Characterization of Surface-Confined α-Synuclein by Surface Plasmon

Langmuir 2006, 22, 13-17

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Characterization of Surface-Confined r-Synuclein by Surface Plasmon Resonance Measurements Taewook Kang, Surin Hong, Hyun Jin Kim, Jungwoo Moon, Seogil Oh, Seung R. Paik, and Jongheop Yi* School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National UniVersity, San 56-1, Shillim, Kwanak, Seoul 151-742, Korea ReceiVed August 22, 2005. In Final Form: October 25, 2005 Urea-driven denaturation and renaturation of surface-bound R-synuclein are monitored by surface plasmon resonance (SPR) spectroscopy. The differential SPR angle shift (∆ΘSPR)Net enables us to estimate the Gibbs free energy change (∆G°) for the denaturation of the supported R-synuclein. ∆G° for the denaturation of the supported R-synuclein, which is indirectly related to its biological activity can be increased significantly by the mixed self-assembled monolayers of 11-mercaptoundecanoic acid and 1,6-hexanedithiol. These SPR measurements of surface-bound biomolecules suggested herein can be further utilized to design effective biological scaffold for biosensor, biocatalyst, and possible diagnosis.

Introduction For the purpose of the possible diagnosis of disease and the development of biosensors and biocatalysts, one of the most straightforward approaches to investigating the physiological function of biomolecules is the in vitro immobilization of object biomolecules on a certain substrate.1-3 An important prerequisite for the success of this approach is that the biomolecules should be immobilized in such a manner that their biological activities as well as stabilities are not altered, compared to that in bulk solution. However, the issue of how biomolecules will behave when grafted to a surface is not predictable. Although the thermodynamic properties of the biomolecules in the free state have been studied systematically,4 our knowledge of these properties, when the biomolecules are immobilized on a solid support, is relatively incomplete.1 For this reason, we have interested in the determination of the fundamental properties of surface-confined biomolecules (e.g., the decrease in Gibbs energy of the structureless protein polypeptide chain when it folds to give a native protein molecule in water from the conformational transition curves induced by chemical denaturants). R-Synuclein, a known natively unfolded protein, has been shown to be a major component of Lewy bodies (LB) in Parkinson’s disease (PD), suggesting that the aggregation of R-synuclein plays a critical role in PD.5 Although significant advances have been made regarding the aggregation and fibrillation6-8 of R-synuclein, the physiological function of the molecule is poorly understood. * To whom correspondence should be addressed. Phone: +82-2-8807438. Fax: +82-2-885-6670. E-mail: [email protected]. (1) Chah, S.; Kumar, C. V.; Hammond, M. R.; Zare, R. N. Anal. Chem. 2004, 76, 2112. (2) (a) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Nano Lett. 2005, 5, 829. (b) Haes, A. J.; Hall, W. P.; Chang, L.; Klein, W. L.; Van Duyne, R. P. Nano Lett. 2004, 4, 1029. (3) Metallo, S. J.; Kang, R. S.; Holmlin, R. E.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 4534. (4) (a) Hasegawa, K.; Ono, K.; Yamada, M.; Naiki, H. Biochemistry 2002, 41, 13489. (b) Gupta, R.; Ahmad, F. Biochemistry 1999, 38, 2471. (5) (a) Lee, D.; Lee, S.-Y.; Lee, E.-N.; Chang, C.-S.; Paik, S. R. J. Neurochem. 2002, 82, 1007. (b) Spillantini, M. G.; Schmidt, M. L.; Lee, V. M.-Y.; Trojanowski, J. Q.; Jakes, R., Goedert, M. Nature 1997, 388, 840. (6) Cole, N. B.; Murphy, D. D.; Grider, T.; Rueter, S.; Brasaemle, D.; Nussbaum, R. L. J. Biol. Chem. 2002, 277, 6344. (7) Sabate, R.; Esterlrich, J. J. Phys. Chem. B 2005, 109, 11027. (8) Lee, H. J.; Choi, C.; Lee, S. J. J. Biol. Chem. 2002, 277, 671.

In the present study, we estimate the thermodynamic parameter for the surface-bound R-synuclein using the urea-induced denaturation surface plasmon resonance (SPR) curve. Because the evanescent field of the surface plasmons decays exponentially from the surface,9-12 the urea-induced denaturation of R-synuclein would lead to a change in the local average refractive index. Consequently, it should be possible to track the denaturation process by monitoring the angle shift in the intensity minimum. In addition, we also propose a way to increase the biological activity of surface-bound R-synuclein. Experimental Section 11-Mercaptoundecanoic acid (MUA), 1,6-hexanedithiol (HDT), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were used as received. Urea, a denaturant, was dissolved in 10 mM potassium phosphate buffer (PBS, pH 7.4) to prepare urea solutions of different concentrations up to 4 M. The incubation buffer for R-synuclein was composed of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5) in the absence of urea. Sulfuric acid and hydrogen peroxide were used to clean the microscope slide glasses on which the gold films were deposited. H2O was purified to above 18 ΜΩ using a Milli-Q water system (Millipore). SPR measurements were performed using an in-house constructed instrument.10b,11,13 Recombinant R-synucleins cloned in pRK172 were overexpressed in Escherichia coli BL21 (DE3). The R-synuclein was extensively purified through heat treatment, DEAE-sephacel anion-exchange, sephacryl S-200 size exclusion, and S-sepharose cation-exchange chromatography steps according to a previously described procedure.5,14 The overall experimental procedures are summarized in Figure 1a. Glass microscope slides were immersed in a piranha solution (H2SO4:H2O2 ) 7:3 v/v) for purification (caution: piranha solution should be handled with extreme care). The glass substrate was rinsed (9) Knoll, W. Annu. ReV. Phys. Chem. 1998, 49, 569. (10) (a) Roy, D.; Fendler, J. H. AdV. Mater. 2004, 16, 479. (b) Chah, S.; Fendler, J. H.; Yi, J. Chem. Commun. 2002, 2094. (11) (a) Kang, T.; Moon, J.; Oh, S.; Hong, S.; Chah, S.; Yi, J. Chem. Commun. 2005, 2360. (b) Chah, S.; Yi, J.; Pettit, C. M.; Roy, D.; Fendler, J. H. Langmuir 2002, 18, 314. (12) (a) Wischerhoff, E.; Zacher, T.; Laschewsky, A.; Rekai, E. D. Angew. Chem., Int. Ed. 2000, 39, 4602. (b) Ekgasit, S.; Thammacharoen, C.; Knoll, W. Anal. Chem. 2004, 76, 561. (13) Kang, T.; Hong, S.; Moon, J.; Oh, S.; Yi, J. Chem. Commun. 2005, 3721. (14) Paik, S. R.; Lee, J.-H.; Kim, D.-H.; Chang, C.-S.; Kim, J. Arch. Biochem. Biophys., 1997, 344, 325.

10.1021/la052276w CCC: $33.50 © 2006 American Chemical Society Published on Web 11/12/2005

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Letters assembled monolayer (SAM) of MUA on the Au thin film was formed by treatment with a 1 mM MUA ethanolic solution for 18 h. The formation of an MUA monolayer was investigated by SPR measurements and auger electron spectroscopy (AES). For the immobilization of R-synuclein on the MUA treated Au thin film by covalent bonding, the MUA treated surface was first activated by 7 min exposure to a 1:1 mixture of 0.4 M EDC and 0.1 M NHS aqueous solution. R-Synuclein in the same buffer solution was then used for the immobilization. At saturation, the surface was rinsed with copious amounts of water and buffer solution.

Results and Discussion

Figure 1. (a) Schematic illustration of the binding reactions involved in the study, including the self-assembly of MUA, the formation of NHS ester, and R-synuclein immobilization (not drawn to scale). It is well-known that, in the MUA system, R-synuclein immobilization is subjected to preactivation of the carboxyl groups with EDC/NHS reagents, the role of which is to activate the carboxyl groups to form O-acylurea intermediates and NHS esters that promote the formation of amide bonds with amino groups on the protein. (b) Incident angle dependent SPR contour plots (in voltage unit) obtained from the MUA-Au surface, after the treatment of NHS/EDC with the MUAAu surface, finally after the immobilization of R-synuclein, using an ambient dielectric of air. For each graph, every fourth point from the collected raw data is plotted to preserve the clarity of the contour plot. The inset figures represent the corresponding surface status. (c) In situ SPR response curves of R-synuclein/Au for an antibody (purified mouse anti-R-synuclein monoclonal ab). The concentrations of the antibody are 2.5 and 0.5 µg/mL. several times with copious amounts of deionized water and ethanol and, then, dried. R-Synuclein was coupled to SAM by amide bond formation as reported in the literatures.15-17 Briefly, the self(15) Su, X.; Wu, Y.-J.; Robelek, R.; Knoll, W. Langmuir 2005, 21, 348. (16) Fung, Y. S.; Wong, Y. Y. Anal. Chem. 2001, 73, 5302.

A schematic diagram describing procedures for the immobilization [the sequential, stepwise assembly of layers of N-hydroxysuccinimide (NHS) ester and R-synuclein] of Rsynuclein onto the carboxyl-terminated SAM (11-mercaptoundecanoic acid, MUA SAM) on a Au thin film is shown in Figure 1a. To follow these assemblies that occur on the surface of the Au thin film, the procedures were monitored during the formation of NHS ester (NHS/Au) and the attachment of R-synuclein (R-synuclein/Au) on the Au thin film by angleresolved SPR. Figure 1b shows angle-dependent SPR contour plots for the sequential assembly of MUA/Au, NHS/Au, and R-synuclein/Au using an ambient dielectric of air. The SPR resonance angle (ΘSPR) increases as a function of both the thickness and the dielectric constant of the layer adjacent to the Au thin film.18,19 Therefore, the substitution by a new bulkier organic layer (order of molecular size: R-synuclein . NHS ester > -COOH) with a similar dielectric constant on the surface of the Au thin film is responsible for the increase in ΘSPR in Figure 1b. Furthermore, the content of “nitrogen” atoms (from auger electron spectroscopy) on NHS/Au compared with MUA/ Au increased dramatically, indicating the successive immobilization of NHS ester. The immobilization of R-synuclein was also evident from in situ SPR measurements (Figure 1c) in antibody experiments.20 The SPR response for the urea-induced denaturation of surfacebound R-synuclein (Figure 2a) shows an increase in maximum reflectance up to a concentration of 3 M urea, followed by no further changes above 4 M urea. The sequential renaturation of R-synuclein by dilution of the urea restored its initial SPR spectra, which suggests that the urea-induced changes are essentially reversible. As a control experiment, we also present data for NHS/Au, which is not denaturated by urea. Two sets of angleresolved SPR curves depending on the urea concentration show that both SPR curves shift proportionally to the increase in urea concentration, as shown in Figure 2b, with identical minimum reflectivity at each ΘSPR. It should be noted that the SPR angle shift in both SPR curves arises from the contributions of both the change in bulk dielectric constant of the urea concentration (see inset in Figure 2b) and the conformational change in R-synuclein that is induced by urea. To differentiate between these two contributions to the SPR angle change, the SPR angle change (∆ΘSPR)Bare at each urea concentration for NHS/Au was subtracted from the SPR angle change (∆ΘSPR)R-Syn for R-synuclein/Au to form the difference, providing a plot of the differential SPR angle change (∆ΘSPR)Net [i.e., (∆θSPR)Net|Urea)x ) (∆θSPR)R-Syn|Urea)x - (∆θSPR)Bare|Urea)x, where x (M) is in the range of 0∼4] versus urea concentration as shown in Figure 3a. (17) Lahiri, J.; Isaacs, L.; Tien, J. Whitesides, G. M. Anal. Chem. 1999, 71, 777. (18) Chen, W. P.; Chen, J. P. Surf. Sci. 1980, 91, 601. (19) Sarkar, D.; Somasundara, P. Langmuir 2004, 20, 4657. (20) A nonspecific reaction was checked by replacing R-synuclein/Au thin film by NHS/Au thin film. No detectable anti-binding to the NHS/Au thin film was found.

Letters

Figure 2. (a) In-situ (time-resolved) SPR measurements of ureadriven denaturation and renaturation for R-synuclein/Au as a function of urea concentration in the range of 0.5∼3.0 M. (b) Angle-resolved SPR curves for R-synuclein/Au as a function of urea concentration of 0, 0.5, 1.0, 1.5, 2, 2.5, 3.0, and 4.0 M. Inset in (b) corresponds to SPR curves for the NHS/Au.

(∆ΘSPR)Net increased gradually as a function of urea concentration up to a concentration of 3 M. The circular dichroism spectrum of unbound R-synuclein in the absence of urea shows a minimum at 198 nm which is characteristic of a high percentage of random coil structure (data not shown). The addition of urea up to 8 M to the unbound R-synuclein resulted in a gradual increase in the molar ellipticity at 222 nm.21-23 This suggests that, contrary to the surface-bound R-synuclein, the denaturation of unsupported R-synuclein continues until the concentration of urea reaches about 6 M. No attempts were made to probe detailed changes in the secondary structure of supported R-synuclein induced by urea. Instead we focused on the detection of changes in the optical properties of the supported R-synuclein layer, as the result of denaturation. During the denaturation, the surface bound R-synuclein produce a partially denatured synuclein layer which consists of the denatured R-synuclein and the natured R-synuclein (that is, inhomogeneous film with two components having different dielectric constants). A simplified approach to deal with this inhomogeneous film is to use the theory of Maxwell and Garnett. Such an inhomogeneous system can then be proved to be equivalent to a homogeneous one with an effective dielectric (21) Davidson, S. W.; Jonas, A.; Clayton, D. F.; George, J. M. J. Biol. Chem. 1998, 273, 9443. (22) Baskakov, I. V.; Legname, G.; Prusiner, S. B.; Cohen, F. E. J. Biol. Chem. 2001, 276, 19687. (23) Greenfield, N. J. Anal. Biochem. 1996, 235, 1.

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Figure 3. (a) Surface plasmon resonance angle shift, ∆ΘSPR versus urea concentration from 0 to 2.5 M. (b) Circular dichroism spectra of unsupported recombinant R-synuclein in the urea-free form and after incubation with 8 M urea. (c) Estimated thermodynamic stability of R-synuclein. Dashed line represents the linear fit to the experimental data. The same experiments were repeated three times (for phenomenological proof).

constant given by eq 1. To correlate (∆ΘSPR)Net to the denaturation process of the surface-bound R-synuclein, the Maxwell and Garnett theory was employed here on the basis of neglecting the possible anisotropy of the supported R-synuclein layer.11,24

(eff - N) (eff + 2N)

) βf where β )

(D - N) (D + 2N)

(1)

Here f is the volume fill fraction (that is, the extent of the denaturation) occupied by the denatured R-synuclein and D and N are the dielectric constants of the fully denatured R-synuclein and R-synuclein in the native state, respectively. From the fact that the optical response in SPR is proportional to the average film thickness (provided the change in the thickness of R-synuclein layer is very small), in this sense, it might be convenient to assume that a partially denatured R-synuclein layer is optically equivalent to a fully denatured R-synuclein layer with a fixed dielectric constant and thickness as a variable. On the other hand, as the denaturation proceeds (f in eq 1 increases), a fixed film (24) Gehr, R. J.; Boyd, R. W. Chem. Mater. 1996, 8, 1807.

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Letters

thickness of the supported R-synuclein layer and a variable dielectric constant could be considered to represent the denaturation of the R-synuclein layer induced by urea in more reasonable way. To test these two assumptions, changes in (∆ΘSPR)Net as a function of coverage (f in eq 1) by the denatured R-synuclein for a fixed thickness and for a fixed dielectric constant were calculated (SPR angle shift for both cases (one is the fixed thickness and the other is the fixed dielectric constant) was calculated using the Fresnel equations). The difference in optical responses of these two cases at the same surface coverage by denatured R-synuclein was found to be indistinguishable and (∆ΘSPR)Net increased linearly as function of f. Therefore (∆ΘSPR)Net could be assumed in a reasonable way to be increased linearly as the denaturation process proceeded (data not shown).25 From the data presented in Figure 3a, subject to the above assumption, we were able to calculate the Gibbs free energy change (∆G°) for the denaturation of the surface-confined R-synuclein as shown in Figure 3b.

Kequilibrium )

[(R-synuclein(s))D]equilibrium

)

[(R-synuclein(s))N]equilibrium

fD fD ) (2) fN 1 - f D

where fD and fN represent the fraction of the denatured and the natured state of R-synuclein. The free energy change for denaturation ∆G° was calculated using

∆G° ) -RT ln Kequilibrium

(3)

It should be noted that unequivocal values of ∆G° for the surfacebound R-synuclein can be obtained if the extrapolation region is reduced to 0 M denaturant concentration. From the linear fit, ∆G° for denaturation of the supported R-synuclein was calculated to be 0.51 ( 0.24 kcal/mol, which is lower by 1 or 2 orders of magnitude than that of similar proteins in the free state.4,26 The further linear fit to data is expressed as follows:27

( ( ))

d log

fD /d(log[urea]) ) n ≈ 1.82, 1 - fD (0.3 M e [urea] e 1.5 M) (4)

The slope in the linear fit of eq 4 indicates that approximately two urea molecules participate in the denaturation of the surfaceconfined R-synuclein under the given conditions.13 These calculations indirectly suggest that a significant loss in bioactivity of the supported R-synuclein can be expected, due to restricted conformational plasticity as the result of the multiple binding (amide bond formation) to the surface. To increase the conformational plasticity of the surface-bound R-synuclein, mixed SAMs composed of MUA and 1,6-hexanedithiol (HDT) were used. When the AFM image (Figure 4a) of an area covered with the mixed SAMs prepared from a χMUA/ χHDT ) 1/10 solution was compared with that of the unmixed sample, the distinctive feature of the mixed SAMs in the crosssectional contours is the appearance of numerous small and isolated islands. The size of these islands, which corresponds to the dimensions of the MUA-rich regime, was found to vary with the MUA/HDT composition. The dimensions of the islands are (25) ∆ΘSPR)Net)Urea)x ) fD‚[(∆ΘSPR)Net]Denaturation, where [(∆ΘSPR)Net]Denaturation is the net SPR angle change by the full denaturation of the supported R-synuclein and fD is the fraction of supported R-synuclein in the denaturated status. (26) Lynch, S. M.; Boswell, S. A.; Colon, W. Biochemistry 2004, 43, 1525. (27) We assumed that the general denaturation equilibrium can be expressed as follows: K ) [(R-synuclein(s))D]/[[(R-synuclein(s))N]]‚[urea(aq)]n], thus yielding log(fD/(1 - fD)) ) n log[urea] + log K.

Figure 4. (a) AFM image of the surface of a Au thin film prepared by a mixed MUA/HDT SAM formed from a χMUA/χHDT ) 1/10 solution. Bar corresponds to 1 µm. Solid arrows represent the MUArich regions and dashed arrows correspond to the HDT-rich regions. (b) Gibbs free energy change versus urea concentration, derived from the experimental data (using the mixed SAM, gray colored dots). Black dots correspond to the unmixed case. Insets in (b) represent the possible surface status (dashed vertical lines represent covalent bonds). All SPR measurements were repeated three times.

comparable to those of the phase-separated mixed SAM systems.28 The angle-resolved SPR measurement for surface-bound Rsynuclein on the mixed SAMs gives a ∆G° value for denaturation in water of 2.0 ( 0.5 kcal/mol, about 4 times larger than that for the unmixed sample (Figure 4b). This suggests that, using mixed SAMs, R-synuclein can be immobilized and that the bioactivity of the immobilized molecule is close to that of a bulk solution. Further studies on the determination of optimum conditions for the increase in the biological activity of supported R-synuclein by the mixed SAMs are currently in progress.

Conclusion We have shown that urea-driven denaturation and renaturation of the supported R-synuclein are quite reversible, as evidenced by monitoring the in situ SPR curve, and the differential SPR angle change (∆ΘSPR)Net allowed us to compare the ∆G° for the denaturation of the supported R-synuclein with that of similar proteins in the free state. ∆G° for the denaturation of the surfacebound R-synuclein was estimated to be 0.51 ( 0.24 kcal/mol and could be increased to 2.0 ( 0.5 kcal/mol by the mixed SAMs method. Since R-synuclein shares similar physiological functions with other natively unfolded proteins, we expect that SPR characterization can be also applied to the estimation of the (28) (a) Chidsey, C. E. D. Science, 1991, 251, 919. (b) Satjapipat, M.; Sanedrin, R.; Zhou, F. Langmuir 2001, 17, 7637. (c) Hobara, D.; Takayuki, S.; Imabayashi, S.-I.; Kakiuchi, T. Langmuir 1999, 15, 5073. (d) Chen, S. F.; Li, L. Y.; Boozer, C. L.; Jiang, S. Langmuir 2000, 16, 9287. (e) Shevade, A. V.; Zhou, J.; Zin, M. T.; Jiang, S. Langmuir 2001, 17, 7566. (f) Li, L.; Chen, S.; Jiang, S. Langmuir 2003, 19, 666.

Letters

fundamental properties of these proteins when grafted to a surface. The findings herein suggest that SPR measurements can be extended for the usage of the characterization of supported biomolecules and can serve as an alternative tool in the design of tailored surfaces because we have successfully monitored the denaturation process of even natively unfolded R-synuclein using SPR. Acknowledgment. This work was supported, in part, by grants fromthe Korean Ministry of Environment through the core environmental technology development project for the next generation (Eco-Technopia-21) as well as the Korean Ministry

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of Science and Technology through the Molecular and Cellular BioDiscovery Research Program [M1-0311-00-0028]. This research was also conducted the Engineering Research Institute (ERI) at Seoul National University. Supporting Information Available: Experimental details (including antibody experiment), Schematic SPR configuration, water contact angle data of the mixed SAMs, and AFM images of the mixed SAMs. This material is available free of charge via the Internet at http://pubs.acs.org. LA052276W