Site-Specific Photocleavage of Hen Egg Lysozyme and Bovine Serum Albumin
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 10361–10366, September 1998 Chemistry Photochemical protease: Site-specific photocleavage of hen egg lysozyme and bovine serum albumin CHALLA V. KUMAR†‡,APINYA BURANAPRAPUK†,GREGORY J. OPITECK§,MARY B. MOYER§,STEFFEN JOCKUSCH¶, AND NICHOLAS J. TURRO¶ †Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060; §Glaxo Wellcome, Inc., Analytical Chemistry, Research Triangle Park, NC 27709; and ¶Department of Chemistry, Columbia University, New York, NY 10027 Contributed by Nicholas J. Turro, July 9, 1998 ABSTRACT Site-specific photocleavage of hen egg ly- sozyme and bovine serum albumin (BSA) by N-(l-phenylala- nine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 6 0.3 3 105 M21 and 6.5 6 0.4 3 107 M21, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, FIG. 1. Structure of protein probe with a specific recognition cobalt(III) hexammine (CoHA), was irradiated at 344 nm. element. The pyrenyl moiety can be photoactivated in the presence of suitable electron acceptors. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was ob- controlling the wavelength of excitation, specific chromophores served in the absence of Py-Phe, CoHA, or light. N-terminal can be selectively activated to high energies, thereby minimizing sequencing of the protein fragments indicated a single cleav- side reactions. age site of lysozyme between Trp-108 and Val-109, whereas the Vanadate, for example, has been used as a photoprobe to cleavage of BSA was found to be between Leu-346 and Arg-347. investigate phosphate-binding sites of proteins (16). Vanadate Laser flash photolysis studies of a mixture of protein, Py-Phe, competes for phosphate-binding sites of proteins (17), and it and CoHA showed a strong transient with absorption centered ' induces photocleavage of proteins with high preference for at 460 nm, corresponding to pyrene cation radical. Quench- serine residues (17–19). Applicability of vanadate is limited to ing of the singlet excited state of Py-Phe by CoHA followed by phosphate-binding proteins (18) and, hence, developing alter- the reaction of the resulting pyrenyl cation radical with the natives to vanadate is challenging. protein backbone may be responsible for the protein cleavage. Two approaches for the design of chemical proteases can be The high specificity of photocleavage may be valuable in distinguished: (i) affinity-based design (1, 5) and (ii) covalent targeting specific sites of proteins with small molecules. linking of redox-active metal complexes to specific residues of the protein, followed by activation of the metal complex to Reagents that can cleave the protein backbone with a high achieve protein cleavage (2–4, 11–14). Coupling chiral recog- specificity (chemical proteases) can be useful as biochemical tools nition elements with a suitable chromophore has been our (1–6). Structure–activity studies of proteins, investigation of approach to design photochemical proteases. N-(l-Phenylala- protein structural domains, and studies of the proximity of nine)-4-(1-pyrene)butyramide (Py-Phe; see Fig. 1), for exam- specific residues in a given protein are some applications of ple, induces site-specific photocleavage of hen egg lysozyme chemical proteases. Rapid proliferation of chemical nucleases for (Lyso), bovine serum albumin (BSA), and carboxypeptidase A DNA cleavage studies suggests how small-molecule-based chem- (CpA), under mild conditions (15). Advantages of using the ical reagents can be of use as tools in molecular biology (7–10). 4-(1-pyrene)butyroyl (Py) chromophore in the design are as Chemical proteases, analogously, could be useful for the manip- follows. (i) Py has high extinction coefficients, and hence, low ulation of proteins (1–6, 11–14). Determining the exposure of concentrations of the probe can capture a significant fraction selected residues of membrane-bound proteins to the aqueous of the incident light. (ii) Py has a long-lived ('110-ns), environment, design of new therapeutic agents, and converting high-energy ('70 kcalymol) singlet excited state that can drive large proteins into smaller fragments that are more amenable to a number of photochemical reactions. Short-lived excited sequencing are additional applications of chemical proteases. states are more likely to waste excitation by rapidly decaying Chemical proteases can be activated by light, and novel pho- to ground state without a fruitful chemical event. (iii)Pyhas tochemical proteases (15) are described here that have (i) high been used to probe the microenvironments of micelles, pro- affinities for selected sites of proteins, (ii) strong absorption bands teins, DNA, and other organized media (20). (iv) Considerable iii in the near visible region, ( ) long-lived singlet excited states that mechanistic information regarding Py excited states and re- are convenient to initiate photoreactions, and (iv) a chromophore 1 1 lated intermediates (such as Py . , PyH , and Py.) is available. that has been used to probe microenvironments. Use of light as (v) Py-Phe shows strong induced circular dichroism bands in a reagent for protein cleavage has distinct advantages. Photore- the 300- to 400-nm region, and these spectra are convenient to actions can be started and sustained with light, providing a sharp monitor the interaction of Py-Phe with proteins. control for initiation and termination of the reaction. Visible light Lyso, BSA, and CpA have been chosen as model proteins for is perhaps the least toxic reagent for environmentally benign photocleavage studies. Lyso is a small ('14-kDa) water- chemical approaches. Photoreactions are amenable for time- soluble enzyme whose activity can be readily monitored. The resolved mechanistic studies to delineate the elementary steps. By x-ray crystal structure of Lyso and its substrate-binding site are The publication costs of this article were defrayed in part by page charge Abbreviations: Py, 4-(1-pyrene)butyroyl; Py-Phe, N-(l-phenylalanine)- payment. This article must therefore be hereby marked ‘‘advertisement’’ in 4-(1-pyrene)butyramide; Lyso, hen egg lysozyme; CpA, carboxypep- accordance with 18 U.S.C. §1734 solely to indicate this fact. tidase A; CoHA, cobalt(III) hexammine trichloride. © 1998 by The National Academy of Sciences 0027-8424y98y9510361-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: cvkumar@ PNAS is available online at www.pnas.org. nucleus.chem.uconn.edu. 10361 Downloaded by guest on September 27, 2021 10362 Chemistry: Kumar et al. Proc. Natl. Acad. Sci. USA 95 (1998) known (21). BSA is a plasma protein that has many physio- spectrometry on a LaserMat instrument (FinniganMat, San Jose, logical functions. Hydrophobic ligands such as fatty acids, CA) with sinapinic acid (Sigma) as the matrix. steroids, and bilirubin bind to BSA (22). Because of its affinity Quantum Yield Measurements. A ferrioxalate actinometer for a variety of biological ligands, the interactions of BSA with (25) was used to determine light intensities at specific wave- specific ligands have been extensively investigated. Several lengths for quantum yield measurements. The entire experi- C-terminal phenylalanine peptides are known to be substrates ment was carried out in a dark room lit only by a yellow for CpA (23). Phenylalanine, therefore, is chosen as the chiral safelight. The actinometer solution (0.006 M) was prepared by z recognition element. Site-specific photocleavage of these pro- dissolving potassium ferrioxalate [K3Fe(C2O4)3 3H2O, 0.14 g] teins by Py-Phe and mechanistic details of protein photocleav- in water (45 ml) and sulfuric acid (0.5 M, 5 ml). Actinometer age are presented here. solutions (200 ml) were irradiated at 344 nm for various lengths of time (0, 5, 10, and 15 min). Aliquots (150 ml) of irradiated MATERIALS AND METHODS actinometer solutions were mixed with excess 1,10-phenanth- 5 5 5 y Lyso (Mr 14,300), BSA (Mr 66,267), and CpA (Mr roline (50 mg 50 ml of H2O, total 1 ml), and concentration of 35,250) were purchased from Sigma. Protein solutions were the ferrous ion was estimated from absorbance at 510 nm. prepared by dissolving the appropriate amounts of the protein Light intensities were measured before and after each irradi- in 50 mM TriszHCl, pH 7.0. Py-Phe was synthesized as de- ation and an average of two measurements was recorded scribed previously (15). All probe solutions were prepared (within 65%). Quantum yields for the photocleavage were fresh, and calibration graphs were constructed by using Beer’s estimated from three separate runs. law. Probe concentrations were restricted to the linear region Laser Flash Photolysis. Light pulses (355 nm, '8mJper of the graph. The molar extinction coefficient of Py-Phe was pulse, 8 ns) from a Continuum Surelite I Nd-YAG laser were estimated to be 42,600 M21zcm21 at 343 nm. used to excite the samples. A pulsed 150-W xenon lamp, Photochemical Protein Cleavage. Photocleavage reactions combined with an ISA (Metuchen, NJ) H10 monochromator, were carried out at room temperature in 50 mM TriszHCl served as the monitoring system. Signals from a Hamamatsu buffer, pH 7.0. The mixtures of protein (15 mM) and Py-Phe R928 photomultiplier tube were terminated into 50 ohms and (15 mM) in the presence of cobalt(III) hexammine trichloride digitized by using a Tektronix 380 (400 MHz) digitizer. The m [Co(NH3)6Cl3; CoHA] (1 mM), in a total volume of 100 l, entire experiment was controlled with a Macintosh Quadra 650 were irradiated at 344 nm by using a 100-W xenon lamp using LABVIEW 3 software as described earlier (26). Transient attached to a PTI (South Brunswick, NJ) A1010 monochro- absorption spectra were produced by obtaining full kinetic mator. A UV cut-off filter (WG-345; 78% transmission at 344 traces at various wavelengths and by sampling the change in nm) was used to remove stray UV light.