮M, 1 h, RT). FRET was measured by exciting at 485 nm (or 520 nm) and monitoring the emission at wavelengths between 500 and 750 nm (or between 530 and 750 nm) in the presence and absence of rapamycin (5 ␮M) using a SPECTRAmax GEMINI fluorescence spectrometer (Molecular Devices Corp.). Purification and sequential labeling of LAGT-DEVD-MAGT was performed as described for FKBP-LAGT and FRB-MAGT. The fusion protein was incubated with caspase 3 (5.8 ␮g/mL; Sigma-Aldrich) at RT overnight or proteinase K (250 ␮g/ml; AppliChem) for 20 min on ice. FRET was measured as described for the FKBP and FRB fusion proteins. Fluorescence Labeling of AGT Fusion Proteins in Living Cells. The construction of expression plasmids for transient expression in mammalian cells and the protocols for transient transfection are given in Supporting Information. Twenty-four hours after transient transfection, AGT-deficient CHO-9-neo-C5 cells and HEK cells in F-12(Ham) medium were incubated with either the substrate PGDF (20 ␮M), BGDF (5 ␮M), or BGSF (5 ␮M) in PBS containing 0.1% (v/v) DMSO for time periods ranging from 5 min to 2 h. In the case of transient co-expression of W160AGT-NLS3 (4) with an LAGT fusion protein, PGDF (20 ␮M) was added to the cells for 1 h, followed by incubation with BGSF (5 ␮M) for 30 min. The cells were then washed three times with PBS to remove excess substrate. Cells were imaged in PBS 30 min after the last washing step with a laser-scanning confocal microscope (Leica TCS SP2 AOBS) using 488-nm argon, 515-nm argon, or 561-nm HeNe laser lines and a 20⫻ water objective. Emission was recorded at wavelengths from 520 to 550 nm and 600 to 700 nm, respectively. DIC optics were used to image nonlabeled cellular structures. Scanning speed and laser intensity were adjusted to avoid photobleaching of the fluorescent probes and damage or morphological changes of the cells. VOL.1 NO.9 • 575–584 • 2006

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Acknowledgments: This work was supported by the Swiss National Science Foundation, the Commission for Technology and Innovation of the Bundesamt für Berufsbildung und Technologie, and the Human Frontier Science Program. The authors would like to thank Beatrice Kunz for technical assistance and Helen O’Hare for critical reading of the manuscript. Supporting Information Available: This material is free of charge via the Internet.

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