Photochemically Induced Nucleation of Ribonuclease A Enhanced by

Photochemically Induced Nucleation of Ribonuclease A Enhanced by a Stable Protein Dimer Produced from the Photochemical Reaction of Tyr Residual ...
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Photochemically Induced Nucleation of Ribonuclease A Enhanced by a Stable Protein Dimer Produced from the Photochemical Reaction of Tyr Residual Groups Kenji Furuta, Hiroaki Horiuchi, Hiroshi Hiratsuka, and Tetsuo Okutsu*

CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 6 1886–1889

Department of Chemistry, Gunma UniVersity, Kiryu 375-8515, Japan ReceiVed October 16, 2007; ReVised Manuscript ReceiVed January 23, 2008

ABSTRACT: We demonstrated the photochemically induced nucleation of ribonuclease A (RNaseA), which lacks Trp residual groups. We have reported the photochemically induced nucleation of the hen egg-white lysozyme and the thaumatin systems, where photochemically induced nucleation brought about the photochemical reaction of the Trp residuals. In the RNaseA system, photochemically induced nucleation brought about the photochemical reaction of the Tyr residuals. The photochemical intermediate radical of the Tyr residue was observed in a transient absorption experiment. The radical produces covalently bonded RNaseA dimers, as observed in a SDS-PAGE experiment. The dimers behaved as the smallest clusters in an early stage of the nucleation process. We carried out the photochemically induced nucleation of RNaseA under metastable conditions. The number of crystals exposed to UV irradiation increased with increasing irradiation time.

1. Introduction Protein crystallization is important for revealing the protein’s 3D structure in X-ray diffraction crystallography. Recently, the photophysical or photochemical light-induced crystallization of proteins has been reported.1–7 The photophysically induced crystallization of proteins using intense femto-second-long laser pulses has been reported.1,2 The laser ablation of the solution is thought to be responsible for the crystallization. On the other hand, we have reported the photochemically induced nucleation of hen egg-white lysozyme.3,4 When the UV light from a Xe lamp was used to irradiate metastable solutions, several crystals appeared only in the irradiated solutions. The mechanism of photochemically induced nucleation of lysozyme has been investigated, and the mechanism is illustrated in Scheme 1a. We revealed in our previous papers that a residual tryptophanyl radical was observed as the photochemical intermediate.3,4 In that paper, we found that enzymatic activity loss by light irradiation showed second-order dependence on the photon fluence. Therefore, the intermediate was through to be denatured by the second photon. Then, we investigate the relationship between the intermediate radical and nucleation.4 When the intermediate radical was selectively excited and denatured, photochemically induced nucleation was inhibited. Therefore, it is concluded that the intermediate grew into a nucleus. We have reported that the addition of poly(ethylene glycol) 4000 (PEG 4000) to the lysozyme solution greatly enhanced the frequency of photochemically induced nucleation.5 The addition of PEG 4000 enhanced the reaction rate of the intermediate radicals. Recently, we observed covalently bonded photochemical dimers in an electrophoresis experiment (SDS-PAGE), and the dimers were concluded to play an important role as the smallest stable clusters in an early stage of the nucleation process.6 The enhancement of protein crystallization due to the covalently bonded dimers is illustrated in Scheme 1b. Molecules gather to form clusters (n ) 2, 3, 4, . . .), which grow into a bulk crystal. When the cluster size is small, the clusters are unstable owing to the surface/volume energy disadvantage. Growth and dis* Corresponding author. Tel.: +81-277-30-1242. Fax: +81-277-30-1242. E-mail: [email protected].

Scheme 1. Mechanism of the Photochemically Induced Nucleation of Lysozyme (a) and Enhancement of Protein Crystallization by a Photochemical Reaction (b)a

a

The photochemical product, the protein dimer, behaved as the smallest cluster of nucleation.

solution take place even in supersaturated solutions. After the cluster size becomes larger than the critical size, the cluster grows into a bulk crystal spontaneously. The first step of the normal nucleation process begins from the formation of the smallest cluster, where n ) 2. The smallest cluster is unstable because the interaction between the molecules is weak (van der Waals, or hydrogen, bonding). Thus, n ) 2 cluster formation is an important step in an early stage of the nucleation process. In contrast, if the covalently bonded dimers behave similarly to the n ) 2 clusters, the dimers grow to n ) 3, 4, . . ., and onward to the critical size. Therefore, if nucleation starts from a stable cluster, i.e., a covalently bonded dimer, the nucleation frequency becomes higher. The next step in our study was to examine whether photochemically induced nucleation is a phenomenon affecting watersoluble proteins in general. The following proteins were selected for this purpose: 1. Let us examine those lysozyme analogue proteins that have Trp residues. Most of proteins contain Phe, Tyr, and Trp residues, which are chromophores of photochemical reactions. Among those three amino acids, the Trp residue has the lowest

10.1021/cg701016p CCC: $40.75  2008 American Chemical Society Published on Web 05/07/2008

Photochemically Induced Nucleation of RNaseA

excited-state energy. If a Phe or Tyr residue absorbs a photon, that excited-state energy transfers to the Trp residue through intramolecular energy transfer. Finally, only the excited state of the Trp residue remains. The photochemically induced nucleation mechanism was expected to be the same as that for lysozyme. As for other lysozyme analogue proteins, we have previously demonstrated the photochemically induced nucleation of thaumatin.7 The crystallization mechanism was concluded to be identical with the mechanism for lysozyme. 2. Proteins that lack Trp residues are interesting and important. In this case, photochemical reactions due to excited-state Phe or Tyr residues are expected. The most appropriate candidate is ribonuclease A. In this study, we demonstrated the photochemically induced nucleation of a prototype protein lacking Trp. For this purpose, bovine pancreatic ribonuclease A (RNaseA) was selected. The crystallization procedure has already been reported by Carlisle et al.8 RNaseA consists of 124 amino acid residues that contain six Tyr and three Phe residues as chromophores.9 The photochemical properties of RNaseA have been reported by Grossweiner et al.10,11 We investigated the photochemical properties of RNaseA by means of steady-state and transient spectroscopy from the viewpoint of photochemically induced nucleation. An electrophoresis experiment for observing the photochemical dimers was also carried out to examine the crystallization mechanism.

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Figure 1. (a) Light-irradiation apparatus used in a photochemically induced nucleation experiment. The light source is a 300 W Xe lamp. A water filter is used to remove IR radiation (light pass length ) 20 mm), and a band-pass filter is used to cut the visible radiation. The UV light falls vertically on the droplets through paraffin oil. (b) Radiation spectra of the 300 W Xe lamp. The dashed and solid lines show the radiation spectra without filters and with filters, respectively.

2. Experimental Section Genomic-research-grade (GR-grade) RNaseA was purchased from Wako Pure Chemicals and used without further purification. Sodium acetate, acetic acid, tyrosine (Tyr), phenylalanine (Phe), sodium chloride (NaCl), and ammonium sulfate (AS), all of them GR-grade, were purchased from Wako Pure Chemicals. Sodium acetate and acetic acid were dissolved in ultrapure water (Milli-Q) and used as the buffer solution (NaAc buffer, 50 mM, pH 5.5). The prepared solution was centrifuged and filtered through a 0.45-µm membrane filter (NALGENE) before the crystallization experiment. The RNaseA concentration was determined using an extinction coefficient of 0.70 L g-1 cm-1 at 280 nm.12 Sample preparation was carried out at room temperature. The steady-state spectra were recorded using a Hitachi F4500 fluorescence spectrometer for the emission measurements and a Hitachi U3300 spectrometer for the absorption measurements. For the measurement of the transient absorption spectra, an Nd3+: YAG laser (Lotis Tii LS-2137U, 266 nm, 30 ns hwfm, 3 mJ pulse-1, 10 Hz) was used as the excitation light source. The sample solutions were flowed through a quartz cavity cell at a flow rate of 40 mL min-1. The transient signals were detected using a photomultiplier tube. The output signals were measured by a digital oscilloscope (Tektronix TDS 380P) and transferred to a personal computer. The detailed experimental setup of the transient absorption experiment has been described in the literature.13 The light source used for the preparation of the irradiation samples and in the photochemically induced nucleation experiment was a Xe lamp (USHIO, UXL 300D, 300 W). Figure 1a shows the lightirradiation apparatus for the photochemically induced nucleation experiment. The light beam from the lamp is passed through water (light pass length ) 20 mm), to cut the near-IR radiation, and through a band-pass filter (Sigma Koki, UVTF-33U), to cut the visible radiation. Figure 1b shows the radiation spectrum of the Xe lamp without filters (dashed line) and with filters (solid line). The SDS-PAGE experiment was carried out using a slab minigel electrophoresis unit (Nihon Eido, NA-1020, CN-1010) with 15% resolving and 0.05% concentrating gels. A Tris-Gly buffer solution (containing 0.4% SDS) was used as the electrode solution. A total of 5 µL of the sample solution was loaded onto each lane of gel. The gel was stained using a silver stain kit. The batch crystallization experiment was carried out in a 72-well micro batch plate purchased from Hampton Research. The micro batch plate was covered with paraffin oil before the addition of the droplets. The droplets were irradiated for 0-300 s by UV light through the

Figure 2. Steady-state electronic spectra of RNaseA (a), Tyr (b), and Phe (c) in a 50 mM NaAc buffer at pH 5.5. The dashed and solid lines show the absorption and emission spectra, respectively. paraffin oil. The irradiated plate was sealed with silicone grease and stored at 20 °C for 1 week.

3. Results and Discussion 3.1. Measurement of Steady-State Electronic Spectra. The steady-state electronic spectra were measured. Figure 2 shows the absorption and emission spectra of RNaseA (a), Tyr (b), and Phe (c). The absorption and emission spectra are indicated by the dashed and solid lines, respectively. Tyr and Phe are aromatic amino acids that are expected to participate in the photochemical protein reaction. Both of these amino acids emit fluorescence. The RNaseA (a) emission spectrum is identical with the Tyr emission spectrum (b) and differs from the Phe emission (c). These results suggest that the excited state of

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Figure 4. Photographs of the gel. Lane 1: molecular weight marker from 14 to 79 kDa. Lanes 2–8: RNaseA solutions. These sample solutions were irradiated with UV light for 0, 3, 5, 15, 30, 60, and 120 min. The irradiation times are indicated below the lane numbers.

Figure 3. Transient absorption spectra of RNaseA (a), Tyr (b), and Phe (c) in a 50 mM NaAc buffer at pH 5.5. The spectra were recorded 64 µs after the laser flash.

RNaseA is actually the excited state of Tyr. Lysozyme emission is known to consist of the emission from excited-state Trp residual groups, which is partly generated through intramolecular energy transfer from Phe or Tyr residuals.14,15 In an RNaseA molecule, six Tyr and three Phe residues are contained as aromatic residues.9 Because the emission from Tyr has a longer wavelength than that from Phe, the excited-state energy of Tyr is lower than that of Phe. If the Phe residual absorbs a photon, the excited-state energy can be transferred to Tyr through intramolecular Förster-type energy transfer. 3.2. Measurement of Transient Absorption Spectra. To observe the photochemical intermediates of RNaseA, transient absorption experiments were carried out and the transient absorptions of Tyr and Phe were compared. Figure 3 shows the transient absorption spectra of RNaseA (a), Tyr (b), and Phe (c) in a 50 mM sodium acetate buffer at pH 5.5. The spectra were measured 64 µs after the laser flash. The triplet state was not detected because the sample solutions were saturated with O2 gas.16 Figure 3a shows the RNaseA transient absorption spectrum with an absorption band shorter than 440 nm having vibrational bands of 410 and 390 nm. Figure 3b shows the Tyr transient absorption spectrum, which is almost identical with the transient absorption spectrum of RNaseA and is the spectrum typically caused by phenoxyl radicals.17,18 Figure 3c shows the Phe transient spectrum with an absorption band shorter than 370 nm, which differs from the absorption bands of RNaseA (a) and Tyr (b). These results indicate that the photochemical intermediate of RNaseA is the residual Tyr radical in which the phenol group is converted into a phenoxyl radical, and this finding agrees with the report by Grossweiner and Usui10 3.3. SDS-PAGE Experiment. Covalently bonded dimers are thought to behave as the smallest stable clusters in an early stage of the nucleation process.6 To detect the presence of photochemically produced covalently bonded RNaseA dimers, a SDS-PAGE experiment was carried out. Figure 4 shows a photograph of the gel. Lane 1 is the molecular weight marker. Lanes 2–8 are the samples irradiated for 0, 3, 5, 15, 30, 60, and 120 min. The irradiation times are indicated below the lane numbers. Lane 2 corresponds to the solution without irradiation and shows an RNaseA monomer band at 14 kDa and a dimer

band at 28 kDa, which resulted from the impurity of the commercially available solution used. In lanes 3–8, corresponding to the irradiated solutions, the dimer band intensities become clear with increasing irradiation time. In lanes 5–8, trimer bands at 42 kDa can be observed, i.e., in samples exposed to more than 30 min of irradiation. These results show that light irradiation produces covalently bonded dimers and trimers, as observed in the lysozyme system. 3.4. Photochemically Induced Nucleation of RNaseA. Photochemically induced nucleation of RNaseA was carried out. Our aim was to induce crystallization by photochemical perturbation where no spontaneous nucleation occurs. We prepared a metastable condition of supersaturation by changing both the protein and precipitant concentrations. As the precipitant, a mixed solution of NaCl and ammonium sulfate (AS) was used, in accordance with the previous reports.19–21 Figure 5a (A-I) shows photographs of RNaseA droplets on a micro batch plate. The RNaseA concentrations were set to 15, 20, and 25 mg mL-1. We made 3 × 3 matrixes of RNaseA and precipitant concentrations. The conditions are labeled A-I in Figure 5a, and the concentrations of RNaseA and the precipitants in the droplets are indicated on the left-hand side and below the photograph, respectively. Four simultaneous experiments for each condition were carried out. In conditions A-E and G, no crystal appeared. In conditions F and H, crystals appeared in 25% of the droplets. In condition H, crystals appeared in all droplets. These results show that experimental conditions F, H, and I induced spontaneous nucleation, whereas conditions A-E and G seem to have been metastable. In this latter condition, photochemically induced nucleation was expected to succeed. We tried to employ experimental condition E. Figure 5b shows photographs of RNaseA droplets exposed to light irradiation for 0 (J), 180 (K), and 300 (L) s. No crystal was seen in the droplet without irradiation (condition J), and none in condition E either. Crystals appeared in the irradiated droplets (K and L), and the number of crystals increased with increasing irradiation time. These results show that photochemically induced nucleation succeeded in the RNaseA system lacking Trp residues.

4. Conclusion We demonstrated the photochemically induced nucleation of RNaseA) which lacks Trp residual groups. We have previously reported photochemically induced nucleation in the hen eggwhite lysozyme and the thaumatin systems, where photochemically induced nucleation brought about the photochemical reaction of the Trp residual groups. In the RNaseA system,

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Acknowledgment. This work was supported by Grants-inAid for Scientific Research (Grants 15033213, 14540527, and 15350004) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government. Support was also given by The USHIO Foundation.

References

Figure 5. Photographs of RNaseA droplets on the micro batch plate. (a) 3 × 3 matrix of RNaseA with various precipitant and RNaseA concentrations. Precipitant concentrations (M): 2.3/1.44, 2.4/1.5 and 2.35/1.47 (NaCl/AS). Protein concentrations: 15, 20, and 25 mg mL-1, respectively. (b) Photochemically induced nucleation of RNaseA in condition E. Irradiation times: 0 (J), 180 (K), and 300 s (L). Scale bar ) 1 mm.

photochemically induced nucleation brought about the photochemical reaction of the Tyr residual groups instead. The photochemical intermediate radical of the Tyr residue was observed in a transient absorption experiment. The radicals produced covalently bonded RNaseA dimers, which were observed by SDS-PAGE. The dimers behaved as the smallest clusters in an early stage of the nucleation process. We carried out the light-induced crystallization of RNaseA under metastable conditions. The number of crystals exposed to UV irradiation increased with increasing irradiation time. These results suggest a new method for controlling nucleation and crystal growth that could be used in fields such as structural genomics and the pharmaceutical industry.

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