Design Consideration and Probes for ... - ACS Publications

352

Bioconjugate Chem. 2000, 11, 352−362

Design Consideration and Probes for Fluorescence Resonance Energy Transfer Studies Michael Sinev,† Pavel Landsmann, Elena Sineva,† Varda Ittah, and Elisha Haas* Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel. Received October 6, 1999; Revised Manuscript Received February 29, 2000

Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-β-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 Å in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.

INTRODUCTION

The experimental approach, based on the phenomenon of fluorescence resonance energy transfer (FRET),1 enables evaluation of distances between relatively remote (10-100 Å) parts of macromolecules, thus extending the scope of spectroscopic determination to the monitoring of large-scale structural transitions and dynamics (Cheung, 1991; Van Der Meer et al., 1994). As such, FRET * To whom correspondence should be addressed. Phone: (972)-3-5318210. Fax: (972)-3-5351824. E-mail: haas@ mail.biu.ac.il. † Current address: Department of Biochemistry and Molecular Biology, The Pennsilvania State University, 201 Alhouse Laboratory, University Park, PA 16802. 1 Abbreveations: Ac O, acetic anhydride; AcOH, acetic acid; 2 AK, adenylate kinase from Escherichia coli or, within all abbreviated names of mutant AK derivatives, cysteine-free (Cys77Ser)-AK mutant with additional Cys and/or Trp mutations; AR, analytical reagent; Ci-AK, single-cysteine AK mutant containing Cys-substitution at position i; AQ4CA, 7-acetamidocoumarin-4-carboxylic acid; Ccai-AK, Ci-AK labeled with I-Cca; CP, chemically pure; D/A pair, donor/acceptor pair of fluorescent probes; DEOA, diethyloxalacetate; DDW, double distilled water; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ET, energy transfer; Et2O, diethyl ether; EtOAc, ethyl acetate; EtOH, ethanol; I-Cca, 7-iodoacetamidocoumarin-4-carboxylic acid; Iac2O, iodoacetic anhydride; I-LY, Lucifer Yellow iodoacetamide; I-Nal, N-iodoacetyl-β-(2-naphthyl)-alanine; I-Sa, 5-iodoacetamidosalicylic acid; IDD, intramolecular distance distribution; FRET, fluorescence resonance energy transfer; LYi-AK, CiAK labeled with I-LY; 2-ME, 2-mercaptoethanol; MeCN, acetonitrile; MEK, methylethyl ketone; MeOH, methanol; NaliAK, Ci-AK labeled with I-Nal; Py, pyridine; Q4CA, 7-aminocoumarin-4-carboxylic acid; Q4CE, 7-aminocoumarin-4-carboxylic acid, ethyl ester; r.t., room temperature; S77-AK, cysteine-free (Cys77Ser)-AK mutant; Sai-AK, Ci-AK labeled with I-Sa; TFA, trifluoroacetic acid; Tris, tris(hydroxymethyl)aminomethane; TR-FRET, time-resolved FRET, Wi-AK, single-tryptophan AK mutant containing Trp-substitution at position i; WiCj-AK, single-tryptophan/single-cysteine AK mutant containing Trpand Cys-substitution at positions i and j, respectively.

spectroscopy, primarily in the time-resolved mode, proves to be a valuable tool for the investigation of the role of structural flexibility in enzyme action, functioning of macromolecular assemblies and protein folding (Cheung, 1991; Haas, 1996). Yet, the application of the FRET method for studies of structure and dynamics of proteins (and other biological macromolecules) might be hindered by practical problems (e.g., the absence of suitable fluorescent probes and involved preparative procedures). In general, a protein sample for use in FRET experiment should have a pair of fluorescent probes (donor and acceptor) covalently attached to predetermined sites in the molecule. Preliminary design of a corresponding protein derivative thus first includes selection of the two sites in the protein molecule to be labeled and then selection of the donor/acceptor (D/A) pair to be attached to these sites. The pair of probes should have spectroscopic characteristics optimized for the range of distances between the two selected sites and the time scale of the process to be studied. It should be noted that the spectroscopic characteristics of a given D/A pair (i.e., the Fo¨rster distance, R0, and the donor fluorescence lifetime, τd) determine the detection limits for measurements of the distances between the two attached probes. The range for reliable distance determination is evaluated as R0 ( R0/2. The time interval available for monitoring protein structural dynamics in FRET experiments may range from subnanoseconds up to milliseconds, depending on the τd values and the mode of measurements (Haas 1996, Van Der Meer et al., 1994, Haas and Steinberg 1984). A single D/A pair attached to a protein derivative can yield a limited amount of data, allowing partial description of intramolecular motions in the protein under study. A set of D/A pairs with adequate spectroscopic characteristics (i.e., R0 and τd) should enable a more detailed characterization of large-scale protein dynamics by the FRET method. The current selection of commercially available fluorescent probes suitable for these FRET experiments is still limited. Recent research activities [(see, e.g.,

10.1021/bc990132l CCC: $19.00 © 2000 American Chemical Society Published on Web 04/21/2000

Design Consideration and Probes for FRET Studies

Terpetschnig et al. (1995), Selvin and Hearst (1994), and Chen and Selvin (1999)] are thus focused on synthesis of novel fluorescent probes with modified spectroscopic characteristics designed for investigation of specific research objectives. Besides spectroscopic properties of fluorescent probes, the properties of the modified protein derivatives and the efficiency of postlabeling purification procedures should be also considered when designing sets of D/A pairs for protein structural studies. The main points to be taken into account are as follows. (a) Site specificity of the chemical modifications, ensuring selective site-directed attachment of fluorescent probes to the desired residues of a protein molecule. Specificity can be achieved by engineering of specific cysteine protein mutants combined with the application of thiol-reactive fluorescent reagents. (Haran et al., 1992; Sinev et al., 1996b; Haas, 1996; Lillo et al., 1997a). (b) Minimal perturbation of the native structure by mutations and by chemical modifications. This implies careful selection of sites to be mutated and design of probes having minimal potential for noncovalent interactions with protein’s side chains or backbone. (c) Maximal rotational freedom of the attached probes. This is of primary importance for correct interpretation of results obtained in an energy-transfer experiment (Cheung, 1991; Van Der Meer et al., 1994; Haas, 1996). (d) The postlabeling purification scheme for doublelabeled protein derivatives. No general procedure is available, and specific schemes should be developed for each set of derivatives. However, the choice of charged probe molecules for protein labeling may essentially simplify postlabeling purification, by means of ionexchange chromatographic isolation of labeled derivative(s) from the unlabeled protein [see, e.g., Sinev et al. (1996a,b) and Lillo et al. (1997a)]. In some cases when a charged probe was used for labeling, ion-exchange chromatography was effective for separation of two singlelabeled derivatives of a two-cysteine protein mutant (these two differed in the site of probe attachment yet had equal total charge). Thereby, individual preparation of these two species and, subsequently, the corresponding site-specific double-labeled derivatives was enabled (Sinev et al., 1996b; Lillo et al., 1997a). Besides that, the presence of a charged/polar group(s) in a probe might diminish undesirable hydrophobic interactions of the attached reagent with a protein. Thus, the use of charged probes provide important practical advantages to be considered in the design of labeling strategies for spectroscopic studies of site-specifically labeled protein derivatives. The present report describes a practical application of the above design considerations, including preparation of two newly synthesized thiol-reactive probes. These reagents were spectroscopically characterized in their conjugates with cysteine residues inserted in mutant protein derivatives. The new probes were used, together with two known fluorescent iodoacetamide reagents and tryptophan residues (native or inserted in the mutant protein), to design a set of seven D/A pairs with R0 values in the range from 15 to 30 Å. Charged groups present in these thiol-reactive probes enabled their efficient conjugation to engineered cysteine residues and facilitated subsequent chromatographic purification of labeled derivatives. The applicability of the newly synthesized dye, 7-iodoacetamidocoumarin-4-carboxylic acid (I-Cca), as an acceptor for tryptophan donor for distance determination in proteins, was verified by distance determination in two Cca-labeled single cysteine/single tryptophan mutants of a model protein of known X-ray structure (Escherichia coli adenylate kinase).

Bioconjugate Chem., Vol. 11, No. 3, 2000 353

Figure 1. Thiol-reactive fluorescent probes used in the present study. I-LY, the commercial Lucifer Yellow iodoacetamide, was purchased from Molecular Probes. I-Sa (5-iodoacetamidosalicylic acid), I-Nal [N-iodoacetyl-β-(2-naphthyl)-alanine], and I-Cca (7iodoacetamidocoumarin-4-carboxylic acid) were prepared by iodoacetylation of the corresponding amines (see Experimental Procedures). EXPERIMENTAL PROCEDURES

Materials. Unless specified otherwise, inorganic reagents and organic solvents used for synthetic preparations were of CP grade and were purchased from BioLab. Synthetic reagents were from Sigma (Aldrich or Fluka). Double-distilled water (DDW), acetonitrile (MeCN), and trifluoroacetic acid (TFA) for HPLC were from Baker. Lucifer yellow iodoacetamide (I-LY) was purchased from Molecular Probes (Eugene, OR). Iodoacetic anhydride (Iac2O) was obtained as a powder (AR) from Aldrich, Sigma. Immediately after opening, it was aliquoted in freshly opened anhydrous dioxane (Aldrich, Sigma) and stored frozen at -20 °C as recommended by Wetzel et al. (1990). Verification of Identity and Purity of Synthetic Preparations. The novel thiol-reactive probes for FRET studies, i.e., I-Nal and I-Cca, were obtained on the base of known chromofores, i.e., commercially available β-(2naphthyl)-(L) alanine (Bachem, California), and 7-acetamidocoumarin-4-carboxylic acid (AQ4CA), which was earlier synthesized and spectroscopically characterized by Besson et al. (1991) from 7-aminocoumarin-4-carboxylic acid (Q4CA). Here, the latter was prepared and iodoacetylated in order to obtain a new iodoacetamide probe, 7-iodoacetamidocoumarin-4-carboxylic acid (I-Cca), which is spectroscopically similar to AQ4CA. Thereby, the identity of all synthetic preparations was verified by comparing their spectroscopic characteristics (1H NMR, UV, fluorescent spectra, quantum yields) to the known ones. The purity of all prepared iodoacetamides was additionally confirmed by estimating their thiol reactivity (Ellmann test, see below) and by examining HPLC purity of their conjugates with thiolic compounds (cysteine, 2-mercaptoethanol). The above verification of purity of synthesized iodoacetamide probes was sufficient for adequate spectroscopic and FRET studies with corresponding protein conjugates presented herein. Synthesis of Thiol-Reactive Fluorescent Probes (see Figures 1 and 2). 7-Aminocoumarin-4-carboxylic acid, ethyl ester (Q4CE) (MW 233.2) was prepared ac-

354 Bioconjugate Chem., Vol. 11, No. 3, 2000

Figure 2. Synthesis of a new thiol-reactive probe I-Cca (7-iodoacetamidocoumarin-4-carboxylic acid) and the reference compound AQ4CA (7-acetamidocoumarin-4-carboxylic acid). (i) Pechmann condensation of 3-aminophenol with diethyloxalacetate (1 h, 110 °C); (ii) 2 N KOH, MeOH, reflux 3 h; (iii) Ac2O, Py, 5 °C, then 24 h, r.t.; (iv) Iac2O, dioxane-water, NaHCO3, 5 °C, 3 h. Synthetic intermediates are 7-aminocoumarin-4-carboxylic acid (Q4CA) and 7-aminocoumarin-4-carboxylic acid, ethyl ester (Q4CE).

cording to procedures (Kanaoka et al., 1984; Besson et al., 1991), with modifications, by Pechmann cyclization of 3-aminophenol (Fluka) with diethyloxalacetate (DEOA). The latter was prepared from its commercially available sodium salt, following the earlier procedure (Riegel et al., 1946). Samples of thus prepared Q4CE for analytical purposes was washed with methanol (MeOH) and vacuumdried. The bulk of the product was used in the next stage without further purification. 7-Aminocoumarin-4-carboxylic acid (Q4CA) (MW 205.2) was prepared from Q4CE by 3 h reflux in methanolic KOH, according to a procedure modified from (Besson et al., 1991). Recrystallization in MeOH/DDW, followed by dissolving in minimal amount of MeOH, dilution with DDW and lyophilizing, yielded the pure Q4CA. The overall yield of the preparations after the above two synthetic steps constituted 60-70%, as related to the amount of 3-aminophenol taken. TLC, performed in EtOAc-CHCl3 1:1, neat methylethyl ketone (MEK), and MEK-AcOH-water 18:1:1 systems, confirmed essential purity of both products thus obtained. 1H NMR, UV, and fluorescence spectra of these intermediate products essentially represented the published data (Kanaoka et al., 1984; Besson et al., 1991). An analytical sample (15 mg) of 7-acetamidocoumarin-4-carboxylic acid (AQ4CA) was prepared according to a procedure modified from (Besson et al., 1991), by acetylation of 20 mg of the crude Q4CA (by ∼5 M excess of Ac2O in dry pyridine, 0-20 °C, 16 h). This AQ4CA served as a stable reference compound representing essential spectroscopic characteristics of the Cca fluorofore to be synthesized (see next paragraph). The 1H NMR, UV (λmax ) 338 nm, in EtOH) and fluorescence spectra of this preparation, as well as its

Sinev et al.

estimated quantum yield (∼0.02, in EtOH), essentially represented the published data (Besson et al., 1991). 7-Iodoacetamidocoumarin-4-carboxylic acid (I-Cca). Example iodoacetylation procedure. All iodoacetylations (final products are depicted in Figure 1), including the following one, were performed according to a procedure modified from (Wetzel et al., 1990; Wood and Wetzel, 1992). A total of 290 mg (1.19 mmol) of previously obtained Q4CA was dissolved in 10 mL of 90% aqueous dioxane (BioLab, AR) and then mixed with 2 mL of 5% NaHCO3 to form clear solution at pH 6-7 (determined upon 1:10 v/v water dilution by indicator paper). The solution was ice-chilled on a magnetic stirrer. To this, 1.2 g of prealiquoted Iac2O (in 14 mL of dry dioxane) was added by 1 mL portions for 20 min, with intensive stirring. The pH of the mixture (determined as above, immediately after adding the next portion of the reagent), was kept at 6-7 during the reagent addition. It was adjusted at need by 1 mL portions of 5% NaHCO3, followed by 2-3 mL portions of dioxane, so that the water content of the mixture would not exceed 30-40%. Afterward, the mixture was stirred an additional 90 min on the ice, with pH continuous monitoring (checked each 10-15 min). It was then diluted with cold water (1:10 v/v), supplied with 10 mL of 1 M NaI, and carefully acidified with 3 M HCl to final pH ∼1 and left overnight in the dark at 4 °C. The yellow precipitate formed was filtered, washed 2 × 50 mL water, dried in air, and then washed with dry Et2O (3 × 2 mL). Subsequent vacuum desiccating (1 Torr, 3 h) left 140 mg of light yellow powder (yield 32%). Its 1H NMR spectra was essentially similar to that of AQ4CA obtained above, differing only in a slight shift of the band corresponding to the amide 1H (δ ) 9.97, instead of 9.67 ppm, broad), and the appeared peak 3.97 ppm (referred to 1H of ICH2CO group). UV spectra (λmax ) 332 nm, in EtOH) were identical to that of AQ4CA (see above). Thiol reactivity of this preparation (as determined by Ellman test for unreacted thiol groups, see corresponding section below) was ∼85%. The purity of thus prepared I-Cca was checked by HPLC, with sample preparations (from 5 mg of I-Cca each) of corresponding thio-conjugates with L-cysteine and with 2-mercaptoethanol (2-ME), as described below for other iodoacetamides. Both Cys- and 2-ME-conjugates of this product were at least 90% HPLC-pure (a single peak). No further purification of the above I-Cca preparations was thus required, though it might be attained with MeOH/water. N-Iodoacetyl-β-(2-naphthyl)-alanine (I-Nal) (MW 383.18) was prepared by iodoacetylation of β-(2-naphthyl)-(L) alanine purchased from Bachem, California. The procedure identical to the above was employed; however, 50 mM sodium phosphate buffer was used instead of 5% NaHCO3. After extensive washings of HCl-precipitated product with water and vacuum-drying performed as above, the crude preparations (average iodoacetylation yield ≈ 50%) were recrystallized from MeOH/DDW. The purity of the recrystallization fractions was checked in sample preparations (5 mg from each recrystallization fraction) of corresponding thio-conjugates with L-cysteine and with 2-mercaptoethanol (2-ME), prepared according to the reaction procedure described below, with cysteine or 2-ME taken at 2:1 molar excess. The conjugate solutions thus obtained were acidified by 1% TFA to quench the reaction and immediately analyzed by reversed-phase HPLC (DDW/0.1% TFA-80% MeCN/0.1% TFA gradient monitored at 280 nm, Waters HPLC binary pump system, 0.5 mL/min flow rate, Protean RP-18 column). Two main recrystallization fractions, that provided essentially HPLC-pure conjugates both with 2-ME


and with cysteine (a single peak, > 90% purity), were combined, dissolved in minimal amount of MeOH, then diluted with excess DDW and lyophilized to leave snowwhite crystalline powder, with overall postpurification yield of ∼20%. The 1H NMR and UV spectra of this preparation essentially corresponded to those of the starting β-(2-naphthyl)-(L) alanine. A quadruplet peak 3.78 in 1H NMR (1H of ICH2CO-group) appeared in both of the main recrystallization fractions. Thiol-reactivity of this preparation determined as above was ∼70% (see the respective section below). 5-Iodoacetamidosalicylic acid (I-Sa) (MW 321.07), which is not commercially available currently (production was discontinued in 1986 by Molecular Probes), was synthesized starting from 5-aminosalicylic acid and purified by recrystallization from MeOH/DDW, with HPLC characterization of sample 2-ME- and L-cysteyl-S-conjugates prepared from recrystallization fractions, in the same way as described above for I-Nal. Combining fractions that yielded HPLC-pure (>90%) thiol-conjugates resulted in a white (yellowish) crystalline powder (overall postpurification yield ≈ 20%). The 1H NMR spectra of this preparation essentially corresponded to those of the starting 5-aminosalicylic acid. UV spectra were identical to those described in the catalog of Molecular Probes (1986). A singlet peak 3.90 in 1H NMR (1H of ICH2CO group) appeared in the main recrystallization fraction. Thiol reactivity of this preparation determined as above approached 100%. Preparation and Analysis of Protein Mutants. Recombinant plasmid pEAK91, containing the intact gene coding for E. coli adenylate kinase (AK) (Reinstain et al., 1988), was gifted by Prof. A. Wittinghofer. The plasmid coding for cysteine-free (C77S)-adenylate kinase mutant (S77-AK) was prepared previously (Sinev et al., 1996a). This plasmid was employed for preparations of mutant plasmids by site-directed mutagenesis method described by Kunkel et al. (1987). Thus, all mutant proteins contained the extra C77S mutation. To confirm the introduced mutations, the full-length AK gene was sequenced for each mutant plasmid. The E. coli strain HB101, transformed by the appropriate plasmid, was used for production of wild-type and mutant AKs. Proteins were purified as previously described (Sinev et al., 1996a) and checked for the presence (or the absence, in the case of cysteine-free mutants) of cysteine residues by the Ellman reaction (Riddles et al., 1979). Reactivity and Specificity of Thiol-Reactive Fluorescent Probes. Reactivity of the used iodoacetamide probes I-Nal, I-Cca, I-Sa, and I-LY (see Figure 1) toward thiol groups was tested with low molecular weight thiols, in 0.1 M Tris-HCl (pH 8.0) containing 1 mM EDTA (the labeling buffer, Bl), each at equivalent ratio of a probe (0.3 mM) and a thiol, L-cysteine (0.3 mM), or DTT (0.15 mM), correspondingly. The reagents were mixed by vortexing and then left for 1 h in the dark at room temperature. The amount of unreacted thiols was determined by the Ellman test (Riddles et al., 1979). The synthesized iodoacetamide fluorescent probes I-Sa, I-Cca, and I-Nal showed almost quantitative SH-reactivity. Both with L-cystein and DTT, the yield was about 100, 85, and 70% for I-Sa, I-Cca, and I-Nal, respectively. Relatively low labeling yield with the commercial I-LY, 40%, might be caused by its known lability under standard SH-labeling conditions (Haugland, 1992). Possible side reactivity of the probes toward nonthiolic groups in a protein was checked using cysteine-free S77AK mutant. The latter was incubated for 1 h in buffer Bl (at 100-130 µM), in the presence of high molar excess


of an iodoacetamide-containing probe (0.5-2 mM). The incubation was stopped by addition of DTT up to 20 mM final concentration. The content was then transferred to 20 mM Tris-HCl (pH 8.0) (the buffer A), by extensive dialysis. An aliquot of the resulting solution was loaded on an ion-exchange (Mono Q HR 5/5) column equilibrated with buffer A and analyzed [Pharmacia FPLC chromatographic system, linear gradient of NaCl concentration (0-0.3 M), 0.5 mL/min], to detect side covalent attachment of charged species (i.e., the tested iodoacetamide probes). None of the four probes was found to have noticeable reactivity toward nonthiolic residues in the protein used. Labeling of Cystein Mutants and Purification of the Labeled Protein Derivatives. Prior to labeling, single-cysteine AK mutants were preincubated for 1 h with 10-20 mM DTT as a reducing agent. The DTT was then removed by gel-filtration using a 10 mL Bio-Gel P-6DG column (Bio-Rad), equilibrated with the buffer Bl. Labeling was performed in the buffer Bl, at 50-80 µM protein concentration using a 10-20 M excess of the thiol-reactive probe. Upon mixing, reagents were incubated for 1-1.5 h in the dark at room temperature; the reaction was then stopped by DTT addition (up to 20 mM final concentration). Further, the labeled derivatives were separated from the unreacted protein according to the procedure modified from (Sinev et al., 1996a,b). In brief, the protein solution was first dialyzed against buffer A, containing 1 mM DTT and 1 mM EDTA, to remove the bulk of the unreacted dye. It was then passed through the 10 mL gel-filtration column (Bio-Gel P-6DG) equilibrated in buffer A (containing 1 mM DTT) to remove the residual dye. (DTT and EDTA were added to the buffers in order to prevent oxidation of the unreacted thiol of cysteine mutants. Without these additives, side elution peaks of charged species derived by oxidation/dimerization of thiol groups in unlabeled protein were observed to interfere with the collected peak of the purified product.) The following anion-exchange separation was performed with Mono Q HR 5/5 column [Pharmacia FPLC chromatographic system, linear gradient of NaCl concentration (0 to 0.3 M), 0.5 mL/min], with total protein amount not exceeding 1 mg. In all separation runs, the difference in charge between the unlabeled mutants and their labeled derivatives (bearing an extra negative charge) was large enough for efficient separation (see Figures 3 and 4). Activity Measurements. Specific enzymatic activities of AK derivatives were determined toward ADP formation (MgATP + AMP T MgADP + ADP) by a spectrophotometric assay employing pyruvate kinase-lactate dehydrogenase coupling system (Rhoads and Lowenstein, 1968). The reaction mixture contained 0.1 M Tris-HCl (pH 7.5), 0.1 M KCl, 10 mM MgCl2, 0.2 mM NADH, 1 mM of phosphoenolpyruvate, the coupling enzymes (3 units/mL for each enzyme), 0.2 mM AMP, and 1 mM ATP (Sinev et al., 1996a). Activity measurements were performed at 25 °C. The values of specific activity were expressed in international units (IU) of enzyme activity per milligram of the protein. One IU defines an amount of enzyme catalyzing the conversion of 1 µmol of substrate/ min under the assay conditions. Spectroscopic Characterization of Fluorescent Probes. Absorption spectra were measured on an Aviv model 17DS UV-vis spectrophotometer (AVIV Assoc., Lakewood, NJ). Fluorescence spectra were recorded on an AVIV ATF-105 spectrofluorimeter, using slit band width of 1 and 3 nm at the excitation and emission monochromators, respectively. All measurements were


Figure 3. Typical separation of a mixture containing singlecysteine AK mutants labeled with N-iodoacetyl-β-(2-naphthyl)alanine (I-Nal) and unlabeled cysteine mutants, by anionexchange chromatography on a Mono Q column. A total of 720 µL of protein solution in 20 mM Tris-HCl (pH 8), containing unlabeled C73-AK (250 µg), Nal73-AK (200 µg), Nal142-AK (150 µg), and Nal203-AK (200 µg) were applied on the Mono Q column (0.5 × 4.5 cm) equilibrated with the same buffer. The column was washed by 5 mL of the buffer and the proteins were eluted with the linear gradient (0-0.3 M) of NaCl concentration (plotted as the dashed line) at flow rate 0.5 mL/min. Positions of peaks related to the unlabeled C73-AK and Nal-labeled AK derivatives listed above were reproducible from the preceding analytical runs of individual compounds. In separate runs under the same conditions, peak position of Nal55-AK was identical to that of Nal203-AK, whereas those of single-cysteine AK mutants C55- and C142-AK were approximately the same as that of C73AK. The position of the peak for C203-AK was shifted by ∼0.5 mL toward higher NaCl concentration as compared to that of C73-AK (not shown).

Figure 4. Typical separation of a mixture containing singlecysteine AK mutants labeled with Lucifer yellow iodoacetamide (I-LY) and unlabeled cysteine mutants, by anion exchange chromatography on a Mono Q column. A total of 580 µL of protein solution in 20 mM Tris-HCl (pH 8), containing C73-AK (37 µg), LY55-AK (37 µg), LY73-AK (26 µg), LY142-AK (37 µg), and LY203-AK (37 µg), were applied on the Mono Q column (0.5 × 4.5 cm) equilibrated with the same buffer. The column was washed by 5 mL of the buffer and the proteins were eluted with the linear gradient (0-0.3 M) of NaCl concentration (plotted as the dashed line) at flow rate 0.5 mL/min. Positions of peaks related to the unlabeled C73-AK and LY-labeled AK derivatives listed above were reproducible from the preceding analytical runs of individual compounds. In separate runs under the same conditions, peak positions of C55- and C142-AK were approximately the same as that of C73-AK, whereas the position of the peak for C203-AK was shifted by ∼0.5 mL toward higher NaCl concentration (not shown).

performed at 25 °C with protein solutions prepared in 0.1 M Tris-HCl (pH 7.5) by size-exclusion chromatography using Superose 12 HR 10/30 column (Pharmacia). Absorption spectra were expressed in the units of molar extinction (M-1 cm-1): (λ) ) [OD(λ) × Mp]/(lc), where OD is the optical density of the protein solution; Mp is the protein molecular mass [evaluated as 23 600 Da, which corresponds to the value of molecular mass for the wild-type AK (Brune et al., 1985)]; l is the optical path length (cm); and c is the protein concentration (g/ L). Concentrations of protein solutions of the labeled derivatives and cysteine-free tryptophan AK mutants

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were determined by the method of Lowry et al. (1951) using wild-type AK as a calibration standard. The protein concentration for wild-type AK was determined by using the absorption coefficient A277nm ) 0.5 (mg/mL)-1 cm-1 (Girons et al., 1987). The Fo¨rster distance (R0) and the average fluorescence lifetime of the donor are the basic spectroscopic parameters of a D/A pair of fluorescent probes, which determine the limits of applicability of a given pair for structural studies by FRET techniques. The Fo¨rster distance corresponds to the interprobe distance at which the rate of energy transfer (from the excited donor molecule to the acceptor) equals the rate of depopulation of the excited state of the donor, in the absence of the acceptor. (In this case, efficiency of energy transfer equals 50%.) The Fo¨rster distance of the donor-acceptor pairs of the fluorescent probes was calculated (in angstroms) using the spectroscopic properties of the probes (Van Der Meer et al., 1994):

R0 ) 0.211(κ2n-4QdJda)1/6

(1)

where Qd is the quantum yield of the donor; κ2 is the orientation factor (taken as 2/3); n is the refractive index of the medium between the probes (taken as 1.34); Jda is the normalized spectral overlap integral (M-1 cm-1 nm4). The overlap integral was calculated as Jda ) [∑ fd(λ)a(λ)λ4∆λ]/[∑ fd(λ)∆λ], using the corrected fluorescence emission spectrum of the donor, fd(λ), and the absorption spectrum of the acceptor, a(λ), respectively. The quantum yield of the donor fluorescent probes, Nal and tryptophan, was determined by the reference method, using solution of degassed naphthalene in cyclohexane [Q ) 0.23 (Berlman, 1971)] and solution of L-tryptophan in water [Q ) 0.144 (Wiget and Luisi, 1978)]. Quantum yields of Sa and Cca were determined using a solution of quinine sulfate in 0.05 M H2SO4 as a reference [Q ) 0.51 (Velapoldi and Mielenz, 1980)]. Measurements of fluorescence lifetimes were performed using the time-correlated single-photon counting system described previously (Haas, 1996). Fluorescence emission was collected through a polarizer oriented at the magic angle relative to the polarization of the exciting beam. Decay of fluorescence emission, I(t), was analyzed by a nonlinear list-squares multiexponential fitting (Grinvald and Steinberg, 1974): I(t) ) ∑ I0i exp(-t/τi). The average fluorescence lifetime was calculated from the parametrized emission decay: 〈τ〉 ) ∑Riτi, where Ri and τi are the relative amplitude (Ri ) I0i/∑I0i) and the lifetime of the ith decay component, respectively. Energy-Transfer (ET) Experiments. Applicability of the Cca as an acceptor for tryptophan donor for distance determination in proteins was tested by timeresolved ET measurements of two single-tryptophan/ single-cysteine AK mutants labeled with I-Cca. Each experiment included measurement of decay of the tryptophan emission in two samples [e.g., W169-AK (the donor without the acceptor) and W169Cca55-AK (the donor in the presence of the acceptor)] prepared in 0.1 M Tris-HCl (pH 7.5) at the same protein concentration (0.5 mg/mL). Tryptophan emission was excited at 297 nm and monitored at 360 nm (band width, 8 nm). Efficiency of ET (E) was calculated from the average lifetime of the donor, 〈τd〉 and 〈τda〉, in the absence and in the presence of the acceptor: E ) 1 - 〈τda〉/〈τd〉. An average interprobe distance, R, corresponding to the measured E-value was calculated [R ) R0 (1/E - 1)1/6] and was compared with the respective CR-CR distance in the crystal structure of the ligand-free AK (Mu¨ller et al., 1996).



RESULTS

Purification of the Labeled Derivatives. To ensure the high yield of the labeled derivatives, single-cysteine AK mutants were reacted for 1-1.5 h with 10-20 M excess of thiol-reactive probes. The Ellman test showed that the extent of the labeling reaction was at least 90% for C55-, C142- and C203-AK mutants. However, with C73and C188-AK, the labeling yields were lower (in several cases ∼50%). In the labeling of C73-AK with LY-I, the yield was as low as 20%. On the other hand, nearly 100% yield with all of the thiol-reactive labels was achieved with C203-AK. Accordingly, in the Mono Q chromatographic profile, the position of the peak corresponding to the unlabeled C203-AK mutant was shifted by ∼0.5 mL toward higher salt concentration, as compared to peak positions corresponding to the unlabeled C55-, C73-, and C142-AK single-cysteine mutants. This shift of the peak position of C203-AK might be related to a larger extent of ionization of the C203-thiol, which is consistent with enhanced reactivity of this mutant. The difference in charge between the labeled and unlabeled protein species was the basis for postlabeling purification of the conjugates by anion-exchange chromatography. A typical chromatogram demonstrating the separation of single-cysteine AK mutants labeled with a single-charged dye (I-Nal), from the corresponding unlabeled mutants, is shown in Figure 3. The lowest (yet quite satisfactory) resolution was accomplished with the labeled derivatives of C203-AK. The position of the peak corresponding to the C142-AK mutant labeled with I-Nal (i.e., Nal142-AK), was slightly shifted toward higher salt concentration, as compared to that of Nal203-AK or Nal55AK. The peak position relating to Nal73-AK was even further shifted. A very similar separation pattern was found with the cysteine AK mutants labeled by the other single-charge reactive dyes I-Sa and I-Cca (Sinev et al., 1996a,b). In the case of the cysteine AK mutants labeled with the double-charged I-LY, the separation pattern was essentially similar to those of the corresponding derivatives conjugated with single-charged probes (see Figure 4). In agreement with the difference in charge, the resolution between the peaks of the LY-labeled derivatives and those of the unlabeled mutants was significantly better. Distinctive feature of the chromatographic pattern of the LY-labeled derivatives was good resolution between the peaks related to LY203- and LY55-AK. On the contrary, in the case of the single-charged probes, the peaks corresponding to the labeled derivatives of C55- and C203-AK mutants were not resolved under the same conditions. Superose 12 size-exclusion chromatography of the labeled AK derivatives did not show any diversity in their elution characteristics. None of the derivatives had elution volume different from that of the wild-type protein. Spectral Properties of the Labeled Derivatives. Conjugates of single cysteine AK mutants with thiolreactive labels were used to determine spectroscopic properties of corresponding fluorescent probes, and further, to match possible donor/acceptor pairs of the probes for FRET studies. Absorption spectra and molar extinction coefficients of the protein conjugates were marginally dependent on the location of the probe attachment. Thus, extinction coefficients determined for cysteine AK mutants labeled with I-Nal were 17 100, 17 100, 17 000, 17 200, and 16 800 M-1 cm-1 for the Nal-AK derivatives labeled at positions 55, 73, 142, 188 and 203, respectively (each value, corresponding to the absorption maximum

Figure 5. Spectral properties of fluorescent probes in conjugates with single cysteine AK mutants. (A) Absorption spectra of unlabeled C203-AK mutant (dashed line) and its conjugates with Nal (bold solid line), Sa (solid line), Cca (bold dashed line), and LY (bold dotted-dashed line). Absorption spectra are expressed in units of molar extinction (M-1 cm-1). (B) Emission spectra of fluorescent probes in conjugates with C142-AK mutant: Nal (1), Sa (2), Cca (3), and LY (4). Emission spectrum of the tryptophan residue in W142-AK is presented by the dashed line. The spectra are corrected for the spectral sensitivity and normalized. Fluorescence emission of tryptophan and Nal was excited at 297 nm. Emission of the other probes was exited at their absorption maximum.

at 278 nm, represents the average of triplicate determinations). Extinction coefficients determined for Sa-AK derivatives were 3230, 3220, 3170, and 3180 M-1 cm-1 (the values correspond to absorption maximum at 310 nm) for the Sa-probe at Cys-55, 73, 142, and 203, correspondingly. For each probe, extinction variations depending on the cysteine mutants selected, did not exceed error limits of individual extinction determinations (estimated as 3%). Emission spectra of the fluorescent probes were, in general, slightly dependent on the probe location. Thus, the emission spectrum of Nal in Nal142-, Nal188-, and Nal203-AK derivatives were practically identical to that of Nal conjugate with free Lcysteine. The spectra of Nal in Nal55- and Nal73-AK were 1.5-2 nm red-shifted relative to the spectrum of the NalL-cysteine. Small variations in emission spectra of the protein conjugates were also observed with the other fluorescent probes. Examples of absorption and fluorescence emission spectra of fluorescent probes, measured for their protein conjugates, are shown in Figure 5. Donor/Acceptor Pairs of Fluorescent Probes. A primary criterion for matching D/A probe pairs for FRET studies is the existence of an essential overlap between fluorescence emission spectrum of one probe (a donor) with absorption spectrum of a second probe (an acceptor). Figure 6 shows overlap of the donor emission spectra (e.g., Nal or tryptophan) and acceptor absorption spectra (e.g., Sa, Cca or LY) of possible D/A pairs. Thus, the excited Nal or tryptophan molecule might be used as an energy donor for nonradiative excitation of Sa, Cca, and


Sinev et al.

The efficiency of FRET was measured for two labeled derivatives, (W142Cca203)-AK and (W169Cca55)-AK, and the apparent mean distances were calculated (Table 3). The values of transfer efficiencies found from these measurements (26.7% and 21.2%) were high enough for reliable determination of the corresponding interprobe distances (30.6 and 29.7 Å, for W142Cca203-AK and W169Cca55-AK, respectively). The apparent mean interprobe distances determined by the ET experiments were in a good agreement with the values between the respective CRatoms (30.8 and 29.3 Å) calculated from the recently published crystal structure of the ligand-free enzyme (Mu¨ller et al., 1996). DISCUSSION

Figure 6. Spectral overlap of fluorescence emission spectra of donor probes and absorption spectra of acceptor probes. (A) The emission spectrum of Nal, conjugate with C142-AK (solid line) and tryptophan, in W142-AK (dashed line), respectively; the absorption spectrum of C203-AK conjugates with Sa (bold line), Cca (bold dashed line) and LY (bold dotted-dashed line), respectively. (B) The emission spectrum of Sa, conjugate with C142-AK (solid line) and the absorption spectrum of C203-AK conjugate with LY. The emission spectra are corrected for the spectral sensitivity and normalized. Absorption spectra are expressed in units of molar extinction (M-1cm-1).

LY. Hence, seven possible D/A pairs for FRET studies might be considered, three pairs of probes with Nal as a donor (with Sa, Cca, and LY as acceptors), three corresponding pairs with tryptophan as a donor, and the Sa/ LY D/A pair. For each of the above pairs, the quantum yield of donor emission (Qd), the average fluorescence lifetime of the donor, and the Fo¨rster distances (R0) of the D/A pair were determined (see Table 1). It should be noted that Qd-values were somewhat dependent on the probe’s location [for Nal donor, Qd ) 0.12, 0.16, 0.16, 0.17, and 0.23 at Cys-188, 203, 55, 142, and 73, respectively; for Sa donor (see reference j in Table 1), Qd ) 0.26, 0.28, 0.31, and 0.33 at Cys-203, 142, 55, and 73, respectively]. Due to the weak dependence of R0 on Qd, since R0 ≈ (Qd)1/6, the above variations in Qd-values have minor effect on the calculated R0-values. Thus, R0-values calculated for the Sa/LY D/A pair ranged between 31 and 32 Å, depending on variations of Qd-values and absorption/emission spectra corresponding to different probe location. When choosing an appropriate fluorescent probe for labeling of a protein, it is strongly desirable to avoid structural alterations caused by the modification. In the case of enzymes, high level of catalytic activity of a labeled protein can provides strong evidence that the original tertiary structure remained essentially intact after the labeling. Thus, specific enzymatic activities of cysteine AK mutants labeled with the thiol-reactive probes studied here were compared with activities of unlabeled mutants. Most of the present derivatives were highly active (see Table 2). Only two out of 23 assayed derivatives (Nal55- and Nal73-AK) showed a major drop in specific activity.

The present study was motivated by the need for multiple D/A pairs of probes which can extend the range of time scales and of intramolecular distances, available for determination when characterizing structural processes in proteins. The set of seven D/A pairs presented here allows probing intramolecular distances in proteins in the range 10-40 Å (Table 1). Thus, the R0 value of the Nal/Sa D/A pair (∼17 Å) enables determination of distances from 10 to 26 Å, whereas the R0 value of the Sa/LY pair (∼31 Å) is suitable for probing a range of larger intramolecular distances, from 20 to 40 Å. Figure 5 shows that for each of the D/A pairs included in the present study, the emission spectra of the corresponding donor and acceptor probes are distinctly resolved (see Figure 5B). This spectral feature allows determination of the contribution of the donor or the acceptor to the emission intensity separately from the contribution of the other probe. The above opportunity, in turn, largely facilitates the separate use of donor and acceptor emission signals for independent evaluation of energy transfer in the double-labeled derivative. Subsequently, this considerably simplifies global analysis of time-resolved ET experiments and, further, simultaneous determination of intramolecular distance distributions (IDDs) and diffusion coefficients (representing rates of structural fluctuations) (Beechem and Haas, 1989; Haas, 1996). Multiple Measurements Using Sets of Donor/ Acceptor Pairs. Three of the four probes studied (Sa, Cca, and LY) can be employed as acceptors when tryptophan or Nal are used as a donor. Both donor probes have similar R0 with each of the three acceptors and thus the donors seem to be interchangeable when probing similar intramolecular distances in proteins. This opens few practical experimental advantages, as follows. (a) The use of the tryptophan donor (either naturally present in a protein or specially introduced by sitedirected mutagenesis) for structural studies of proteins by FRET spectroscopy extensively simplifies the preparation of the “double labeled” protein derivatives. When a Trp/Cys mutant is used, only one chemical modification is needed. This makes the use of derivatives with Trpdonor useful in preliminary FRET studies of major structural transitions associated with protein functioning (e.g., ligand binding) or folding. (b) The fluorescence lifetime of the Nal donor is 10fold larger than that of Trp and offers a time window for more reliable detection of segmental motions in the nanosecond time scales (which are difficult to detect using Trp as a donor). Specifically, as shown in Table 1, substitution of Nal for Trp in each of the present D/A pairs does not lead to a significant shift in R0 values. The Trp-containing D/A pairs, with τd of Trp (3-5 ns), are to be preferred for monitoring faster structural fluctuations



Table 1. Spectroscopic Properties of Donor-Acceptor Pairs of Fluorescent Probesa donor propertiesb λmax (nm) EM

donor Nal

333

W

353

τd

Qdd (( 0.01) 0.17

acceptor propertiesc

τi (Ri) 33.0 (0.62) 40.3 (0.38) 7.6 (0.06) 5.0 (0.87) 0.5 (0.07)

0.24

(ns)e,f 〈τd〉 (χ2)

acceptor Saj

35.8 (1.1) 4.84 (1.2)

ag (M-1 cm-1)

R0 (Å)i

λmax (nm)

τa

(ns)e,h

donor

ABS

EM

τi (Ri)

〈τa〉 (χ2)

Nal

W

9.15 (0.25) 4.51 (0.75) 1.27 (0.62) 0.65 (0.38)

5.67 (1.3)

17.0

15.6

1.03 (1.0)

26.5

25.9

8.14 (0.87) 5.01 (0.13)

7.73 (1.1)

20.4

24.9

3200

310

437

Ccak

18 700

332

455

LY

12 600

428

534

a All spectroscopic measurements were performed in 0.1 M Tris-HCl (pH 7.5) at 25 °C. b The data refer to spectroscopic properties of β-(2-naphthyl)alanine (Nal) and tryptophan (W) donor in single cysteine C142-AK mutant labeled with N-iodoacetyl-β-(2-naphthyl)alanine (I-Nal) and in single tryptophan W142-AK mutant, respectively. c Measurements were performed using C203-AK single cysteine mutant labeled with 5-iodoacetamidosalicylic acid (I-Sa), 7-iodoacetamidocoumarine-4-carboxylic acid (I-Cca) and lucifer yellow iodoacetamide (I-LY), respectively. d Qd is the quantum yield of the donor. e Multiexponential analysis of the time-resolved fluorescence emission. Decay parameters of the fluorescence emission τi and Ri are the lifetime and the relative amplitude of the ith decay component, respectively; 〈τ 〉 is the average (amplitude-weighted) lifetime (χ2-values are presented in parentheses). Errors for the average lifetimes are ( 1% of the presented values. f Fluorescence emission of the donors (excited at 297 nm) was recorded at 350 and 360 nm for Nal and W, respectively. g Molar extinction of the acceptor corresponding to the last absorption maximum (λ h max). Acceptor fluorescence was excited at 310 nm for Sa and Cca, and at 400 nm for LY. Fluorescence emission was recorded at the wavelength corresponding to the acceptor emission maximum. i The Fo ¨ rster distances of the donor-acceptor pairs. j Sa can be as well considered as a donor for LY acceptor; see data given in Results. k Quantum yield of the newly synthesized Cca as measured with the Cca-203-AK was 0.11 ( 0.01.

Table 2. Enzymatic Activities of AK Mutants and Their Derivatives Labeled with Thiol-Reactive Fluorescent Probesa specific activity (IU/mg)b single cysteine mutantse

tryptophan-containing mutantse

probe

wtc

S77d

C55

C73

C142

C188

C203

W142

W142C203

W169

W169C55

none Nal Sa Cca LY

960

930

920 360 710 750 650

960 130 550

920 750 780

940 820

840

940

1190

1100

810

710

950 900 960 980 940

890 830

900 700

a Enzymatic activities were determined toward ADP formation (MgATP + AMP T MgADP + ADP). Measurements were performed at 25 °C in 0.1 M Tris-HCl (pH 7.5) containing 0.1 M KCl, 10 mM MgCl2, 0.2 mM AMP, and 1 mM ATP. b Specific activities are expressed in international units of enzyme activity (IU) per milligrams of the protein. The values presented are the means of experiments done in triplicates. (As tested separately, reproducibility of the measurements was within 5% of the presented values.) c Wild-type enzyme. d Cysteine-free (C77S)-AK mutant. e All mutant proteins contained the extra C77S mutation. Mutant cysteine and tryptophan positions in AK are denoted with indexed symbols Ci or Wi, correspondingly.

Table 3. Determination of Interprobe Distances in (W142C203-Cca)-AK and (W169C55-Cca)-AK Derivatives by Time-Resolved Energy Transfer Measurementsa multiexponential analysis of time-resolved fluorescence of the tryptophan donor τd (ns)b protein W142Cca203-AK

W169Cca55-AK

τi (Ri)

τda (ns)c 〈τd〉

(χ2)

W142-AK 7.58 (0.06) 4.84 (1.20) 4.97 (0.87) 0.26 (0.07) W169-AK 6.65 (0.10) 2.78 (1.22) 2.35 (0.90)

τi (Ri)

〈τda〉 (χ2)

W142Cca203-AK 5.22 (0.39) 3.55 (1.30) 3.57 (0.38) 0.71 (0.23) W169Cca55-AK 4.74 (0.12) 2.19 (1.24) 1.84 (0.88)

Ed (%)

Re (Å)

Rcrystf (Å)

26.7 ((1.0)

30.6 ((0.3)

30.8

21.2 ((1.1)

29.7 ((0.4)

29.3

a Each set of energy transfer (ET) experiments included measurements of the time-resolved fluorescence of the tryptophan donor in two protein samples (e.g., W142-AK and W142Cca203-AK) prepared in 0.1 M Tris-HCl (pH 7.5) at equal protein concentration (∼0.5 mg/ mL). Tryptophan fluorescence was excited at 297 nm and detected at 360 nm, with 8 nm slit bandwidth of the emission monochromator. b Decay parameters of tryptophan donor emission τ and R are the lifetime and the relative amplitude of the ith decay component, i i respectively; 〈τd〉 and 〈τda〉 are the average (amplitude-weighted) lifetimes of the donor in the absence of the Cca-acceptor (χ2-values are presented in parentheses). Errors for the average lifetimes are (1% of the presented values. c Decay parameters of tryptophan donor emission τi and Ri are the lifetime and the relative amplitude of the ith decay component, respectively; 〈τd〉 and 〈τda〉 are the average (amplitude-weighted) lifetimes of the donor in the presence of the Cca-acceptor (χ2-values are presented in parentheses). Errors for the average lifetimes are (1% of the presented values. d Efficiencies of ET were calculated from the average lifetimes of the donor: E ) 1 〈τda〉/〈τd〉. e Interprobe distances were calculated using the values of the Fo¨rster distance (R0) determined for each derivative: R ) R0(1/E - 1)1/6. R0 values were 25.9 and 23.9 Å for W142Cca203-AK and W169Cca55-AK, respectively. f The average distance between the respective CR-atoms calculated from the crystal structure of the ligand-free AK from Escherichia coli (Mu¨ller et al., 1996).

in a protein, whereas Nal-containing pairs (τd ) 30-50 ns) would be more appropriate when turning to slower fluctuations in the same protein. The synthetic Nal-probe is thereby complementary to Trp as a donor, enabling

selection of the proper time scale for studying different structural fluctuations in proteins. (c) The accuracy of global analysis of time-resolved ET experiments for characterization of protein structural


fluctuations in each of its equilibrium states is enhanced in the case of D/A pairs with increased ratio of the lifetimes of the donor and the acceptor, τd/τa (Beechem and Haas, 1989). A high τd/τa ratio enables improved resolution of the ET-induced acceptor emission (excited through energy transfer) and the intrinsic acceptor emission (induced by direct excitation of acceptor probes). This allows improved accuracy in the simultaneous determination of distribution (IDD) parameters and rates of fluctuations related to intramolecular distances, by means of global analysis of the time-resolved fluorescence of both the donor and the acceptor. (d) Further enhancement of statistical significance in determinations of IDD parameters (particularly in case of wide IDDs) and rates of fluctuations is made possible by series of measurements of each intramolecular distance, using derivatives with different donor and/or acceptor component of the D/A pair. In this way, variation of the optimal time and distance ranges (according to the R0 and τd values) over the range of interest is achieved. This allows additional overdetermination and enhanced statistical significance of the global analysis (Beechem and Haas, 1989). The present series of probes enable either variation of time scale with the distance range fixed (by selecting either Nal or Trp) or variation of distance scales (by using one donor with each one of the three acceptors, i.e., Cca, LY, and Sa). Thiol-Reactive Charged Probes. So far, the most general and versatile methodology allowing site-specific attachment of various external probes to selected sites on protein’s surface is based on the combination of sitedirected insertion of cysteine residues (by mutagenesis) and alkylation reaction (thiol-directed labeling) (Mchaourab et al., 1996; Haas, 1996; Sinev et al., 1996a,b; Lillo et al., 1997a,b). This implies the use of probes containing a reliable thiol-reactive linker (that would ensure selective and stable probe conjugation to the cysteine residues in the protein), e.g., iodoacetamide (Haugland, 1992). All the thiol-reactive probes included in the set presented here are negatively charged at pH >5 and have reactive iodoacetamide linkers. The charged substituents make them water-soluble (at pH >5) and that enables large molar excess of the probe to be used in homogeneous reaction, resulting in high conjugation yields (see Results). The charged groups also diminish undesirable interactions with hydrophobic parts of the protein and thereby reduce the probability for perturbations in the native protein’s structure. In the present study, a model protein with enzymatic properties (AK from E. coli) was used for tests of the conjugation reactions. This provided a convenient test for overall structural integrity of the labeled protein derivatives, by measurements of the enzymatic activity and comparison with that of the native protein (Table 2). Furthermore, hydrophobic interactions of a covalently linked probe with neighboring protein environment may hinder rotational dynamics of the probe and increase uncertainty in interpretation of the FRET data due to the effect of the dipole orientations (Dale et al., 1979; Van Der Meer et al., 1994). The presence of a polar/charged groups in the probes can enhance the orientation averaging of a D/A pair. Another advantage of the design and use of charged probes is the enhanced resolution of chromatographic separation of labeled products. High purity of doublelabeled protein derivatives is of special concern in applications of FRET spectroscopy for determinations of intramolecular distances and their distributions, where contamination of the double labeled derivatives with

Sinev et al.

single-labeled species might cause problems in processing the experimental data (Lakowicz et al., 1991). Preparation of Double-Labeled Derivatives. The use of two-cysteine mutants of a protein to be studied provides flexible and efficient means for selection of pairs of sites on the protein surface for attachment of the two probes and for preparation of derivatives labeled by different D/A pairs. This warrants the predominant use of two-cysteine double-labeled derivatives in FRET studies, though labeling of two-cysteine mutants appears to be much more involved as compared to that of the corresponding Trp/Cys mutants. The practice of double-labeling procedure includes several steps. The first step, labeling of a two-cysteine mutant with one of the probes (either donor or acceptor, depending on separation efficiency of the mono-labeled derivatives obtained), yields a mixture of four products: (1) two single-labeled derivatives which differ in the labeling sites (C-X and X-C), (2) unlabeled protein mutant (C-C), and (3) protein labeled with the first probe in both sites (X-X) (where X is the conjugated probe and C is the free thiol). In general, subsequent separation of such a mixture might be a formidable task, which often requires a multistep purification scheme. However, for charged probes (X), ion-exchange chromatography of this mixture may yield three separate fractions: C-C (“unlabeled”), a still unresolved mixture of C-X and X-C (“single-labeled”), and X-X (“twice labeled”). The second separation step (i.e., the separation of single-labeled species) can be bypassed by labeling a nonseparated C-X/ X-C mixture with a second label (denoted as Y). This would yield a mixture Y-X/X-Y (which might contain residual C-X and X-C). The mixture Y-X/X-Y could be useful for FRET experiments by using the original C-X/X-C mixture as a reference, provided that either the extent of labeling of both single-labeled derivatives by the Y probe is close to 100% or at least the rate of labeling for the both of them is essentially the same (and hence the two mixtures have exactly the same molar ratio of the two components, correspondingly). Yet in many cases, neither of these two conditions is fulfilled. The latter thus requires separate use of Y-X and X-Y derivatives in FRET experiments. Site-dependent variations of spectroscopic properties of the X and Y probes commonly suggest that either C-X (in case of Y-X) or X-C (in case of X-Y) derivative should be used as a spectroscopic reference when evaluating energy transfer in the corresponding double-labeled derivative. Apparently, the above could be essentially easier to attain if the corresponding single-labeled species C-X and X-C were available as individual compounds, following efficient separation of the C-X/X-C mixture after the first labeling step. At first glance, the use of a charged fluorescent probe X for the first labeling of a two-cysteine mutant, though allowing efficient ion-exchange purification of the C-X/ X-C mixture from the differently charged postlabeling components (that is C-C and X-X), could hardly assist to the separation between the equally charged C-X and X-C species. However, the electrostatic interaction of a charged protein derivative with an ion-exchange matrix is effected not only by the protein’s total charge but also by spatial charge distribution over the protein molecule. The latter, in case of the charged probe attached to the protein, depends on the location of the probe. Indeed, in certain cases, single-labeling of a two-cysteine mutant with a charged probe allowed subsequent separation of


the resulting C-X/X-C mixture by means of ionexchange chromatography (Sinev et al., 1996b; Lillo et al., 1997a). In the present study, the effect of the labeling site, in case of a charged probe, on the elution volume of the corresponding single-labeled derivative of the protein in ion-exchange chromatography, was observed with singlecysteine AK mutants. Thus, anion-exchange chromatography of a mixture of cysteine AK mutants, C55-, C73-, C142-, and C203-AK, labeled with the single-charged Nalprobe, yielded three peaks, the first corresponding to coeluted Nal203- and Nal55-AK, the second to Nal142-AK, and the third to Nal73-AK, respectively (Figure 3). When the same cysteine mutants were labeled with the LY probe, which has an extra negative charge as compared to the Cca, Nal, or Sa probes, complete separation of the corresponding derivatives was attained under the same conditions (Figure 4). The latter suggests that the resolution between single-labeled species tends to improve when the charge on the label is increased. The observed effect might be thus practically employed in separating a mixture of single-labeled species C-X and X-C, by choosing, if possible, charged probes for the first labeling of double mutants. Thereby, the use of charged probes is apparently of advantage when preparing samples of double-labeled protein derivatives suitable for FRET experiments. However, the available probes having the desired spectroscopic characteristics may lack the charged group(s) required for the efficient chromatographic separations of the corresponding protein conjugates. Design of a special charged thiol-reactive reagent (“molecular handle”), which could be reversibly attached to free cysteine residues in the protein, could be of use in the case, e.g., a sulfhydryl similar to the known Ellmann reagent, capable of forming mixed disulfide bonds and charged under chromatographic conditions, could be thus employed for treatment of the corresponding C-X/X-C mixture (where X is an uncharged probe). The resulting mixture of the correspondingly modified (cysteyl-disulfide) protein species, bearing extra charge(s), could be then chromatographically separated and afterward reduced by a thiolic agent (e.g., 2-mercaptoethanole or DTT) in order to detach the handle, thus enabling the subsequent labeling with a second probe. To summarize, spectroscopic methods that employ labeled protein derivatives, such as time-resolved FRET spectroscopy, fluorescence anisotropy measurements, or electron spin resonance spectroscopy, complement the structure determination methods (X-ray crystallography and NMR) in elucidation of protein structural dynamics (Haas, 1996; Sinev et al., 1996a,b; Lillo et al., 1997a,b; Mchaourab et al., 1996). A preferred set of experimental molecular tools allowing efficient applications of FRET techniques in complex protein studies appears to comprise two major components: first, an appropriately selected set of D/A pairs having both proper spectroscopic characteristics and molecular properties (polarity, charge); second, a corresponding, properly designed set of Cys/ Cys (Trp/Cys) mutants. The methodology based on the new FRET probes described above and the procedures of site-specific labeling applied in the present study allows facile site-specific conjugate preparations and wide range of distance determinations. This forms a basis for systematic study of structural transitions in proteins over a wide range of distances. Finally, the Trp/Cca donor/ acceptor pair of fluorescent probes, involving newly synthesized cysteinyl-S-linked Cca fluorophore, was successfully used in trial energy transfer experiments for


interprobe distance determination, with two Cca-labeled Trp/Cys mutants of E. coli AK, W142C203 and W169C55 (Table 3). Agreement of the distance data obtained here, with the corresponding X-ray parameters (Mu¨ller et al., 1996) provides a good practical illustration verifying the suggested design approach. ACKNOWLEDGMENT

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