Chemistry of Sulforhodamine−Amine Conjugates - Bioconjugate

For experiments on the interaction of myosin subfragment 1 with 12−15, ..... 8.33 and 8.18 (purine H), 6.60−6.99 (m, xanthene H-1' to H-8'), 3.50â...
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Bioconjugate Chem. 2001, 12, 186−194

Chemistry of Sulforhodamine-Amine Conjugates John E. T. Corrie,* Colin T. Davis, and John F. Eccleston National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. Received July 6, 2000; Revised Manuscript Received November 30, 2000

Commercially-available sulforhodamine sulfonyl chlorides contain two isomeric monosulfonyl chlorides. Conjugates of these isomers with amines have different properties because the sulfonamide formed from one isomer can undergo ring-closure to a colorless sultam. This chemistry has been examined for a model conjugate with methylamine and for a bioconjugate with 2′(3′)-O-[N-(2-aminoethyl)carbamoyl]ATP. The interaction of each isomer of the latter conjugates with myosin subfragment 1 has been characterized. Significant differences between the two isomers are observed in these interactions.

INTRODUCTION

Rhodamine dyes are used extensively for conjugation with biomolecules because of their excellent fluorescence properties. Among the most commonly used reactive dyes for amine labeling are the sulforhodamine chlorides, principally Sulforhodamine B1 sulfonyl chloride 1a and Texas Red sulfonyl chloride 2a. We became interested

in these dyes for preparation of ribose-modified fluorescent nucleotides (1, 2) but were intrigued to observe that treatment of commercial SRB-SC2 1a with an excess of a primary amine reduced the intensity of the visible absorption band by ∼50%. The color change was reversible upon acidification. For the product obtained from reaction with methylamine, the color was highest at pH 4 and lowest at pH 9. To explain this observation, it is necessary to consider the structure of the starting sulfonyl chloride. Compounds 1a and 2a are prepared from the corresponding bis-sulfonates 1b and 2b, typically by treatment with POCl3 or PCl5. The original report (3) of Texas Red sulfonyl chloride recognized the probability that two isomeric monosulfonyl chlorides were present. SRB-SC dates from a 1958 paper (4) in which isomerism * To whom correspondence should be addressed. Phone: (+44) 20 8959 3666 (ext 2276). Fax: (+44) 20 8906 4419. E-mail: [email protected]. 1 Sulforhodamine B 1b is also known as Lissamine Rhodamine B. 2 Abbreviation: SRB-SC, sulforhodamine B sulfonyl chloride; ED-ATP, 2′(3′)-O-[N-(2-aminoethyl)carbamoyl]ATP.

Scheme 1. Formation and Ring-Chain Tautomerism of Sulforhodamine-Amine Conjugates

was not considered, although more recent reports (5, 6) have discussed the presence of two isomers. The presence of a sulfonyl chloride ortho to the xanthylium ring system, as in isomer 1c, presents the opportunity for spirosultam formation upon reaction with a primary amine (Scheme 1) and the spirosultam (as shown), in which the extended rhodamine chromophore is absent, is expected to be colorless. However, these rhodamine spirosultams are capable of ring-opening under appropriate pH conditions. In fact, these properties of sulforhodamine spirosultams were first recorded in qualitative terms 100 years ago (7), and the color change in either direction has been exploited in patents related to photographic (8) and thermal (9) imaging. Despite these precedents, the origin of this pH-dependent behavior continues to be subject to misinterpretation (e.g., ref 10), evidently based on an assumption that amine conjugation takes place only on the sulfonyl group para to the xanthylium ring, i.e., reaction with isomer 1d. We now describe a detailed study of these phenomena, with particular focus on the properties of amine conjugates derived from the different isomers and the consequences of the different structures for biological applications of these dyes. The investigation covers a conjugate of SRBSC with methylamine as a convenient model, a full characterization of the chemistry of the recently described Rhodamine Red-X reagent 7 (11), and studies of ribose-modified ATP conjugates derived from the two different isomers of SRB-SC. The results with the ATP conjugates (and with similar GTP conjugates not reported

10.1021/bc0000785 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/02/2001

Chemistry of Sulforhodamine−Amine Conjugates

Bioconjugate Chem., Vol. 12, No. 2, 2001 187

here) are particularly relevant to our previous work in which these analogues were used in fluorescence energy transfer measurements (1, 2). In the earlier of those studies (1), we had assumed that only the conjugate formed from isomer 1d was present in the material used for fluorescence studies (i.e., that it was homogeneous and not subject to variations of fluorescence induced by pH changes). The present work confirms that two isomers are formed in the synthesis but are well separated by the purification protocol. The interactions reported here of the ATP conjugates with myosin subfragment 1 reveal differences in fluorescence responses for the two isomers. We note that the ring-chain tautomerization chemistry described here is relevant only to conjugates with primary amines, as similar conjugates with secondary amines cannot cyclize. MATERIALS AND METHODS

SRB-SC 1a (lots 1141-1 and 1161-2) and Rhodamine Red-X succinimidyl ester 7 (lot 4462) were from Molecular Probes, Eugene OR. ED-ATP2 11 was prepared as described (12). Myosin subfragment 1 was prepared by the method of Weeds and Taylor (13). Reduced Triton X-100 and DEAE-cellulose anion exchanger were from Sigma. Silica gel for flash chromatography was Merck type 9385. All other materials were from Aldrich and used without further purification. TLC was performed on Merck 60F silica gel plates. NMR spectra were recorded on a Bruker AM400 spectrometer for solutions in CDCl3 (unless otherwise specified) and referenced to internal tetramethylsilane. Electrospray mass spectra were determined at low resolution on a VG Platform instrument and the FAB mass spectrum at high resolution was determined on a VG ZAB-SE instrument. UV-Vis spectra were determined on a Beckman DU70 instrument. Analytical reversed-phase HPLC was carried out on a Novapak C18 column (3.9 × 150 mm) eluted isocratically with 100 mM potassium phosphate, pH 6.8, containing 25% acetonitrile (v/v) at 0.5 mL min-1. Steady-state fluorescence intensity measurements were made on an SLM 8000S fluorimeter. Excitation was at 572 nm and emitted light was monitored through a monochromator at 585 nm. Fluorescence anisotropy titrations were carried out on the same instrument but with the use of Glan-Thompson polarizers. The data were fitted to eq 1:

Aobs ) AL + (AEL - AL) where

[EL]

)

0.5[(K

+

[EL] [LT]

[ET]

+

(1) [LT]2)

(

x(K + [ET] + [LT] - 4[ET][LT], Aobs ) observed anisotropy, AL ) anisotropy of free ligand, AEL ) anisotropy of enzyme-ligand complex, [ET] ) total enzyme concentration, [LT] ) total ligand concentration, and K ) equilibrium dissociation constant. Stopped-flow measurements were made on a Hi-Tech Scientific MX61 instrument operating in absorbance mode for studies of pH changes with compound 3 (wavelength 567 nm) or fluorescence mode for studies with the conjugates 12-15 (excitation at 530 nm; emission monitored through a Wratten 21 cutoff filter that passed light above 540 nm). For experiments on the interaction of myosin subfragment 1 with 12-15, all concentrations quoted are those after mixing so the syringe concentrations were twice these values. Data were analyzed using Hi-Tech software. Mean values of rate constants and amplitudes with their standard devia2

tions for stopped-flow data were obtained from analysis of 4-6 reactions but single data sets are shown in the Figures. Buffer solutions for measurements in aqueous ethanol were prepared from water-ethanol (3:1 v/v) solutions of appropriate acids at the molarities specified and adjusted to the required pH values by addition of NaOH. pH values were determined with a glass electrode referenced to buffer solutions in 100% aqueous solution. NMR Spectroscopic Characterization of SRB-SC (1a). Two batches of commercial material were examined. In the first sample (lot 1141-1), two major families of signals were distinguishable because their relative intensity ratios were ∼6:4. Selected single frequency decoupling experiments confirmed the assignment to one or other set for some of the signals. The first set of signals (A) were as follows (multiplicities are consistent within each set): δ 9.05 (d, J ) 2.1 Hz, 1H), 8.12 (dd, J ) 8.0 and 2.1 Hz, 1H), 7.36 (d, J ) 8.0 Hz, 1H), 7.22 (d, J ) 9.6 Hz, 2H), 6.83 (dd ) 9.6 and 2.4 Hz, 2H), 6.74 (d, J ) 2.4 Hz, 2H), 3.58 (q, J ) 6.5 Hz, 8H) and 1.29 (t, J ) 6.5 Hz, 12H). The second set of signals (B) had δ 9.00 (d, J ) 1.5 Hz, 1H), 8.55 (dd, J ) 7.8 and 1.5 Hz, 1H), 7.41 (d, J ) 7.8 Hz, 1H), 7.08 (d, J ) 9.5 Hz, 2H), 6.90 (dd, J ) 9.5 and 2.3 Hz, 2H), 6.79 (d, J ) 2.3 Hz, 2H), 3.64 (q, J ) 6.5 Hz, 8H) and 1.35 (t, J ) 6.5 Hz, 12H). A third, minor set of signals (C) was present in this sample, but was much more prominent in a second batch of material (lot 1161-2). These signals, for which the line shapes were broader than those of the other two sets, were at δ 8.93 (d, J ) 1.6 Hz, 1H), 8.77 (dd, J ) 8.0 and 1.6 Hz, 1H), 8.27 (d, J ) 8.0 Hz, 1H), 7.04 (d, J ) 9.2 Hz, 2H), 6.97 (dd, J ) 9.2 and 2.1 Hz, 2H) and 6.87 (d, J ) 2.1 Hz, 2H). The signals from the ethyl side chains were not resolved from those of the other two components. In this sample the relative intensity of the signals in sets A, B, and C was approximately 18:44:38. Reaction of SRB-SC with Methylamine: Compounds 3 and 4. A solution of SRB-SC (lot 1141-1; 100

mg, 0.17 mmol, in acetone (100 mL) was mixed with 40% aq. methylamine (20 mL) and concentrated under reduced pressure to remove most of the acetone. The remaining solution was diluted with water to ∼50 mL, filtered to remove some insoluble material and lyophilized. The residue was dissolved in 50% aqueous ethanol (20 mL) and quantified by spectrophotometry of a solution at pH 4. The total rhodamine concentration was 7.08 mM (83% recovery) but fell to 4.12 mM after storage for 48 h at 4 °C because part of the solute precipitated. In this mixture the absorbance ratio at 567 nm in solutions at pH 4 and 9 was 3.6:1, which was taken to indicate that the relative proportion of compounds 3 and 4 present in solution was 2.6:1. This material was used without further processing for most of the experiments described below. For experiments in later work where pure 3 was required, a portion of the stock solution, containing 45 µmol mixed rhodamines, was lyophilized and the residue

188 Bioconjugate Chem., Vol. 12, No. 2, 2001

was dissolved in MeOH and absorbed onto silica gel (1 g). This was added to a packed flash chromatography column, that was eluted with a step gradient of CHCl3MeOH (from 97.5:2.5 to 92:8; total volume 1200 mL). Fractions were analyzed by UV-vis spectroscopy at pH 4 and 9 and the main pH-responsive fraction (26 µmol) was combined and evaporated. This material retained a trace contaminant (∼3%; λmax 558 nm) that was not decolorized at pH 9 and interfered with the required measurements. It was removed by repeated preparative TLC (silica gel; CHCl3-MeOH 85:15) to give pure 3 (16.5 µmol); 1H NMR (MeOH-d4-NaOD) δ 8.18 (d, J ) 1.3 Hz, 1H), 7.90 (dd, J ) 8.2 and 1.3 Hz, 1H), 6.99 (d, J ) 8.2 Hz, 1H), 6.65 (d, J ) 8.5 Hz, 2H), 6.32-6.35 (m, 4H), 3.29 (q, J ) 7.0 Hz, 8H), 2.32 (s, 3H), and 1.07 (t, J ) 7.0 Hz, 12H). The addition of NaOD was made to drive the compound into its colorless sultam form. In MeOH-d4 alone the spectrum showed a mixture of the open and closed forms in ∼1:1 ratio. ES-MS, m/e (M + H) calcd for C28H33N3O6S2 + H, 572; found, 572. Ring Opening and Closing Kinetics of 3. For stopped-flow measurements starting from high pH, the 3+4 mixture was diluted to 9 µM (i.e., 6.5 µM 3, 2.5 µM 4) in 5 mM sodium borate, pH 9.0, and for measurements in the opposite direction it was diluted in 5 mM sodium citrate, pH 4.0. In each case, these starting solutions were mixed in the stopped-flow apparatus with an equal volume of 100 mM MOPS, pH 7.0. All concentrations are premixing values. Buffer solutions were prepared in water-EtOH (3:1 v/v) as described above and experiments were conducted at 4 °C. The observation light traversed a 1-cm path length. Transmission data were converted to absorbance and analyzed as the best fit to single exponentials. 3′,6′-Bis(diethylamino)-2-(2,3-dihydroxypropyl)spiro[1H-isoindole-1,9′-[9H]xanthen]-3(2H)-one (6).

Corrie et al.

total rhodamine concentration of 8.3 µM in various buffers. Aqueous ethanolic buffers (each 25 mM in buffer salt) were acetate, pH 4, phosphate, pH 7, and borate, pH 10. For measurements in the presence of detergent, the buffers (each 25 mM) were citrate, pH 2.1, and borate, pH 9, each containing 0.1% reduced Triton X-100 but without ethanol. In each case, solutions were mixed in various proportions to generate ∼20 solutions at different pH values. The pH of each solution was measured and the absorbance spectrum was recorded (10 mm path length). The 567 nm absorbance values were fitted to a single pK equation over the pH range 4-9. For the rhodamine-ATP conjugates 12 and 13 (see below), measurements were performed similarly in either aqueous or aqueous ethanol solutions, using 0.1 M buffers prepared from MES (pH 6.5), Tris (pH 8 and 8.5), CHES (pH 9.0, 9.5 and 10.0), CAPS (pH 10.5), and sodium phosphate (pH 12.0). Absorbance data were fitted to a single pK equation. For the titration of 6, 16.7 µM solutions of the lactam were prepared in either hydrochloric acid, pH 1 or in 25 mM sodium phosphate, pH 7, each of which contained 25% ethanol (v/v) and ∼30 solutions were prepared by mixing the two stock solutions in varying proportions. The pH values were determined as above and absorbance spectra were recorded over the range 220-400 nm. Absorbance values at 275 and 320 nm were least-squares fitted to eq 2, using the Marquardt algorithm with software written in-house by Dr. S. R. Martin.

Absorbance ) H2L[H2L2+] + HL[HL+] + L[L]

(2)

where H2L, HL, and L are absorption coefficients for the diprotonated (H2L2+), monoprotonated (HL+), and unprotonated (L) forms, respectively. The concentrations were calculated using eq 3:

[L] )

LTotal 1 + KL[H+] + KLKHL[H+]2

(3)

where KL) [HL+]/([L][H+]) and KHL) [H2L2+]/([HL+][H+]). Reaction of SRB-SC with Methyl 6-Aminohexanoate: Compounds 8, 9, and 10. A mixture of methyl

A mixture of Rhodamine B (330 mg) and SOCl2 (5 mL) was kept at room temperature for 18 h and excess reagent was removed under reduced pressure. The residue was dissolved in CHCl3 (50 mL), cooled in ice and treated with a solution of 1-aminopropane-2,3-diol (1 g) and triethylamine (1.5 g) in DMF (50 mL). The solution was stirred at room temperature for 2 h, by which time most of the color was discharged. Solvents were evaporated in vacuo and a solution of the residue in CHCl3 was washed with water, dried and evaporated. Flash chromatography (EtOAc-hexanes 80:20) and crystallization (×3 from benzene-hexanes) gave lactam 6 (104 mg), mp 222-224 °C; UV (EtOH) λ/nm (/M-1 cm-1) 240 (56 900), 273 (33 400), and 314 (13 100); 1H NMR (CDCl3 after D2O exchange) δ 7.88-7.92 (m, 1H), 7.36-7.50 (m, 2H), 7.11-7.13 (m, 1H), 6.36-6.40 (m, 4H), 6.25 and 6.28 (2 × dd, J ) 8.8 and 2.4 Hz for each, 2 × 1H), 3.40 (dd, J ) 12.2 and 3.2 Hz, 1H), 3.29-3.37 (m, 10H), 3.15 (dd, J ) 14.4 and 6.1 Hz, 1H), 3.04-3.09 (m, 1H) and 1.16 and 1.17 (2 × t, J ) 9 Hz, 12H); FAB-MS, m/z (M + H) calcd for C31H38N3O4 + H, 516.2880; found, 516.2880. Spectroscopic Titrations. For sultam 3 the mixture of 3 + 4, prepared as described above, was diluted to a

6-aminohexanoate hydrochloride (80 mg, 0.44 mmol) and triethylamine (0.12 mL) in chloroform (8 mL) was stirred in an ice-bath and SRB-SC (lot 1161-2; 200 mg, 0.35 mmol) was added in portions over 5 min. The solution

Chemistry of Sulforhodamine−Amine Conjugates

was allowed to warm to room temperature and stirred overnight, then diluted to 50 mL with chloroform. For initial analysis, an aliquot was diluted in ethanol (×50) and further diluted into buffers at pH 4 and 9. The absorbance ratio (567 nm) at low and high pH was 1.54. The chloroform solution was washed with water (3 × 30 mL) and dried, and the solvent was removed. TLC analysis (CHCl3-MeOH 95:5) showed three main spots, Rf 0.70, 0.25, and 0.10. Flash chromatography with a step gradient from 0 to 7.5% MeOH in CHCl3 (total volume 850 mL) gave the first two components essentially pure (TLC), while the third was contaminated with a trace of slightly less polar material. Visible spectroscopy at pH 4 showed recoveries of 56, 33, and 24 µmol for the highest, middle and lowest mobility compounds, respectively. The total recovery based on the starting rhodamine was 32%. At pH 9, the 567 nm absorption of the highest and lowest mobility compounds was essentially zero, while that of the middle fraction was the same as at pH 4. The highest mobility fraction was 3′,6′-bis(diethylamino)-2-(5-methoxycarbonyl-1-pentyl)-6-{[(5-methoxycarbonyl-1-pentyl)amino]sulfonyl}spiro[1,2-benzisothiazole3(2H)-9′-[9H]xanthene]-1,1-dioxide 8; 1H NMR δ 8.34 (d, J ) 1.8 Hz, 1H), 7.88 (dd, J ) 8.2 and 1.8 Hz, 1H), 7.13 (d, J ) 8.2 Hz, 1H), 6.82 (d, J ) 9.7 Hz, 2H), 6.35-6.37 (m, 4H), 4.58 (t, J ) 6.7 Hz, 1H), 3.66 (s, 3H), 3.60 (s, 3H), 3.35 (q, J ) 7.1 Hz, 8H), 3.01 (q, J ) 6.7 Hz, 2H), 2.91 (t, J ) 7.3 Hz, 2H), 2.30 (t, J ) 7.3 Hz, 2H), 2.11 (t, J ) 7.6 Hz, 2H), 1.49-1.63 (m, 6H), 1.31-1.41 (m, 6H) and 1.18 (t, J ) 7.1 Hz, 12H); ES-MS, m/z (M + H) calcd for C41H56N4O9S2 + H, 813; found 813. The middle fraction was 3,6-bis(diethylamino)-9-({4[(5-methoxycarbonyl-1-pentyl)amino]sulfonyl}-2-sulfophenyl)xanthylium, inner salt 9; 1H NMR (CDCl3-MeOH-d4 9:1) δ 8.68 (d, J ) 1.8 Hz, 1H), 8.04 (dd, J ) 7.9 and 1.8 Hz, 1H), 7.29 (d, J ) 7.9 Hz, 1H), 7.19 (d, J ) 9.4 Hz, 2H), 6.87 (dd, J ) 9.4 and 2.4 Hz, 2H), 6.75 (d, J ) 2.4 Hz, 2H), 3.69 (s, 3H), 3.57-3.64 (m, 8H), 3.02 (t, J ) 7.0 Hz, 2H), 2.35 (t, J ) 7.5 Hz, 2H), 1.66 (quintet, J ) 7.7 Hz, 2H), 1.60 (quintet, J ) 7.6 Hz, 2H), 1.38-1.45 (m, 2H) and 1.33 (t, J ) 7.1 Hz, 12H); ES-MS, m/z (M + H) calcd for C34H43N3O8S2 + H, 686; found, 686. The least mobile fraction was 3′,6′-bis(diethylamino)2-(5-methoxycarbonyl-1-pentyl)spiro[1,2-benzisothiazole3(2H)-9′[9H]xanthene]-1,1-dioxide-6-sulfonic acid 10; 1H NMR δ 8.68 (d, J ) 1.7 Hz, 1H) 8.35 (dd, J ) 7.8 and 1.7 Hz, 1H), 7.24 (d, J ) 7.8 Hz, 1H), 7.19 (d, J ) 9.4 Hz, 2H), 6.88 (dd, J ) 9.4 and 2.1 Hz, 2H), 6.72 (dd, J ) 2.1 Hz, 2H), 3.60 (s, 3H) superimposed on 3.57-3.66 (m, 8H), 2.89 (t, J ) 6.5 Hz, 2H), 2.22 (t, J ) 7.4 Hz, 2H), 1.50 (quintet, J ) 7.4 Hz, 2H), 1.43 (quintet, J ) 7.0 Hz, 2H), 1.32 (t, J ) 7.0 Hz, 12H) and 1.18-1.26 (m, 2H); ES-MS, m/z (M + H) calcd for C34H43N3O8S2 + H, 686; found, 686. Rhodamine-ED-ATP Conjugates 12 and 13. The procedure here amplifies that given previously (2) and describes in detail the isolation of isomers 12 and 13. A solution of ED-ATP (0.25 mmol) in 50 mM NaHCO3 (25 mL) was mixed with a solution of SRB-SC (lot 1141-1; 0.5 mmol) in acetone (25 mL) and kept for 2 h at 20 °C. A control reaction without ED-ATP was set up in parallel, and, at the end of the reaction time, each solution was analyzed by TLC [2-propanol-water-NH3(aq) 7:2:1]. The control reaction had a single red spot, Rf 0.9, also present in the reaction mixture, but the latter had two additional spots, one at the origin and the other with Rf 0.2. The latter spot only became colored as ammonia evaporated from the TLC plate. The reaction mixture was concentrated under reduced pressure to remove the acetone, then diluted with 1.5 vol of water and applied to a DEAE-

Bioconjugate Chem., Vol. 12, No. 2, 2001 189

cellulose column (34 × 150 mm, bicarbonate form). The column was eluted with a linear gradient of 0-1 M triethylammonium bicarbonate (total volume 3 L) and the eluate was monitored at 254 nm. Unreacted ED-ATP eluted at 0.27 M followed by two well-resolved peaks at 0.36 and 0.60 M that both contained the rhodamine fluorophore. On reverse-phase HPLC each peak eluted as partly resolved doublets because of isomerism arising from linkage through the 2′- or 3′-hydroxyl group (12, 21). The peak that eluted at 0.60 M TEAB (later assigned as 12) had tR 10.2 and 12.0 min while the peak at 0.36 M TEAB (later assigned as 13) had tR 7.9 and 8.6 min. The 2′/3′-isomers were not separated on a preparative scale in this work. Fractions containing the resolved components 12 and 13 were separately pooled and concentrated by rotary evaporation followed by further evaporation from methanol (3 × 30 mL) to remove buffer salts. Each residue was redissolved in water (5 mL) and the solutions were analyzed by TLC as above. The first and second fluorescent peaks eluted from the DEAE-cellulose column had Rf 0 and 0.2, respectively, and the latter spot was reversibly bleached on the TLC plate by exposure to ammonia vapor. By comparison with the behavior of the methylamine conjugate 3, this was assigned structure 12 while the spot that remained at the origin was assigned as structure 13. The yields were 13.5 and 66 µmol for 12 and 13 respectively (overall yield 32% based on ED-ATP). For 1H NMR characterization, the triethylammonium salts were converted to Na+ salts (Dowex 50) and the spectra were interpreted with the aid of 2D TOCSY and COSY spectra to correlate overlapping spin systems that arise from the presence of unresolved 2′- and 3′-isomers, together with previous data for related ED-ATP conjugates with Cy3 (21). However, complete assignment for each isomer was not possible. Reported intensities are consistent within the data for a particular isomer. For 13 (500 MHz, D2O, acetone ref.), the 2′-isomer had assignable signals at δ 8.56 (d, J ) 1.8 Hz, 1 H, xanthene H-3), 8.34 (dd, J ) 7.5 and 1.8 Hz, 1 H, xanthene H-5), 7.92 (d, J ) 7.5 Hz, 1 H, xanthene H-6), 6.04 (d, J ) 2.0 Hz, 1 H, H-1′), 5.24 (dd, J ) 5.6 and 2.0 Hz, 1 H, H-2′), 4.71-4.73 (m, 1 H, H-4′), 4.21-4.28 (m, 2 H, H-5′). The signal for H-3′ was obscured under the HOD peak. The 3′-isomer had δ 8.55 (d, J ) 1.8 Hz, 1 H, xanthene H-3), 8.29 (dd, J ) 8.0 and 1.8 Hz, 1 H, xanthene H-5), 7.59 (d, J ) 8.0 Hz, 1 H, xanthene H-6), 5.78 (d, J ) 6.0 Hz, 1 H, H-1′), 5.14-5.16 (m, 1 H, H-3′), 4.81-4.84 (m, 1 H, H-2′), 4.25-4.45 (m, 1 H, H-4′), 4.13-4.26 (m, 2 H, H-5′). Signals not assignable to a specific isomer were at δ 8.33 and 8.18 (purine H), 6.60-6.99 (m, xanthene H-1′ to H-8′), 3.50-3.66 (m, NCH2CH3), 3.20-3.46 (m, NCH2CH2), and 1.21-1.29 (NCH2CH3). The spectrum of isomer 12 in pure D2O was complex. This was shown to be due to noncovalent rhodamine dimerization at the concentration of the NMR sample, as revealed by absorption spectroscopy (0.1 mm path length) in the range 500-600 nm, that showed a spectrum typical for rhodamine dimers (22). Dimerization was not detected by absorption spectroscopy of 13. Addition of 50% methanol to the aqueous solution of 12 abolished the dimer absorption spectrum, and therefore, the 1H NMR spectrum was run in D2O-methanol-d4 (1:1 v/v). The 2′-isomer had assignable signals at δ 6.09 (d, J ) 2 Hz, 1 H, H-1′), 5.31 (dd, J ) 5 and 2 Hz, 1 H, H-2′), 4.71 (t, J ) 5 Hz, 1 H, H-3′). The 3′-isomer had δ 6.02 (d, J ) 5 Hz, 1 H, H-1′), 5.21 (d, J ) 4 Hz, 1 H, H-3′), 4.94 (dd, J ) 5 and 4 Hz, 1 H, H-2′). Signals not specifically assignable to either isomer were at δ 4.05-4.18 (m, H-4′,5′), 3.35-3.60 (m, NCH2CH3), 3.05-3.30 (m, NCH2-

190 Bioconjugate Chem., Vol. 12, No. 2, 2001

Corrie et al.

CH2N), and 1.10-1.13 (m, NCH2CH3). The xanthene protons appeared as broad envelopes in the ranges δ 7.38.8 (H-3,5 and 6) and 6.4-7.1 (H-1′ to H-8′), evidently because of exchange broadening between the open and closed forms of the rhodamine sultam, while sharp singlets at δ 8.62, 8.47, 8.19, and 8.14 were assignable to the purine protons, that in this case were distinct in each isomer. These ATP analogues were converted to the corresponding ADP analogues by incubating solutions of 34 µM analogue with 3.4 µM myosin subfragment 1 in 50 mM Tris‚HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2, and 1 mM DTT for 5 min at 20 °C. HPLC analysis showed quantitative conversion of both analogues to the ADP derivatives 14 (tR 12.1 and 16.6 min; see above for the cause of double peaks) and 15 (tR 8.8 and 10.1 min). RESULTS

The 1H NMR spectrum of one commercial sample of SRB-SC 1a indicated the presence of two major components, presumed to be isomeric species 1c and 1d. The spectrum of a second sample used in later work revealed an additional component, inferred from the products of its reaction with an amine to be the bis-sulfonyl chloride 1e (see below). The 567 nm absorbance of the mixture obtained by treatment of the first sample of 1a with excess methylamine was ∼2-fold greater at pH 4 than at pH 9. This result is consistent with the presence of the pH-responsive compound 3 and its isomer 4 in ∼1:1 ratio. The absorbance (or fluorescence) of 4 is not expected to vary with pH changes in this range, as the sulfonamide at the para-position cannot interact with the xanthylium system. Marchesini et al. (5) have previously reported the separation of 3 and 4 and described their 1H NMR spectra. Our studies were mostly conducted on a mixture of 3 + 4, in which 3 and 4 were present in ∼2.6:1 ratio because a proportion of 4 selectively precipitated from the stock solution after initial preparation. Later measurements not described here required pure 3, which was isolated from the mixture by repeated chromatography. The colorless form of 3 was favored at alkaline pH and the apparent pKa for ring-chain tautomerization was 7.37, determined from 567 nm absorbance measurements in 25% ethanol-75% aqueous buffer. Solutions could not be accurately manipulated without this concentration of ethanol, presumably because of losses by surface adsorption of the more hydrophobic ring-closed form. Nevertheless, an estimate of the apparent pKa in fully aqueous solution was obtained by measuring absorbance values immediately after dilution into appropriate aqueous buffers. This gave a value of approximately 6.95, suggesting that the presence of 25% ethanol did not greatly perturb the measurement. In contrast, the presence of detergent (5) had a significant effect and with 0.1% reduced Triton X-100 in fully aqueous solution, the measured pKa was 5.36. This difference is considered in the Discussion. Rates of interchange between the colored and colorless forms were investigated with pH changes imposed by rapid mixing in stopped-flow experiments. At 4 °C and in the region of pH 7, observed rate constants for the color change were ∼200 s-1 whether approached from the colored or colorless form (data not shown). A full data set covering the pH range 0-9.5 will be described elsewhere with an account of the detailed mechanism operating in the ring-opening and ring-closing reactions. We considered whether an amine conjugate of a carborhodamine would show analogous ring-chain tautom-

Figure 1. UV absorption spectra of lactam 6 as a function of pH. The figure shows superimposed traces from 30 individual solutions at various pH values between the extremes specified.

erism. The N-methyl lactam 5 derived from Rhodamine B has been reported to remain colorless “to about pH 4” (14) but was insufficiently soluble in water-ethanol (3:1) for accurate investigation. However, the more hydrophilic diol 6 was sufficiently soluble for accurate titration. This compound had no visible chromophore at any pH in the range 2-7, but its UV spectrum showed significant changes (Figure 1) associated with sequential protonation of the two diethylamino groups. The mean fitted pKa values (using separate fits to data at 240, 275, and 320 nm where the absorbance changes were greatest) were 2.97 and 4.52. Our clarification of the chemistry of 3 and 4 enabled us to reinvestigate aspects of a recent report that describes amine conjugates of 1a and 2a as improved reagents for fluorescent labeling of proteins (11). For the Rhodamine Red-X conjugate derived from 1a, the structure was shown as 7, leaving open the possibility that the sulfonamide and sulfonate groups were present as a mixture at positions 2 and 4. However, visible spectra of aqueous solutions of a commercial sample of Rhodamine Red-X succinimidyl ester were identical at pH 4 and 9, implying that the compound was a single isomer with the sulfonamide para to the xanthylium system. This result was unexpected in view of other results presented here, so the first stage of preparation of the Rhodamine Red-X reagent was repeated, i.e., reaction of 1a with methyl 6-aminohexanoate (11). This experiment used a batch of 1a for which the NMR spectrum showed three main species. TLC analysis of the crude reaction mixture also showed three major products, that were isolated by flash chromatography and characterized by visible, mass, and NMR spectroscopy. The least polar spot (50% of total rhodamines isolated by chromatography) was sulfonamide sultam 8, the middle spot (29% of recovered rhodamines) was monosulfonamide 9, and the most polar spot (21%) was sultam 10. Both 8 and 10 showed pH dependence of their visible spectra, similarly to the other sultams investigated here, while the spectrum of 9 was unaffected in the pH range 4-9. Isolation of the doubly derivatized compound 8 confirms the presence of bissulfonyl chloride 1e in the starting material used for this experiment. Consequences of these results are considered in the Discussion. Last, the influence of structural isomerism in sulforhodamine derivatives was examined for the conjugates

Chemistry of Sulforhodamine−Amine Conjugates

Bioconjugate Chem., Vol. 12, No. 2, 2001 191

Figure 2. Titration of the rhodamine-ED-ATP analogues 12 (O) and 13 (9) in aqueous solution as a function of pH. The solid line is a least-squares fit to the data of 12 for a single pKa.

12 and 13 derived by coupling SRB-SC 1a with ED-ATP 11. The reaction produced two species that were readily Figure 3. Stopped-flow fluorescence records of the interaction of the rhodamine-ATP analogues 12 (panels a and b) and 13 (panels c and d) with excess myosin subfragment 1. The reactions were monitored over two time scales.

separated analytically by reverse-phase HPLC and preparatively by anion exchange chromatography. Structures were assigned by inference from the known starting materials, the reactivity with myosin subfragment 1 (see below), the 1H NMR spectra and the response of each compound to pH changes. The visible absorption of 12

was pH dependent as for the methylamine conjugate 3, with apparent pKa 9.12 in fully aqueous solution (Figure 2), that reduced to 8.24 in solutions containing 25% ethanol (v/v). The absorption spectrum of 13 was invariant over the pH range 4-12 (Figure 2).

The interactions of ATP conjugates 12 and 13 with myosin subfragment 1 were investigated under single turnover conditions (i.e., [protein] > [ATP analogue]) at pH 7.5, 20 °C in 50 mM Tris‚HCl, 100 mM KCl, 5 mM MgCl2, 1 mM DTT. A 0.5 µM solution of either isomer was rapidly mixed with 2.5 µM subfragment 1 in a fluorescence stopped-flow instrument and the fluorescence was recorded with time (Figure 3). With both isomers, there was a fast initial decrease in intensity followed by a slower recovery. These phases correspond to binding of the analogue to subfragment 1 and the ratelimiting release of inorganic phosphate after hydrolysis (see Discussion). With 12 the initial fluorescence decrease was to 69.6 ( 0.6% of the starting value with subsequent recovery to 73.4 ( 0.4% of the starting value. For 13, the initial decrease was to 57.6 ( 0.6% of the starting value followed by recovery to 81.5 ( 1.3% of the starting value. Thus, both isomers give a similar fluorescence decrease on binding to subfragment 1 but the fluorescence recovery is smaller for the pH-responsive isomer 12 than for 13. Rate constants of the above two processes were 2.0 ( 0.1 and 0.050 ( 0.003 s-1 for 12 and 1.07 ( 0.08 and 0.042 ( 0.001 s-1 for 13. Although a concentration dependence of the binding reactions were not made, the rate constants of the faster processes represent second-order association constants of 8.0 × 105 and 4.3 × 105 M-1 s-1 for 12 and 13, respectively. These calculations depend on the subfragment 1 being 100% active and so represent lower limits of these rate constants. These single turnover experiments were repeated at pH 8.5, i.e., closer to the pK value for the color change of 12, so perturbation of this pK on binding to subfragment 1 might have been observed. In practice, neither isomer showed differences between pH 7.5 and 8.5 in the amplitudes of either phase of the fluorescence changes (data not shown). Dissociation rate constants of the isomeric ADP conjugates 14 and 15 from subfragment 1 were measured in displacement experiments by rapid mixing of a solution containing 2.5 µM subfragment 1 and 0.5 µM 14 or 15 with a solution containing 100 µM ATP (Figure 4). With 14 there was 41.4 ( 0.9% exponential increase in fluorescence with a rate constant of 1.15 ( 0.07 s-1 while 15 showed a 27.2 ( 0.9% exponential increase in fluorescence with a rate constant of 0.75 ( 0.10 s-1. Association rate constants, determined by mixing 0.5 µM 14 or

192 Bioconjugate Chem., Vol. 12, No. 2, 2001

Figure 4. Stopped-flow fluorescence records of the displacement of rhodamine-ADP analogues 14 (panel a) and 15 (panel b) from myosin subfragment 1 by excess ATP.

Figure 5. Equilibrium binding of rhodamine-ADP analogues 14 (O) and 15 (b) to myosin subfragment 1, monitored by fluorescence anisotropy. The solid lines are best fits to eq 1, with values for Kd and limiting anisotropy as shown in the Results.

15 with 2.5 µM subfragment 1, were 1.9 s-1 and 1.3 s-1, respectively (data not shown). The equilibrium dissociation constants of 14 and 15 from subfragment 1 were investigated by fluorescence anisotropy titrations. Increasing concentrations of subfragment 1 were added to a solution of 1 µM 14 or 15. The anisotropies of 14 and 15 free in solution were 0.070 and 0.060, respectively, and, as the subfragment 1 concentration increased, both isomers showed a hyperbolic increase in anisotropy (Figure 5). Fitting these data to eq 1 gave values for the limiting anisotropy of 0.168 and 0.294, and equilibrium dissociation constants of 2.62 ( 0.26 and 2.27 ( 0.60 µM for 14 and 15, respectively. DISCUSSION

The pH-dependent reversible cyclization of rhodamine sultams has consequences for experiments that use sulforhodamine dyes. First, the apparent pKa for the colored to colorless transition of 3 varies markedly with changes in solution conditions. In contrast to the value of 7.37 determined here by absorbance measurements of water-ethanol solutions, Marchesini et al. (5) reported an apparent pKa of ∼4.6 from fluorescence measurements in fully aqueous solutions that contained 0.1% reduced

Corrie et al.

Triton X-100. In our hands, absorbance measurements under the latter conditions gave a value of 5.36. Thus, the apparent pKa is substantially lower in the presence of reduced Triton X-100. The critical micellar concentration of this detergent is 0.25 mM (15), substantially below the 0.1% level used by Marchesini et al. [1.6 mM based on an average molecular weight of 625 (16)]. We suggest the lipophilic sultam form of 3 would partition preferentially into micelles and the resultant stabilization of the conjugate base is responsible for enhanced acidity of the open form. The earlier proposal (5) that 3 could be used as a pH indicator for cellular compartments of acidic pH is therefore problematic. However, introduction of hydrophilic substituents on one or more of the nitrogen atoms in 3 could generate a new family of fluorescent pH indicators. Modulation of the pKa by variable substitution of the aromatic amino groups in compounds related to 3 has been qualitatively described in the patent literature (8a). In the present work, the sensitivity of the apparent pKa to changes in substituents is demonstrated by the values obtained for the ATP conjugate 12, i.e., 9.12 in fully aqueous solution and 8.24 in solutions containing 25% ethanol. The overall shift to a higher value can be rationalized by the inductive effect of the β-nitrogen in the substituent on the sulfonamide, that reduces the nucleophilicity of the sulfonamide nitrogen, i.e., the driving force for ring closure is reduced. The apparent pKa values for 3 and 12 appear to change in opposite directions when measured in water or ethanol-water. Complex solvation effects are well-known to influence acid and base strengths (17) and probably underlie these differences. A second point is that the kinetics of the ring opening and closing reactions of the model sultam 3 at pH values near neutrality are in the millisecond time domain, i.e., comparable to rates of many biological processes. Thus small variations of pH could themselves generate rapid signals from probes that incorporate the sultam structure. Since the kinetics of the color change are slow compared to most indicators, these rhodamine sultams would not be suitable to measure very rapid pH changes. Third, the ability to separate compounds 9 and 10 means that rhodamine probes of this type, insensitive or sensitive respectively to the effects of pH and suitable for covalent attachment to biomolecules, can be considered as accessible. In particular, the Rhodamine Red-X reagent (11) has been shown to be insensitive to pH changes and therefore must have a structure derived from isomer 9. The interactions of the isomeric ATP conjugates 12 and 13 with myosin subfragment 1 demonstrate the potential for the different isomers of these and other sulforhodamine conjugates to report differently on underlying biological processes. In single turnover experiments when 12 and 13 were mixed with excess subfragment 1, both isomers showed an initial exponential decrease in fluorescence followed by a slower increase in fluorescence. Such behavior has previously been observed when ATP was mixed with subfragment 1 and intrinsic tryptophan fluorescence monitored (18) or when fluorescent analogues of ATP were mixed with subfragment 1 (19-23). The results can be interpreted in light of a simplified scheme for the subfragment 1 ATPase mechanism (24). 1

2

3

M + ATP 98 M.ATP a M.ADP.Pi 98 4

M.ADP + Pi h M + ADP

Chemistry of Sulforhodamine−Amine Conjugates

The initial process is associated with the essentially irreversible binding of ATP (or an analogue thereof) to subfragment 1 (step 1) to form the M‚ATP state, that is in rapid equilibrium with M.ADP.Pi (step 2). This is followed by the rate-limiting inorganic phosphate release step of the overall hydrolysis process (step 3), that is also essentially irreversible, and the cycle terminates with the release of ADP (step 4). The rate constants given in the Results section for the initial fluorescence decrease when 12 or 13 were mixed with subfragment 1 show that 12 binds at over twice the rate of 13. However, for the subsequent slow increase of fluorescence that corresponds to the rate-limiting step of the overall hydrolysis (step 3), rate constants for the two isomers were identical within experimental error and similar to that of the subfragment 1 ATPase (18). As shown in Figure 3, the amplitudes of the slow fluorescence recovery, that corresponds to step 3 above, were markedly different for the two isomers. The much smaller amplitude of the slow phase for analogue 12 could result from its ADP form 14 binding more tightly to subfragment 1 than the isomeric ADP analogue 15 and/ or that the enhancement of fluorescence on binding to subfragment 1 is greater for isomer 14. The displacement of these ADP analogues from subfragment 1 by excess ATP gave a larger increase in fluorescence for 14 than for 15, which again might be due to tighter binding or higher fluorescence change of 14. The rate constant for dissociation of 14 was almost twice that for 15. However, the equilibrium titrations based on fluorescence anisotropy measurements show that, within experimental error, the two ADP analogues bind to subfragment 1 with the same affinity, suggesting that 14 has a faster secondorder association rate constant than 15, as reported in the Results. Thus the ATP and ADP analogues are similar in that in each case the pH-responsive isomer shows more rapid binding. The differences highlight the importance of using defined isomers of these rhodamine conjugates. A particular source of potential error could have arisen in our previous FRET-based distance measurements (1, 2) using these conjugates if the pHresponsive isomer were present and underwent some degree of cyclization upon binding to a protein. The results now reported and related data not shown confirm that sulforhodamine conjugates with both GTP and ATP used in previous work were solely the nonresponsive isomers because the pH-responsive isomers were wellresolved by preparative anion exchange chromatography. The results described in this work put the chemistry of sulforhodamine-amine conjugates on a sound foundation and clarify some previous reports. Future challenges are to define in detail the mechanism of the ring-chain tautomerization process and explore uses of these conjugates, particularly the pH-responsive forms, as probes in biophysical research. ACKNOWLEDGMENT

We thank Drs. S. Howell and K. Welham for the low and high resolution mass spectral data, respectively, Dr. S.R. Martin for some spectroscopic pH titrations and Drs. V.R.N. Munasinghe and G. Kelly for recording NMR spectra. We are grateful to the MRC Biomedical NMR Centre for access to facilities. LITERATURE CITED (1) Watson, B. S., Hazlett, T. L., Eccleston, J. F., Davis, C., Jameson, D. M., and Johnson, A. E. (1995) Marcromolecular arrangement in the aminoacyl-tRNA‚elongation factor TuGTP

Bioconjugate Chem., Vol. 12, No. 2, 2001 193 ternary complex. A fluorescence energy transfer study. Biochemistry 34, 7904-7912. (2) Sawyer, W. H., Chan, R. Y. S., Eccleston, J. F., Davidson, B. E., Samat, S. A., and Yan, Y. (2000) Distances between DNA and ATP binding sites in the TyrR-DNA complex. Biochemistry 39, 5653-5661. (3) Titus, J. A., Haugland, R., Sharrow, S. O., and Segal, D. M. (1982) Texas Red, a hydrophilic red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J. Immunol. Methods 50, 193-204. (4) Chadwick, C. S., McEntegart, M. G., and Nairn, R. C. (1958) A trial of new fluorochromes and the development of an alternative to fluorescein. Immunology 1, 315-327. (5) Marchesini, S., Gatt, S., Agmon, V., Guidici, M. L., and Monti, E. (1992) A novel fluorescent pH indicator for the acidic range. Biochem. Int. 27, 545-550. (6) Kang, H. C. (1998) Conjugates of sulforhodamines with enhanced fluorescence, U.S. Patent 5,846,737. (7) Monnet, P., and Koetschet, J. (1897) Les sacchare´¨ınes, nouvelles matie`res colorantes de´rive´es du sulfimide benzoı¨que. Bull. Soc. Chim. Fr. 17, 691-702. (8) Foley, J. W., Locatell, L., and Zepp, C. M. (1981) Novel xanthene compounds and photographic products and processes employing the same, U.S. Patent, 4,258,118. (b) Carlier, P. M., Filosa, M. P., and Lockshin, M. P. (1993) Sulfonated xanthene dyes and photographic products and processes employing these dyes, U.S. Patent, 5,187,282. (9) Borror, A. L., Ellis, E. W., and McGowan, D. A. (1985) New heat fragmenting compounds and heat-sensitive materials containing them give multicolour images with excellent resolution sharpness and stability, European Patent 174,054. (b) Borror, A. L., and Ellis, E. W. (1986) Thermal imaging material for preparation of mono and polychrome images has colourless di- or tri-arylmethane compounds: electrophilic compounds which are contacted on heating, PCT Patent 86/ 07313. (c) Ellis, E. W. (1989) Heat sensitive recording element, U.S. Patent, 4,818,742. (d) Marshall, J. L., Takiff, L. C., and Telfer, S. J. (1995) Imaging medium and process for producing an image, U.S. Patent, 5,441,850. (10) Pitner, J. B., and Hoke, R. A. (1995) Fluorescent pH indicators, U.S. Patent 5,393,514. (11) Lefevre, C., Kang, H. C., Haugland, R. P., Malekzadeh, N., Arttamangkul, S., and Haugland, R. P. (1996) Texas Red-X and Rhodamine Red-X, new derivatives of sulforhodamine 101 and Lissamine Rhodamine B with improved labeling and fluorescence properties. Bioconjugate Chem. 7, 482-489. (12) Jameson, D. M., and Eccleston, J. F. (1997) Fluorescent nucleotide analogues: synthesis and applications. Methods Enzymol. 278, 363-390. (13) Weeds, A. G., and Taylor, R. S. (1975) Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin. Nature 257, 54-56. (14) Cincotta, L., and Foley, J. W. (1981) 3,6-(Dialkyl/phenyl)amino-9-carboxamidophenylxanthates, U.S. Patent, 4,290,955. (15) Tiller, G. E., Mueller, T. J., Dockter, M. E., and Struve, W. G. (1984) Hydrogenation of Triton X-100 eliminates its fluorescence and ultraviolet-light absorption while preserving its detergent properties. Anal. Biochem. 141, 262-266. (16) Sigma Chemical Company Product Information Sheet CKV/012196/T6878.gen. (17) March, J. (1985) Advanced Organic Chemistry. Reactions, Mechanisms and Structure, 3rd ed., pp 234-236, Wiley, New York. (18) Bagshaw, C. R., and Trentham, D. R. (1973) The reversibility of adenosine triphosphate cleavage by myosin. Biochem. J. 133, 323-328. (19) Woodward, S. K. A., Eccleston, J. F., and Geeves, M. A. (1991) Kinetics of the interaction of 2′(3′)-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes. Biochemistry 30, 422-430. (20) Bagshaw, C. R., Eccleston, J. F., Trentham, D. R., Yates, D. W., and Goody, R. S. (1972) Transient kinetic studies on

194 Bioconjugate Chem., Vol. 12, No. 2, 2001 the Mg2+ dependent ATPase of myosin and its proteolytic subfragments. Cold Spring Harbor Symp. Quantum Biol. 37, 127-136. (21) Oiwa, K., Eccleston, J. F., Anson, M., Kikumoto, M., Davis, C. T., Reid, G. P., Ferenczi, M. A., Corrie, J. E. T., Yamada, A., Nakayama, H., and Trentham, D. R. (2000) Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives. Biophys. J. 78, 30483071. (22) Selwyn, J. E., and Steinfeld, J. I. (1972) Aggregation equilibria of xanthene dyes. J. Phys. Chem. 76, 762-774.

Corrie et al. (23) Conibear, P. B., Jefferys, D. S., Seehra, C. K., Eaton, R. J., and Bagshaw, C. R. (1996) Kinetic and spectroscopic characterization of fluorescence ribose-modified ATP analogues upon interaction with skeletal muscle myosin subfragment 1. Biochemistry 35, 2299-2308. (24) Geeves, M. A., and Holmes, K. C. (1999) Structural mechanism of myosin contraction. Annu. Rev. Biochem. 68, 687-728.

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