Protein Diffusion Probed By the Transient Grating Method With a New Type of Photochromic Molecule[Dagger]

By Eitoku, Takeshi Terazima, Masahide

ABSTRACT A new type of photochromic molecule that can be used for diffusion coefficient (D) measurements of various proteins in solution is described. The absorption spectrum of this molecule is changed upon photoexcitation by the trans-cis isomerization reaction. Target proteins were labeled by this photochromic molecule in the dark and the translational motion of the proteins was detected by the transient grating (TG) method. The TG signal was simple enough to determine D accurately and was stable even for long- time irradiation by the laser light. The TG method using this probe molecule improves many drawbacks of the other techniques.

INTRODUCTION

The diffusion coefficient (D) is one of the useful physical properties for characterizing the nature of molecules in solution phase. This value reflects not only the molecular size or solution viscosity but also intermolecular interactions between a diffusing molecule and water molecules (1). Recently, it has been demonstrated that D also reflects a conformation of a protein (2-7). because the hydrogen bond networks, i.e. intermolecular interactions, are sensitive to the conformation. Indeed, this D detection method has been used to reveal conformational change during photoreactions of a variety of photosensor proteins, e.g. PYP, phototropins, etc. in time domain (2-7). These studies demonstrated that protein researches monitoring D will be a powerful technique. However, it is still an immature field; it is desirable to accumulate data and develop a technique that can be used for measuring the D of many proteins under various conditions.

Various techniques, such as Taylor dispersion (8,9), NMR (10), fluorescence correlation method (11) and dynamic light scattering (DLS) (12) have been developed so far. Each method has its merits and demerits but in general sensitivity and time-resolution have been the major problems for these methods. Our group has been using the transient grating (TG) method for measuring D of various photoreactive proteins (2-7,13,14). This technique detects photoinduced refractive index change with a high sensitivity and a high time-resolution, so that we can investigate the D of even unstable reaction intermediates. However, one of the drawbucks of this method is that this TG technique is difficult to apply to photochemically stable proteins, because the change in the refractive index cannot be induced by light irradiation. If this technique can be used for many proteins, it would be very powerful for establishing the relationship between D and protein conformation.

In order to overcome this difficulty. Baden and Terazima previously reported a method to measure D of photochemically stable proteins using a probe molecule HSAB (N-hydroxysulfosuccinimidyl-4- azidobenzoate) (15). Upon photoexcitalion of this molecule, the nitrogen molecule is dissociated and this dissociation reaction produces the TG signal. This probe molecule can indeed be applied to D measurements of many proteins and DNA (15). However, there are some drawbacks even in this method; that is, this probe molecule is decomposed and consumed by photoexcitation. The sample solution should be replaced with fresh solution after every 10 laser shots (the exact number depends on the experimental conditions, e.g. the sample volume, laser power, etc.). Further, the dissociated molecules produce additional signals which sometimes complicate the grating signal. Due to this complex feature, it is sometimes difficult to avoid large ambiguity in determined D.

We searched for a more appropriate probe molecule for improving the TG method. In this study, we report a new type of photochromic probe molecule for D measurements of a variety of proteins under various conditions by the TG method. We chose 4-acctoamido-4′- isothiacyanatostilben-2,2′-disulfonic acid Na^sub 2^ salt (SITS) (Fig. 1), which exhibits the trans-cis isomerization upon photoexcitation (16). The -N = C = S group of this molecule reacts with the -NH^sub 2^ group of proteins. The -SO^sub 3^Na group is necessary for increasing the solubility in water. A significant merit of using this probe molecule is that, as the probe molecule is not decomposed, we can use this molecule continuously without sample exchange. This advantage is particularly important under a condition such that the sample solution cannot be replaced frequently. Further, as D values of the reactant and the product of this probe molecule are almost identical, the observed TG signal is very simple compared with the previous HSAB probe method, and ambiguity for determination of D is reduced significantly. This probe molecule will open a large field to which the TG technique can be applied. Some examples of the D measurements are presented and the advantages discussed.

Figure 1. (A) Illustration of reaction schemes of SITS and a protein. The relative size of the protein is significantly reduced compared with the size of SITS. (B) Absorption spectra of SITS (a) in dark and (b) after photoirradiation of the YAG laser pulses (355 nm) (300 shots).

PRINCIPLE OF MEASUREMENT

In this study, a photoisomerization reaction of SITS was used for producing the refractive index modulation. This molecule can bind with the -NH^sub 2^ group at the N-terminal of proteins (Fig. 1A) (16). Upon photoexcitation of this molecule, the trans-cis isomerization reaction takes place. The absorption spectra (without binding with a protein) before and after light irradiation are shown in Fig. 1B. (The absorption spectrum in the visible wavelength does not change by binding with a protein.) As the absorption spectra of the trans- and cis-forms are different, the refractive indices are different from each other, and the concentration modulation leads the refractive index modulation to produce the TG signal. From the decay rate of the species grating signal, D of the reactant and the product can be determined.

EXPERIMENTAL SETUP

The experimental setup for the TG signal was similar to that reported previously (17-19). Briefly, a laser beam from the third harmonics of a Nd:YAG laser (Spectra Physics, GCR170l lambda = 355 nm; pulse width ~10 ns) was split into two with a beam splitter and crossed in a sample solution by a lens (focus length = 20 cm) to create the TG. The TG was detected as a diffraction of a CW probe beam (a He-Ne laser) that was led into the TG region with the angle satisfying the Bragg condition. The diffracted beam (TG signal) was detected by a photomultiplier tube. The signal was averaged by a digital oscilloscope (Tektronix TDS-5054).

SITS-labeled proteins were prepared by a method similar to that described in a previous study (16). A solution containing the target protein of 6~8 mg mL^sup -1^ was mixed with a SITS solution of ~3 mg mL^sup -1^. The mixed solution was incubated for 24 h in the dark at room temperature (23[degrees]C). The protein labeled by SITS was purified by filtering through a Sephadex G-25 column to remove excess reagent. The number of labeled molecules per protein molecule was determined by the absorption spectrum, and the number for all proteins we investigated was about ~1. The solution was diluted to ~1 mg mL^sup -1^ and dusts in the solution were removed by a membrane filter (0.2 [mu]m; Millipore) before the measurement.

The DLS experiments were carried out using a green diode laser (532 nm, 100 mW) as a probe light. The scattering angle was 60[degrees] for all measurements. Incident laser power was manually controlled with an equipped ND optical filter. The scattered light was detected with a cooled photomultiplier via an optical fiber, and self-correlation of its intensity fluctuation was calculated by a correlator (FDLS-3000; Otuka Electronics, Japan). NMR sample tubes (5 mm diameter) were used for measurements at room temperature (298 K). Prior to the experiments, sample solutions were filtered with a 0.2 [mu]m syringe filter (GHP membrane filter, PALL). Measured correlation profile was analyzed with a software (Igor Pro; Wave Metrics).

RESULTS AND DISCUSSION

TG signal of SITS

Figure 2. Typical TG signals after photoexcitation of (a) SITS (100 [mu]M [= 0.05 mg mL^sup -1^]) and (b) SITS-labeled ubiquitin (1 mg mL^sup -1^). The best fitted lines with Eq. (5) for (a) and Eq. (6) for (b) are shown by solid lines.

Previously, the TG signal of another probe molecule, HSAB was reported (15). Compared with the TG signal of HSAB, which was reproduced by a 4-exponential function, the temporal profile of SITS is much simpler. The complicated profile of HSAB was due to the photo-decomposition reaction. Not only the reactant but also the photo-decomposed compounds (nitrogen molecule and nitrene) contributed to the signal. Compared with this complex behavior of the HSAB probe, the simple feature of the TG signal of SITS is desirable for analyzing the TG signal.

TG signal of proteins with SITS

It is important to note that the TG signal corresponding to the protein diffusion was observed as expected. This TG signal was averaged 100 times with a repetition rate of 2 Hz. At the beginning of the average step, the TG signal was relatively strong, and it gradually became weaker by successive irradiations. This reduction in the intensity is due to the consumption of the trans-form. However, the TG signal did not vanish completely even after 100 shots of the laser pulses. This is probably because the photoirradiated volume is small and unphotoexcited molecules were diffused into that region from the bulk phase during the repetition period of the laser pulse (0.5 s). Appearance of the SITS signal was unexpected. We do not know the exact reason as to why this signal appeared, but it might be that unbound SITS with the protein was not completely removed by the column filter or photoexcited SITS was dissociated from the protein. In any case, as this contribution is weak and D^sub SITS^ can be accurately determined from the SITS solution as described previously, this rising component does not cause any problem for determination of D of the protein.

We observed similar TG signals from the other proteins we investigated. Some examples are shown in Fig. 3. The concentration of these protein solutions was adjusted to about 1 mg mL^sup -1^. (The signal of ubiquitin was larger than those of the other proteins, probably because the molecular weight of this protein is smaller than those of the other proteins, i.e. the concentration of ubiquitin solution is larger than the other ones.) The signals were fitted by Eq. (6). As D^sub SITS^ was fixed to the value determined from the previous section during the signal fitting, there was almost no ambiguity in the determined parameter. The D values of proteins determined by this method are listed in Table 1.

Strictly speaking, D^sub p^ is the diffusion coefficient of a protein with SITS. However, this value should be very close to that of the protein without SITS, because the molecular size of the proteins is much larger than that of SITS, and labeling ratio per one protein molecule is small enough (~1), so that the difference between the D of protein and the protein with SITS should be negligible. In fact, the D values of proteins determined by this method (Table 1) agree well with those reported previously. Therefore, we conclude that this photolabeling technique should be reliable in determining the D of proteins using the TG method.

Merits of this method

There are several advantages in this method. As this SITS probe method is based on the TG technique, some of the advantages are similar to those of the HSAB probe method. For example, the time response of this method is excellent. The TG signal completely decays within 100 ms in Fig. 1. If we used a much larger grating wavenumber, the decay rate should be much faster so that D measurement should be faster.

Figure 3. Some examples of the TG signal from proteins labeled using SITS. The best fitted lines with Eq. (6) are shown by solid lines.

Table 1. Diffusion coefficients (D/10^sup -11^ m^sup 2^ s^sup – 1^) of some proteins measured by the SITS-TG method and the literature values. Accuracy of the values is +- 10%.

Second, the sensitivity of this TG method is very good. The TG signal can be observed even below a protein concentration of 1 mg mL^sup -1^. For comparison, we measured a DLS signal of these proteins with the same concentration (1 mg mL^sup -1^). A typical example of the signal of beta-lactoglobulin is shown in Fig. 4. This signal was recorded after averaging 300 times, which took about 20 min. Comparing with the TG signal in Fig. 2, which was obtained after averaging 100 times (about 1 min), one may immediately notice that the S/N ratio of the TG signal is higher than that of the DLS data.

Figure 4. Typical autocorrelation of DLS signal (dotted line) of beta-lactoglobulin. The best fitted curve by Eq. (7) is shown by the solid line.

Compared with the HSAB probe method, there are two apparent merits for using this SITS probing. First, as HSAB has been developed as a photoactive cross-linking reagent, it creates a chemically reactive species upon photoexcitation and it could be linked with another protein in the solution. Hence, if the protein concentration is high, this reagent may produce a dimer of the protein. In fact, D of a dimer of cytochrome c was observed before (6). On the other hand, the photoproduct of SITS is the cis-form and it is a stable molecule, and we do not expect dimerization or unstable species upon photoexcitation.

Second, as HSAB is decomposed by photoexcitation, the TG signal becomes very weak by successive photoirradiation. After every 10 shots (the exact number depends on the experimental conditions), the sample solution should be refreshed. On the other hand. SITS is not decomposed. Hence, we can continuously use the same solution. This merit is especially important when the sample solution cannot be replaced frequently. One such example is the D measurement at high pressures. Recently, our group developed a high pressure cell for measurement of the TG signal (20). The high pressure cell should be sealed completely to prevent pressure leakage, and it is not practical to replace the sample solution frequently. Further, the sample cell was quite small (0.2 cm^sup 3^). Hence, it should be impossible to use HSAB as the probe molecule for the measurement with this cell. However, even under this condition, we could measure D of proteins using SITS. The pressure dependence of D of proteins upon pressure-induced conformational change will be reported in the future.

SUMMARY

A new type of photochromic molecule was used for application to the diffusion measurement of proteins by the TG technique. The probe molecule SITS exhibits a trans-cis photoisomerization reaction. The TG signal of SITS indicates that the trans and cis forms possess the same D so that the observed TG signal is very simple. This simplicity is very useful for D measurement of proteins. We demonstrated that D of some proteins can be measured accurately. As this molecule does not consume even after successive laser irradiations, we can continuously use this SITS probed protein without replacing with a fresh solution.

Acknowledgement-A part of this study was supported by the Grantin- Aid (13853002 and 15076204) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

[dagger] This invited paper is part of the Symposium-in-Print: Photoreceptors and Signal Transduction.

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Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan

Received 5 December 2007, accepted 7 January 2008, DOI: 10.1111/ j.1751-1097.2008.00315.x

* Corresponding author email: mterazima@kuchem.kyoto-u.ac.jp (Masahide Terazima)

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