Sulfhydryl-Reactive, Cleavable, and Radioiodinatable Benzophenone

Apr 29, 2005 - University of Wisconsin. § Both authors have equally contributed to this work. | Isfahan University of Technology. ‡ Harvard Medical...
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Bioconjugate Chem. 2005, 16, 685−693

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Sulfhydryl-Reactive, Cleavable, and Radioiodinatable Benzophenone Photoprobes for Study of Protein-Protein Interaction Lian-Wang Guo,*,†,§ Abdol R. Hajipour,†,|,§ Monica L. Gavala,† Marty Arbabian,† Kirill A. Martemyanov,‡ Vadim Y. Arshavsky,‡ and Arnold E. Ruoho† Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, Pharmaceutical Laboratory, College of Chemistry, Isfahan University of Technology, Isfahan, 84156, I.R., Iran, and Department of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114. Received January 24, 2005; Revised Manuscript Received March 17, 2005

The major task in proteomics is to understand how proteins interact with their partners. The photocross-linking technique enables direct probing of protein-protein interaction. Here we report the development of three novel sulfhydryl-reactive benzophenone photoprobes of short “arm” length, each with a substitution of either amino, iodo, or nitro at the para-position, rendering the benzophenone moiety directly radioiodinatable. Their potential for study of protein-protein interaction was assessed using the inhibitory subunit of rod cGMP phosphodiesterase (PDEγ) and the activated transducin R subunit (GRt-GTPγS) as a model system. These photoprobes proved to be stable at neutral pH and dithiothreitol-cleavable in addition. The PDEγ constructs derivatized at the C-terminal positions with these probes could be readily purified, had unaltered PDEγ functional activity, and were shown to photo-cross-link to GRt-GTPγS with an efficiency as high as 40%. Additionally, the amino benzophenone probe was radioiodinated, facilitating sensitive detection of label transfer. The uniquely combined features of these benzophenone photoprobes promise robust and flexible methods for characterization of protein-protein interaction, either by mass spectrometry when a nonradioactive label is available or by autoradiography when using radioiodinated derivatives.

INTRODUCTION

The signaling pathway for visual excitation in rod photoreceptors represents a prototypical G-protein cascade comprising the seven-transmembrane receptor (rhodopsin), the heterotrimeric G-protein (transducin), and the effector (phosphodiesterase, PDE61). The interaction between the trandsducin R subunit (GRt) and the inhibitory γ subunit of PDE6 (PDEγ or Pγ) is a key step. Upon activation by photoexcited rhodopsin, GTP-loaded GRt activates PDE6 by binding to PDEγ. This signaling event eventually leads to the generation of the electrical response of the photoreceptor cell (1). On the other hand, the GRt/PDEγ interaction is also required for rapid termination of the rod photoresponse (2). The conversion of the activated transducin to its inactive state is greatly accelerated by the ninth member of the regulators of G-protein signaling family (RGS9), whose action requires the association of transducin with PDEγ (2, 3). Therefore, * To whom correspondence should be addressed. Tel: 608 263 3980. Fax: 608 262 1257. E-mail: [email protected]. † University of Wisconsin. § Both authors have equally contributed to this work. | Isfahan University of Technology. ‡ Harvard Medical School. 1 Abbreviations: PDE6, rod phosphodiesterase; PDEγ, PDE6 inhibitory subunit; GRt, transducin R subunit; MTS, methanethiosulfonate; MS, mass spectrometry; NMR, nuclear magnetic resonance; ABM, 4-methanethiosulfinate methyl-4′-aminobenzophenone; IBM, 4-methanethiosulfinate methyl-4′iodobenzophenone; NBM, 4-methanethiosulfinate methyl-4′nitrobenzophenone; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography.

information concerning the GRt/PDEγ interaction is critical for understanding the molecular mechanisms of visual transduction, and, most likely, other G-protein pathways. Much has been learned about regulation of visual transduction. The atomic structures of activated GRt alone (4, 5) and complexed with the PDEγ C-terminal half and the catalytic domain of RGS9 (6) have greatly enriched our knowledge about interactions of these two signaling proteins. However, due to a lack of atomic structure of the complex of GRt and full-length PDEγ, it remains a challenging task to uncover important details of a “real” signaling state of the GRt/PDEγ interaction. While X-ray crystallography and nuclear magnetic resonance (NMR) can offer detailed information about protein-protein interactions, the stability issue and large size of a protein complex add extra difficulty for solving its atomic structure. Some alternative approaches such as photo-cross-linking, however, offer a powerful approach for discovering the missing pieces of the puzzle. A radiolabeled cleavable photoprobe, [125I]-ACTP ([125I]N-[(3-iodo-4-azidophenyl propionamido-S-(2-thiopyridyl)]cysteine), which contains an azide photophore, has been applied for the structural study of GRt (7), GRt/PDEγ interaction (8), and PDE6 Rβ subunits/PDEγ interaction (9). Azides have been conventionally taken as the primary choice of photoreactive group by many researchers since it can be prepared very easily. However, shorter UV wavelengths required for activation of azide photoprobes sometimes cause significant damage to proteins, and azide photoprobes are not efficient since the singlet phenylnitrenes are short-lived (10). A photoprobe with an aryldiazonium moiety is more efficient than its azide

10.1021/bc050016k CCC: $30.25 © 2005 American Chemical Society Published on Web 04/29/2005

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derivative but less stable (11). In contrast, the benzophenone photophore has distinct advantages. It can be activated in a reversible manner via excitation-relaxation cycles at 350-360 nm, avoiding UV damage, and only C-H bonds within 3.1 Å of the carbonyl oxygen are modified. Moreover, the benzophenone group is stable to common protic solvents, reacts only with target proteins, and generally leads to highly efficient single-site photocovalent modification (12). Noncleavable benzophenone photoprobes have been used for defining protein-protein interactions of GRt/PDEγ (13, 14) and other proteins (1518) by photo-cross-linking and mass spectrometry (MS). The proteins cross-linked with the noncleavable photoprobes cannot be separated, resulting in complexity in identification of photoinsertion sites. Cleavable benzophenone photoprobes suited for photo-cross-linking/ label transfer experiments are therefore highly desirable. Here we report the development of three cleavable benzophenone photoprobes each with a methanethiosulfonate (MTS) group, which is highly sulfhydryl-reactive (Figure 1). To illustrate the use of these probes, they were employed to derivatize the single-cysteine PDEγ constructs for cross-linking with GRt-GTPγS. Their potential for study of protein-protein interactions was assessed using PDEγ/GRt-GTPγS as a model system. As an additional feature, the amino-benzophenone probe was shown to be radioiodinatable. A noniodinatable benzophenone derivative has been reported for immobilization of ion channels (19, 20), but it is not suitable for radiolabel transfer. To our knowledge, the photoprobes developed in this study are the only reported sulfhydrylreactive, cleavable, and radioiodinatable benzophenone photoprobes that are optimized for protein-protein interaction studies through a photo-cross-linking/label transfer methodology. EXPERIMENTAL PROCEDURES

General. All yields refer to isolated products after purification. Products were characterized by spectroscopy data (IR, 1H NMR spectrum, CHN analyses, melting and boiling point). 1H NMR spectra were recorded at 300 MHz. The spectra were measured in CDCl4 unless otherwise stated, relative to TMS (0.00 ppm). BPMTS (benzophenone-4-carboxamidocysteine methanethiosulfonate) was purchased from Toronto Research Chemicals. Na125I was from Perkin-Elmer as carrier-free radioiodide (2200 Ci/mmol, 5 mCi/20 µL in 0.1 N NaOH). All other chemicals and reagents were from Sigma-Aldrich unless stated elsewhere. Synthesis of 4-Methyl-4′-nitrobenzophenone (1). In a 500 mL round-bottomed flask to a stirred solution of toluene (300 mL) and 4-nitrobenzoyl chloride (20 g, 108 mmol) was added aluminum chloride (18.7 g, 140 mmol). The resulting red solution was stirred at room temperature for 90 min, and then 5 mL of water was added dropwise and the mixture was stirred for 20 min. The reaction mixture was washed with water (2 × 100 mL) and 10% NaHCO3 (2 × 100 mL) and dried (Mg SO4). The solvent was evaporated under reduced pressure with a rotary evaporator to a pale yellow solid (22.3 g). The resulting residue was recrystallized from methylenechloride/hexane to produce 19.8 g of compound 1 (80%) as light yellow needles. Mp: 119-120 °C. 1H NMR: δ 8.36 (d, J ) 8.8 Hz, 2 H), 7.9 (d, J ) 8.1 Hz, 2 H), 7.70 (d, J ) 8.0 Hz, 2 H), 7.31 (d, J ) 8.0 Hz, 2 H), 2.50 (s, 3 H). Anal. Calcd for C14H11NO3: C, 69.70; H, 4.60; N, 5.81. Found: C, 69.80; H, 4.70; N, 5.80. Synthesis of 4-Bromomethyl-4′-nitrobenzophenone (2). In a 250 mL round-bottomed flask to a stirred

Figure 1. Schematic strategy of protein-protein interaction analysis by photo-cross-linking/label transfer using cleavable benzophenone photoprobes. PDEγ/GRt interaction is presented as a model system. Solid line, PDEγ. Bar, GRt. Box, photoinsertion-containing proteolytic fragment of a target protein. DTTreversed sulfhydryl (underlined) can be made available for addition of a biotin or fluorescent tag.

solution of CCl4 (100 mL) and compound 1 (2.4 g, 10 mmol) were added N-bromo succinimide (NBS) (2.0 g, 11 mmol) and 2,2′-azobisisobutyronitrile (AIBN) (10 mg). The resulting mixture was refluxed for 38 h. After cooling, the white precipitate of succinimide was filtered and the solvent was evaporated to a brown solid. The resulting residue was purified by column chromatography on silica gel (CH2Cl2/hexane, 1:1 v/v) to produce 2.1 g of compound 2 (66%). Mp: 124-125 °C. 1H NMR: δ 8.40 (d, J ) 8.8 Hz, 2 H), 7.9 (d, J ) 7.9 Hz, 2 H), 7.70 (d, J ) 8.1 Hz, 2 H), 7.55 (d, J ) 8.1 Hz, 2 H), 4.54 (s, 2 H). Anal. Calcd for C14H10BrNO3: C, 52.52; H, 3.15; N, 4.38. Found: C, 52.60; H, 3.20; N, 4.30. Synthesis of Sodium Methanethiosulfonate (MeSO2SNa). A mixture of sodium methanesulfonate (MeSO2Na) (5 g, 42.0 mmol) and sulfur (1.56 g, 48.8 mmol) in methanol (300 mL) was treated under reflux conditions for 30 min, at which time all of the sulfur had

Sulfhydryl-Reactive Benzophenone Photoprobes

dissolved. Removal of the solvent gave sodium methanethiosulfonate as a white powder. IR (KBr): 1320, 1220, and 1100. 1H NMR (D2O): δ 3.30 (s, 3 H). Synthesis of 4-Methanethiosulfinate Methyl-4′nitrobenzophenone (NBM) (3). In a 100 mL roundbottomed flask to a stirred solution of methanol (30 mL) and compound 2 (0.44 g, 1.46 mmol) was added sodium methanethiosulfonate (0.2 g, 1.7 mmol). The resulting mixture was refluxed for 3 h until compound 2 disappeared on TLC (toluene/diethylamine, 20:1 v/v). After cooling, the precipitate was filtered and the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography on silica gel (toluene/diethylamine, 20:1) to produce 0.39 g of compound 3 (80%). Mp: 156-158 °C. 1H NMR: δ 8.40 (d, J ) 8.8 Hz, 2 H), 7.9 (d, J ) 7.9 Hz, 2 H), 7.70 (d, J ) 8.1 Hz, 2 H), 7.55 (d, J ) 8.1 Hz, 2 H), 4.42 (s, 2 H). Anal. Calcd for C15H13NO5S2: C, 51.28; H, 3.70; N, 3.99; S, 18.23. Found: C, 51.20; H, 3.80; N, 4.10; S, 18.30. Synthesis of 4-Methanethiosulfinate Methyl-4′aminobenzophenone (ABM) (4). In a 100 mL roundbottomed flask a stirred solution of methanol (30 mL) and compound 3 (0.35 g, 1.0 mmol) and 10% Pd-C (80 mg) was reduced with hydrogen gas at room temperature for 10 h until compound 3 disappeared on TLC (toluene/ diethylamine, 4:1). After cooling, the precipitate was filtered and the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography on silica gel (toluene/diethylamine, 4:1) to produce 0.31 g of compound 4 (98%). Mp: 178-180 °C. 1H NMR: δ 7.9 (d, J ) 7.9 Hz, 2 H), 7.55 (d, J ) 8.1 Hz, 2 H), 7.30 (d, J ) 8.1 Hz, 2 H), 6.90 (d, J ) 8.8 Hz, 2 H), 4.42 (s, 2 H), 3.52 (s, 2 H, NH2). Anal. Calcd for C15H15NO3S2: C, 56.07; H, 4.67; N, 4.36; S, 19.94. Found: C, C, 56.20; H, 4.60; N, 4.50; S, 20.10. Synthesis of 4-Methanethiosulfinate Methyl-4′iodobenzophenone (IBM) (5). In a 50 mL roundbottomed flask a cooled stirred solution of water (30 mL) and compound 4 (0.16 g, 0.5 mmol) was treated with NaNO2 (52 mg, 0.75 mmol) and 1 mL of concentrated HCl. The resulting mixture was stirred at 0 °C for 30 min and then with NaI (113 mg, 0.75 mmol). After 30 min the product was extracted with ethyl acetate (3 × 10 mL). The extracts were pooled and dried with MgSO4, and the solvent was evaporated into an orange oil with a rotary evaporator. The resulting residue was purified by column chromatography on silica gel (toluene/diethylamine, 4:1) to produce 140 mg of compound 5 (65%), as an oil. 1H NMR: δ 7.9 (d, J ) 7.9 Hz, 2 H), 7.55 (d, J ) 8.1 Hz, 2 H), 7.30 (d, J ) 8.1 Hz, 2 H), 7.25 (d, J ) 8.8 Hz, 2 H), 4.42 (s, 2 H). Anal. Calcd for C15H13IO3S2. C, 41.67; H, 3.01; S, 14.81. Found: C, 41.80; H, 3.20; S, 14.70. Preparation of the Benzophenone-PDEγ Photoprobes. The constructs for expressing the full-length wild type (wt) PDEγ (single cysteine at position 68) (14) and the single-cysteine mutants were generated as described previously (9). The plasmids were transformed into E.coli strain BL21 DE3 cells (Novagen) for overexpression. Induction by 1 mM IPTG was initiated at an optical cell density of ∼1.5 (OD600) and continued for an additional 4-6 h at 30 °C. Expressed PDEγ constructs were first purified through a column of chitin beads (New England Biolabs), and then further purification was achieved (>95%) by reversed-phase HPLC (Waters) using a self-packed column of POROS 20 R2 resin (PerSeptive Biosystems). The lyophilized pure PDEγ after POROS column purification was used for derivatization. Benzophenone

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photoprobes were dissolved in acetonitrile to prepare stock solutions immediately before the derivatization reactions. A typical reaction includes 150 µM PDEγ, 50 mM NaH2PO4, pH 6.7, 60% acetonitrile, and 3 mM benzophenone compound. The reaction was incubated at room temperature under argon for 1 h and quenched with 0.1% TFA, then loaded to a Vydac C4 column for reversed-phase HPLC. A gradient of 1% acetonitrile per 8 min with 0.1% TFA was used to separate the derivatized PDEγ from the underivatized population at a flow rate of 1 mL/min. All the steps were carried out in dim room light. The benzophenone-PDEγ product eluted at ∼47% acetonitrile was lyophilized and stored at -80 °C. Functional Assay of the Benzophenone-PDEγ Photoprobes. Transducin GTPase activity was determined by using a single-turnover technique as described previously (21). The assays were conducted at room temperature (22-24 °C) in a buffer containing 25 mM Tris-HCl (pH 8.0), 140 mM NaCl, and 8 mM MgCl2. The urea-treated ROS membranes, lacking endogenous activity of RGS9, were used as a source for the photoexcited rhodopsin required for transducin activation. The reactions were initiated by the addition of 10 µL of 0.6 µM [32P] GTP (∼105 dpm/sample) to 20 µL of urea-treated ROS membranes (20 µM final rhodopsin concentration) reconstituted with transducin heterotrimer (1 µM) and recombinant RGS9-Gβ5 complex (0.5 µM). The reactions were performed in either the absence or presence of PDEγ derivatives (1 µM). The reaction was stopped by the addition of 100 µL of 6% perchloric acid. The 32Pi formation was measured with activated charcoal. The assays were conducted in the absence of reducing agent because of the disulfide linkage between the benzophenone group and PDEγ. Photo-Cross-Linking of the Benzophenone-PDEγ Photoprobes with Grt-GTPγS. GRt-GTPγS was prepared as previously described (8). Briefly, rod outer segment membranes were prepared from frozen darkadapted bovine retinas (from J. A. & W. L. Lawson Co.), and the GRt-GTPγS subunit was released using GTPγS and purified on Blue Sepharose CL-6B, as described by Kroll et al. (22). Coomassie Blue staining demonstrated that the GRt subunit was more than 95% pure on SDSpolyacrylamide gel electrophoresis (PAGE). Photo-cross-linking reactions were carried out in HEPES buffer (10 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2) containing GRt-GTPγS (5 µM) and benzophenone-PDEγ derivative at a desired concentration. The reaction mixture contained in a 1.7 mL ultraclear microcentrifuge tube (Axygen) was dark-incubated on ice for 15 min and then illuminated at 350 nm and ∼4 °C in a RPR-100 Rayonet photochemical reactor (Southern New England Ultraviolet) for various times (5 min dark interval after each 10 min UV light). The cross-linked proteins were separated from the un-cross-linked by nonreducing 15% SDS-PAGE. Preparation of Radioiodinated BenzophenonePDEγ Photoprobes. [125I] IBM was synthesized based on the Sandmeyer method for preparation of [127I] IBM (compound 5) as described above. In standard scale reactions, NaNO2 (3 mg, 43 µmol) was dissolved in 20 µL of H2O, and 6 µL of HCl (1 N) was added. Into this solution was added ABM (0.3 mg, 1.0 µmol) in acetonitrile immediately after HCl addition. The mixture was stirred at room temperature for 10 min, and then a 15 µL NaOH solution (0.1 N) containing Na127I (0.3 mg, 2 µmol) and 0.5 mCi Na125I was added and incubated for 30 min at room temperature with occasional vortexing. The reaction mixture was then extracted three times with 200 µL of

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ethyl acetate. The extracts were pooled and purified by TLC (silica gel, toluene/diethylamine, 20:1 v/v). The radioactive band that comigrated with [127I] IBM on the TLC plate was extracted with ethyl acetate, dried under N2 gas, and redissolved in 50 µL of acetonitrile. A 10 µL portion of this solution was used to derivatize wt-PDEγ (50 µg, 5 nmol) in 50 mM phosphate buffer for 1.5 h at pH 6.7 and room temperature. The [125I] IBM-PDEγ derivative was purified using an AutoSeq G50 spin column (Pharmacia). An alternative radioiodination that yields 125I-ABM was carried out using chloramine T. For a standard reaction, ABM (1 mg, 3 µmol) in 30 µL of methanol was mixed with 20 µL of sodium acetate (0.5 M, pH 5.6). Into this solution was added 2 µL of Na125I (0.5 mCi) followed by 2 µL of HCl (0.1 N), and then 30 µL of chloramine T (30 µg, 0.13 µmol) was immediately added. The reaction was incubated at room temperature for 10 min and then stopped by adding 100 µL of Na2S2O5 (0.5 mg, 2.6 µmol). Product extraction, purification, and PDEγ derivatization were performed as described in the above [125I] IBMPDEγ preparation. Photolabeling of GRt-GTPγS by 125IABM, as essentially described in “Photo-Cross-Linking of the Benzophenone-PDEγ Photoprobes with GRtGTPγS” above, was detected on a reducing SDS-PAGE 15% gel by PhosphorImager (445 SI, Molecular Dynamics). Other Methods. Electrospray ionization mass spectrometry (ESI MS) of the HPLC-purified PDEγ samples was performed in the Mass Spectrometry Facility of the Chemistry Department at the University of WisconsinMadison. The mass spectra were obtained with a Micromass (Beverly, MA) LTC mass spectrometer. This instrument uses a time-of-flight analyzer. Aliquots with added methanol were sprayed with a sample cone set at 20 V. The samples in a piece of glass capillary were inserted directly into the source, and the resulting vapor was bombarded with 70 eV electrons. Perfluorokerosene was used for calibration. For Western blotting, proteins were separated by nonreducing 15% SDS-PAGE and transferred to a PVDF membrane (Millipore, 0.45 µm) and analyzed using antiPDEγ (N-terminus) (Affinity BioReagents), horseradish peroxidase (HRP)-conjugated IgG, and enhanced chemiluminescence (ECL) reagents obtained from Pierce. The same blot was stripped and reprobed using anti-GRt (Nterminus) (Affinity BioReagents). GRt concentration was measured by the Bradford method using BSA as a standard. PDEγ concentration was also measured by the Bradford method but corrected based on the 280 nm spectrophotometric measurement and extinction coefficient of 6990. Protein amount on the scanned Coomassie gels was quantified using NIH Image 1.62. RESULTS AND DISCUSSION

Practical Considerations. Photo-cross-linking is a straightforward technique to investigate protein (or peptide)-protein interaction. In recent years, the interest in this technique has been raised again as a result of the development of more efficient photophores and the emergence of proteomics (10, 12, 23-25). Combined with other techniques such as modern mass spectrometry, the photo-cross-linking technique is useful not only for identifying protein interaction sites but also for obtaining structural information of proteins (26-29). The principle of a photo-cross-linking/label transfer approach for characterization of protein-protein interac-

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tion is illustrated in Figure 1, using PDEγ/GRt as a model system. A benzophenone probe is used to derivatize a single cysteine that is site-specifically engineered on a bait protein, PDEγ (Step 1). The benzophenone-derivatized bait protein is mixed with its interaction target protein, GRt, to form a complex, and subjected to UV light to cross-link the two proteins (Step 2). The photoinserted target protein is separated from the bait protein by reducing with DTT (dithiothreitol) and then proteolyzed (Step 3). The photoinsertion site on the target protein is identified, either by MS when a nonradioactive label is available or by autoradiography when using a radiolabeled photoprobe (Step 4). In this strategy, label transfer following photo-cross-linking is advantageous because the bait protein that is cross-linked to the target protein can be removed by DTT reversal, and the contamination of the bait protein in the analysis of the photoinsertion site on the target protein can thus be avoided. Furthermore, the free sulfhydryl of the photoprobe released by DTT reversal can be used for adding a tag, such as a biotin group, permitting affinity purification of the photoinserted proteolytic fragments for MS analysis (30) (Grant, J. E. and Ruoho, A. E., 2005, unpublished data obtained using IBM-derivatized PDEγ). Since the tag is added to the photoprobe after photolabel transfer, a possible impairment of the tag on the function of bait protein can be avoided. Therefore, a photoprobe favored for protein-protein interaction studies is expected to be not only stable and efficient but also sulfhydryl-reactive, cleavable, and tagged with a label convenient for purification or detection yet exerting minimum impairment on the function of bait protein. Several comparative studies have shown that reliable and reproducible high-efficiency labeling of target proteins is obtained using tetrafluorophenyl azides, trifluoromethylphenyl diazirines, and benzophenone photoprobes, but the benzophenone photophore performs the best (12, 25, 31). We thus have chosen benzophenone as the photophore for our photoprobes. Of the sulfhydrylreactive groups, MTS is the most rapidly reacting. MTS is also distinguished by its high selectivity for cysteinyl sulfhydryls, its ability to effect quantitative and complete conversion to the mixed disulfide, and the general reversibility of the disulfide bond (32). For radiolabel transfer experiments, an 125I-labeled benzophenone photoprobe may be prepared since 125I has often been used as a radiolabel due to its high sensitivity and relatively small size. Since the PDEγ C-terminal half has been suggested to be the major domain interacting with activated GRt (6, 8, 13, 33-35), we linked the benzophenone probes to single cysteines engineered at the PDEγ C-terminal positions for cross-linking with GRt-GTPγS. High-Yield Chemical Synthesis of the Bezophenone Photoprobes. As shown in Scheme 1, 4-methyl4′-nitrobenzophenone (compound 1) was prepared by Friedel-Crafts reaction of 4-nitrobenzoyl chloride with toluene in the presence of AlCl3 at room temperature with an 80% yield. Compound 1 was converted to 4-bromomethyl-4′-nitrobenzophenone (compound 2) by NBS in CCl4 in the presence of a catalytic amount of AIBN as a free radical initiator. Reaction of compound 2 with sodium methanethiosulfinate in refluxing methanol produced NBM (compound 3) with an 80% yield. NBM was reduced to the corresponding amine derivative ABM (compound 4) by Pd-C in methanol at room temperature with a 98% yield and then converted to IBM (compound 5) with a 65% yield by treating with NaNO2/HCl and then with NaI at room temperature.

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Sulfhydryl-Reactive Benzophenone Photoprobes Scheme 1. Synthetic Scheme of the Benzophenone-MTS Photoprobesa

a

NBS: N-bromo succinimide; AIBN: 2,2′-azobisisobutyronitrile.

The Benzophenone-PDEγ Photoprobes Were Readily Prepared, Purified, and Fully Functionally Active. Stability of photoprobes is an important issue for their biochemical applications. Importantly, all three benzophenone-MTS compounds with a para-position substitution of an amino (ABM), iodo (IBM), and nitro (NBM) group, respectively (Figure 1), were found to be stable at pH 6.7; for example, IBM stayed as a single peak on the HPLC chromatogram even after overnight incubation in phosphate buffer at pH 6.7 and room temperature. BPMTS, however, showed a significant breakdown product after 1 h incubation (data not shown). We thus carried out the PDEγ derivatization reactions using the benzophenone compounds at pH 6.7, taking advantage of their superb stability. As indicated by the chromatograms in Figure 2A, B, and C, after a 1 h roomtemperature incubation with PDEγ (73C), reactions were ∼70% completed with all three compounds, as estimated by the underivatized and derivatized PDEγ peaks. In fact, the derivatization reaction can be 100% completed after 2.5 h at room temperature, as demonstrated by the reaction using wt-PDEγ and IBM (Figure 2E). The benzophenone-PDEγ derivatives were well separated from the underivatized PDEγ population and unreacted free compounds by reversed-phase HPLC with a Vydac C4 column. Derivatization of PDEγ with all three benzophenone compounds shifted the PDEγ peak to ∼4 min later than underivatized PDEγ, although these compounds have different hydrophobicity, as evidenced by different HPLC retention times (Figure 2A, B, and C). An additional beneficial feature of the benzophenone probes is their UV absorption property, which is very different than PDEγ. As is evident in the HPLC chromatograms (Figure 2), the ratio of absorption by the benzophenone group at 215 nm versus 280 nm was approximately 1. In contrast, the 215 nm/280 nm absorption ratio of PDEγ was greater than 20, but the benzophenone adduct to PDEγ molecule changed this ratio to 20 (Figure 2D). Furthermore, when the molecular mass of the derivatized PDEγ products was determined by ESI MS, the major population, as shown in the mass spectrum of either IBM73C or IBM-87C, was highly consistent with the pre-

Figure 2. HPLC purification of PDEγ derivatized with the benzophenone photoprobes. The HPLC (Vydac C4 column) chromatograms are shown as absorptions at 215 nm (upper, solid) and 280 nm (lower, dashed). AU, relative absorption unit. A, B, and C: Purification of PDEγ (73C) derivatized with ABM, NBM, and IBM, respectively. The underivatized PDEγ (73C) fractions were eluted at 29.5 min; the derivatized products were at ∼33.5 min. D: DTT reversal of IBM-73C, which was taken from a portion of the 33.5 min peak in C (asterisk-marked). IBM73C was incubated with 50 mM DTT at room temperarure and pH 7.5 for 1 h and then applied to HPLC under the same conditions as in C. E: Purification of the IBM-68C derivative. The derivatization reaction was conducted at room temperature for 2.5 h, with 150 µM PDEγ, 50 mM NaH2PO4, pH 6.7, 60% acetonitrile, and 3 mM benzophenone compound.

dicted mass (Figure 3A). A minor population, with the measured mass lower than the major population by 131, is very likely the PDEγ population with the N-terminal methionine excised by the ribosome-associated methionyl-aminopeptidase during the posttranslational processing in E.coli (36). The N-terminal methionine-removed PDEγ population had unaltered function (data not shown). Taken together, the ESI MS data further verified that

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Figure 3. Characterization of the benzophenone-PDEγ photoprobes. A: ESI mass spectra of IBM-73C (upper panel) and IBM-87C (lower panel). The intensity of the major MS peak is taken as 100%. The predicted masses were calculated using PeptideMass of ExPASy (Swiss Institute of Bioinformatics). A measured mass is an m/z value multiplied by charge, as indicated in parentheses. B: Functional activity of IBM-73C. The RGS9 stimulated GTPase activity of transducin was measured in single-turnover assays in the absence of PDEγ (NoPDEγ) and in the presence of either wild-type PDEγ (wt-PDEγ) or IBM-derivatized PDEγ (IBM-73C). The rate constants of GTP hydrolysis (khydr) were determined from the single-exponential fits of the reaction time course and plotted as bars. Error bars represent SE of exponential analysis.

the benzophenone-PDEγ derivatives were highly purified and homogeneous. One great concern for the application of photoprobes is whether the addition of a photoreactive group to a bait protein affects its function. Since PDEγ is known to be an essential component for accelerating the GTP hydrolysis activity of GRt (1, 37), the functional integrity of the benzophenone-derivatized PDEγ preparations was assayed by analyzing their abilities to potentiate the activity of RGS9/Gβ5 in stimulating GRt-GTPase. Our data indicate that the derivatization of PDEγ at position 73 with IBM did not affect the function of PDEγ (Figure 3B). This was most likely due to the short length of the benzophenone photoprobe and the structural similarity of the benzophenone moiety to the phenylalanine residue at position 73 on wt-PDEγ. The Benzophenone-PDEγ Photoprobes Specifically Cross-Linked to Grt-GTPγS with High Efficiency. The photo-cross-linking reactions using benzophenone-PDEγ photoprobes and GRt-GTPγS were

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Figure 4. Specific photo-cross-linking of the benzophenonePDEγ photoprobes with GRt-GTPγS. Following UV photolysis, the reaction contents were separated by 15% SDS-PAGE in the absence of DTT and then stained with Coomassie Blue R-250. Percentage of cross-linking was calculated as the ratio of the protein amount in the cross-linked band versus total protein present in both the cross-linked and the un-cross-linked bands. A: Photo-cross-linking time course. The cross-linking reactions were performed with IBM-73C and GRt-GTPγS (molar ratio 1.5: 1) under UV light (as described in the “Experimental Procedures”) for various times as indicated on top of the Coomassiestained SDS-PAGE. The background from the dark control (0 min) is subtracted. XL, cross-linking. B: Western blotting of the cross-linked GRt/PDEγ complex. Photo-cross-linking reactions were performed under conditions similar to A, using PDEγ that was not derivatized (lane a) or derivatized (lane b) with IBM. After 20 min UV photolysis, the reaction was subjected to Western blotting, as described in the “Experimental Procedures”. C: Photo-cross-linking was conducted under UV for total 30 min with GRt and 73C (lane 1), IBM-73C (lane 2), and IBM73C after 10 min preincubation (on ice) of wt-PDEγ in 5-fold excess (lane 3). The ratio of IBM-73C/GRt was 1.5:1. D: PDEγ/ GRt molar ratio dependence of photo-cross-linking. The crosslinking reactions were performed at various IBM-87C/GRtGTPγS ratios under UV light for a total of 30 min. E: Photocross-linking of GRt-GTPγS with different benzophenone-73C photoprobes. The cross-linking reactions were performed under UV for a total of 30 min with a PDEγ/GRt-GTPγS molar ratio of ∼1.5:1.

conducted to test the specificity and cross-linking efficiency of the photoprobes (Figure 4). As shown in Figure 4A, UV photolysis of the GRt/PDEγ reaction mixture generated a band on the Coomassie-stained gel with a molecular size of approximately 50 kDa, the sum of GRt (∼40 kDa) and PDEγ (∼10 kDa). This cross-linked complex was confirmed to contain both GRt and PDEγ by Western blotting (Figure 4B). The cross-linked species was not observed in the dark control (0 min) (Figure 4A), nor with the underivatized 73C construct (Figure 4B), and was inhibited by preincubation of wt-PDEγ with GRt (Figure 4C), indicating specificity of crosslinking. Moreover, the cross-linking of IBM-73C with GRt-GTPγS was saturable with regard to photolysis time (Figure 4A) and photoprobe concentration (Figure 4D), further demonstrating the cross-linking specificity.

Sulfhydryl-Reactive Benzophenone Photoprobes

The maximum efficiency of cross-linking between the benzophenone-PDEγ photoprobes and GRt-GTPγS was as high as 40% (Figure 4A and D); a comparison of the 73C derivatives of the three benzophenone photoprobes indicated similar cross-linking efficiencies (Figure 4E). The efficient cross-linking of the PDEγ C-terminal positions with GRt-GTPγS further supports the previous observations that the PDEγ C-terminal half is a major domain interacting with GRt-GTPγS (33-35). The high efficiency of specific photo-cross-linking of the benzophenone photoprobes is a valuable feature, since a common problem using the photo-cross-linking approach is the low efficiency of the photolysis event that produces only trace amounts of labeled peptides after proteolysis. This often means that a number of unlabeled fragments mask the specifically labeled fragments and result in difficult purification of these complex digestion mixtures (10). The exceptionally high cross-linking efficiency of the benzophenone probes used in this study is likely to circumvent this problem. Besides the high photoreactivity of the benzophenone photophore, the short arm length (estimated 8-10 Å from the modified cysteinyl R-carbon) of these photoprobes is likely to be an additional factor leading to high cross-linking efficiency. These probes are probably the shortest stable benzophenone photoprobes that can be synthesized, considering that they have only one carbon between the disulfide bond and the benzophenone moiety. A similar but shorter benzophenone compound that we synthesized with no carbon between the sulfur and the benzophenone moiety (4-methanethiosulfonate benzophenone) turned out to be unstable at neutral pH (data not shown). A shorter arm length is usually favorable for cross-linking studies since the photoinsertion site reflects a more precise proteinprotein interaction position, a property not held by BPMTS, which has a longer arm length. The Directly Radioiodinated Amino-Benzophenone Photoprobe Offered a Sensitive Detection of Label Transfer to Grt-GTPγS. While the benzophenone photoprobes proved to be substantially useful in probing photoinsertion sites on GRt-GTPγS from derivatized PDEγ, through a biotin tag added to the photoprobe after photolysis (Grant, J. E. and Ruoho, A. E., 2005, unpublished data), detection of label transfer through autoradiography is a convenient approach under some circumstances. We therefore explored this possibility for extended applications of these benzophenone photoprobes. Since the nonradiolabeled iodobenzophenone photoprobe ([127I] IBM) was synthesized using ABM as starting material, we radioiodinated ABM in order to generate a radiolabeled benzophenone photoprobe. One approach that was used was to follow the [127I] IBM synthesis protocol described in the Experimental Procedures to generate [125I] IBM (Figure 5A). As shown in Figure 5B, the purified radiolabeled product comigrated on TLC with the standard [127I] IBM control, indicating the product to be [125I] IBM. In addition, PDEγ derivatized with the radioiodinated IBM was disulfide cleavable by DTT (Figure 5C), further characterizing it as [125I] IBM. A second approach was through the use of a mild oxidizing agent, chloramine T, by which Na125I was “activated” and added ortho to the amino-benzophenone group of ABM, yielding the product 125I-ABM (Figure 5D). This probe derivatized a single-cysteine PDEγ construct in a manner that was sensitive to DTT reversal (data not shown). The PDEγ derivatives of the 125I-labeled benzophenone probes were readily purified from free Na125I by size-exclusion

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Figure 5. Preparation of radioiodinated benzophenone-PDEγ photoprobes. A: Molecular structure of [125I] IBM. B: Autoradiogram of TLC purification of [125I] IBM. Left lane, [125I] IBM reaction; right lane, comigration of the [125I] IBM reaction and the pure [127I] IBM control whose position is indicated by the dashed ring. C: Autoradiogram of [125I] IBM-PDEγ in the dark on a SDS-PAGE gel run without and with DTT. D: Molecular structure of 125I-ABM. E: Autoradiogram of a SDS-PAGE gel showing the purification of 125I-ABM-PDEγ with a spin column (right lane) as compared with the sample before purification (left lane). F: Photolabel transfer to GRt-GTPγS (2 µM) from 125IABM-68C (3 µM). The proteins after photolysis (lane 1) or dark incubation on ice (lane 2) were separated by 15% reducing SDSPAGE. Upper panel, Coomassie-stained gel; lower panel, autoradiogram.

chromatography using spin columns (Figure 5E), which offers a sensitive detection of label transfer to GRt-GTPγS from derivatized PDEγ (see Figure 5F). An indirect way of photoprobe radiolabeling has been reported previously which involved derivatization of the DTT-reversed sulfhydryl of a reversible thiol-reactive photoprobe using [3H]-N-ethylmaleimide, but this method suffers from the need to block unlabeled free sulfhydryls first and an unfortunate problem involving a high radioactive background (11). Another way of indirect photoprobe radiolabeling was to radioiodinate a peptide at a site away from a benzophenone photoprobe incorporated in the peptide, as previously reported (38). The radiolabel, however, can be lost if unwanted proteolytic cleavage separates the radioiodinated residue from the benzophenone cross-linking site. The direct radiolabeling of benzophenone reported here is therefore advantageous. While biotin and fluorescent groups may provide alternate labeling approaches, their bulkiness often greatly impairs the function of derivatized bait proteins, thus limiting their applications. The 125I radiolabel, on the other hand, is exceptionally sensitive yet relatively small and can be directly added to the benzophenone moiety with minimal effects on the function of PDEγ. Summary. The data presented in this study indicate that the novel benzophenone photoprobes that have been developed in our laboratory are suitable for proteinprotein interaction analysis through a photo-cross-linking/label transfer strategy. These probes are short in length, stable at neutral pH, and DTT-cleavable. The PDEγ constructs derivatized with these probes could be readily purified and had unaltered function (as demonstrated by the IBM-73C derivative). Photo-cross-linking of PDEγ and GRt-GTPγS through these photoprobes was

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highly efficient. In addition, the amino-benzophenone photoprobe (ABM) was radioiodinatable, facilitating sensitive detection of photoinserted proteins, and this probe is potentially a module for adding additional functional groups to the amino on benzophenone. These combined features favor convenient identification of photoinsertion sites. Since these benzophenone probes can be linked through a disulfide bond to an engineered cysteine on a given protein in a site-specific manner, they may have broad applications for investigation of protein-protein interactions. ACKNOWLEDGMENT

This work was supported by an NIH Grant (NIH GM33138) to A.E.R. and an NIH Grant (EY12859) to V.Y.A. We thank Dr. Martha M. Vestling, director of the Mass Spectrometry Facility, Department of Chemistry, University of Wisconsin-Madison, for performing ESI MS and data processing. Thanks also go to Dr. Jennifer E. Grant in the laboratory of A.E.R. for the wt-PDEγ/ intein construct. LITERATURE CITED (1) Arshavsky, V. Y., Lamb, T. D., and Pugh, E. N., Jr. (2002) G proteins and phototransduction. Annu. Rev. Physiol. 64, 153-187. (2) Tsang, S. H., Burns, M. E., Calvert, P. D., Gouras, P., Baylor, D. A., Goff, S. P., and Arshavsky, V. Y. (1998) Role for the target enzyme in deactivation of photoreceptor G protein in vivo. Science 282, 117-121. (3) Chen, C. K., Burns, M. E., He, W., Wensel, T. G., Baylor, D. A., and Simon, M. I. (2000) Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1. Nature 403, 557-560. (4) Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) The 2.2 A crystal structure of transducin-alpha complexed with GTP gamma S. Nature 366, 654-663. (5) Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) GTPase mechanism of G proteins from the 1.7-A crystal structure of transducin alpha-GDP-AIF4-. Nature 372, 276-279. (6) Slep, K. C., Kercher, M. A., He, W., Cowan, C. W., Wensel, T. G., and Sigler, P. B. (2001) Structural determinants for regulation of phosphodiesterase by a G protein at 2.0 A. Nature 409, 1071-1077. (7) Dhanasekaran, N., Wessling-Resnick, M., Kelleher, D. J., Johnson, G. L., and Ruoho, A. E. (1988) Mapping of the carboxyl terminus within the tertiary structure of transducin’s alpha subunit using the heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine. J. Biol. Chem. 263, 17942-17950. (8) Liu, Y., Arshavsky, V. Y., and Ruoho, A. E. (1996) Interaction sites of the COOH-terminal region of the gamma subunit of cGMP phosphodiesterase with the GTP-bound alpha subunit of transducin. J. Biol. Chem. 271, 26900-26907. (9) Guo, L.-W., Grant, J. E., Hajipour, A. R., Muradov, K., Arbabian, M., Artemyev, N. O., and Ruoho, A. E. (2005) Asymmetric interaction between the rod phosphodiesterase inhibitory gamma subunits and the alpha and beta catalytic subunits. J. Biol. Chem. In press. (10) Hatanaka, Y. and Sadakane, Y. (2002) Photoaffinity labeling in drug discovery and developments: chemical gateway for entering proteomic frontier. Curr. Top. Med. Chem. 2, 271-288. (11) Teixeira-Clerc, F., Michalet, S., Menez, A., and Kessler, P. (2003) A cysteine-linkable, short cleavable photoprobe with dual functionality to explore protein-protein interfaces. Bioconjug. Chem. 14, 554-562.

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