Cytochrome c Superstructure Biocomposite Nucleated by Gold

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Biomacromolecules 2005, 6, 3030-3036

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Cytochrome c Superstructure Biocomposite Nucleated by Gold Nanoparticle: Thermal Stability and Voltammetric Behavior Xiue Jiang, Li Shang, Yuling Wang, and Shaojun Dong* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, ChineseAcademy of Sciences, Graduate School of the Chinese Academy of Sciences, Changchun, Jilin, 130022, China Received May 22, 2005; Revised Manuscript Received July 27, 2005

The thermal stability of cytochrome c (cyt c) after Au-nanoparticle-directed association has been studied by various spectroscopic (electronic absorption, resonance Raman, and circular dichroism) and electrochemical methods. The results show that the thermal stability of the Au-cyt c superstructure biocomposite formed by the electrostatic and hydrophobic interactions among the associated proteins increases significantly. It is mainly caused by strong hydrophobicity of the associated cyt c in Au-cyt c superstructure at high temperature, which results from the compact secondary structure and the packing of hydrophobic side chains around the Trp 59 and heme. In addition, the formation of bis-His configuration of heme is facilitated by the tightly self-associated state of cyt c in the Au-cyt c superstructure. The electrostatic coupling of the opposite charges among shells of the adsorbed proteins due to the formation of the superstructure biocomposite can reduce repulsions among the same charges in protein. These factors are also important for enhancing the stability of the associated cyt c. Furthermore, the voltammetric behavior of Au-cyt c at DNA modified glassy carbon electrode has been investigated for extending the application of Au-cyt c. Introduction Recently, many researches have been focused on studying the structure and function of biological molecules adsorbed onto nanoscale materials.1-4 These bioconjugated nanomaterials could produce advances in molecular diagnostics, biosensors, material science, and cell biology.5-11 Biofunctionalized nanoparticles have been used to probe pathogenic bacteria,7 control protein structure and function,8,10 prepare protein gradients,12 and capture target substance at ultralow concentration.13 The most common particles used for protein conjugation are gold and silver nanoparticles. Many properties of bioconjugated complex are distinctly different from those of individual protein molecules due to some unique properties of nanoparticles, such as enhanced stability.14 Using this property, a biocomposite silica aerogel has been prepared successfully.15,16 Nevertheless, the thermal stability mechanism of the bioconjugated nanoparticle complex has never been studied systematically despite wide interest and practical importance of this phenomenon. In this study, we investigated the thermal stability mechanism of cytochrome c (cyt c) after Au-nanoparticle-directed association. Cyt c was used as a model protein because its conformational transitions from native to the unfolded state under the influence of temperature, pH, and ionic strength have been studied extensively.17-21 The effects of polyanions22,23 and macromolecular crowding24-27 on the stability of cyt c also have been established. Based on these results, we investigated the thermal stability of the Au-cyt c * To whom correspondence should be addressed. Fax: 86-431-5689711. E-mail: [email protected].

superstructure biocomposite by electrochemistry and spectroscopy. By comparison of the spectra of RR and CD between free cyt c and Au-cyt c, we discussed the thermal stability mechanism of Au-cyt c. We studied also the electrochemical behavior of Au-cyt c at DNA modified glassy carbon electrode (GCE). This is expected to probe the potential of the Au-cyt c superstructure biocomposite in new applications. Experimental Section Materials and Methods. Horse heart cytochrome c (cyt c, purity 100% based on H2O content 7.8%), purchased from Sigma (used as received without further purification), was prepared in 0.1 M or 10 mM phosphate buffer solution (PBS, pH 7.2). The concentration of the cyt c solution was measured spectrophotometrically using a molar absorptivity of 106 100 mol-1 cm-1 at 410 nm.28 PBS was prepared by Na2HPO4‚12H2O and NaH2PO4‚2H2O (both purities g 99%, Beijing chemical reagents Co.). Doubly purified water was from Milli-Q system. Chloroauric acid trihydrate (HAuCl4‚ 3H2O, g 99%) was purchased from EM Sciences. All of the other chemicals were of analytical grade and were used as received. Preparation of Colloidal Au and Colloidal Au-Cyt c Superstructure Biocomposite. All glassware used was cleaned in a bath of freshly prepared 3:1 HCl:HNO3 and rinsed thoroughly in H2O prior to use. Colloidal Au solution of ca. 18 nm in diameter was prepared by citrate reduction of HAuCl4 as previously described.29 Cyt c solution (100 µL, cyt c 10 mg/mL, in a 0.1 M PBS, pH 7.2) was then

10.1021/bm050345f CCC: $30.25 © 2005 American Chemical Society Published on Web 08/26/2005

Cytochrome c Superstructure Biocomposite

added into the stock colloidal Au solution (1 mL). The mixture was stirred very gently at 4 °C for about 30 min and no separation techniques (i.e., centrifugation or filtration) were employed in order to prevent shear forces from disrupting the soft superstructure.15,16 Thus, the Au-cyt c superstructure biocomposite was prepared, referred as Aucyt c. Adsorption of Cyt c and Au-Cyt c at DNA Modified Glassy Carbon Electrode. The GCE was mechanically polished with 1.0, 0.3, and 0.05 µm R-Al2O3 slurry, successively, and washed ultrasonically in doubly distilled water followed by ethanol for a few minutes at each step and dried. Then, the electrode was modified immediately by transferring 10 µL of DNA solution (3 mg/mL) onto the surface, followed by air-drying overnight. The electrode was then soaked in doubly distilled water for about 4 h to remove any unadsorbed DNA.30 The DNA modified GCE was then obtained, referred as DNA/GCE. The DNA/GCE was scanned over the potential range of -0.2 to +0.4 V at 20 mV s-1 in pH 7.2 PBS containing 1 mg/ml cyt c and Au-cyt c, respectively, until a constant voltammogram was obtained. Then, cyt c or Au-cyt c was immobilized on DNA modified GCE successfully, referred as cyt c/DNA/GCE or Au-cyt c/DNA/ GCE. Instruments and Methods. Cyclic voltammograms were recorded by using a Mode 630 electrochemical analyzer (CH Instrument, U.S.A.) with a homemade electrochemical cell. A pretreated GCE with cyclic scanning in the potential range of 0.0-1.5 V at 100 mV s-1 for 50 cycles in 0.5 M H2SO4 solution, cyt c/DNA/GCE, and Au-cyt c/DNA/GCE was used as the working electrode, a twisted platinum wire was used as the auxiliary electrode, and Ag/AgCl (saturated KCl) was used as the reference electrode. The temperaturedependent electrochemical measurements were controlled by using a water bath system (Varian Co., U.S.A.). A Cary 500 UV-vis-NIR spectrometer (Varian Co., U.S.A.) was used for UV-visible spectra measurements, and the temperature was controlled by a water bath system (Varian Co., U.S.A.). CD spectra were measured by using an AVIV 62A DS circular dichroism spectrometer (AVIV Co., U.S.A.) equipped with a thermoelectric cell holder (AVIV) to control the temperature. A 0.1-cm cuvette was used for UV-visible and CD spectra measurements. RR spectra were measured by a J-Y T64000 laser Raman instrument (German) with a 514-nm excitation line of a Spectra Physics 2017 argon ion laser and a CCD detector cooled by liquid nitrogen. The temperature was controlled by a thermoelectric cell holder (German). The accumulation time was 50 s for each RR spectrum, and the power of the incident laser beam was 20 mW. Atomic force microscopy (AFM) images were obtained by using a Nanoscope IIIa instrument operating in the tapping mode with standard silicon nitride tips. Typically, the surface was scanned at 2 Hz with 256 lines/image resolution and 1.0-2.0 V setpoint. Transmission electron microscopy (TEM) image was obtained by using JEOL 2010 TEM operated at 200 kV. Data Analysis (Singular Value Decomposition Least Squares Analysis (SVDLS)). To ascertain the different

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Figure 1. (A) Electronic absorption spectra of 95 µM cyt c (solid line) and 95 µM Au-cyt c (dotted line) in pH 7.2, 10 mM PBS. (B) AFM tapping mode image of Au-cyt c superstructure biocomposite. (C) TEM image of Au-cyt c superstructure biocomposite.

conformations of cyt c and Au-cyt c presented over a range of the solution temperatures, the CD spectral data were evaluated by SVDLS analysis. In this analysis, several CD spectra were measured with the same m (m ) total wavelengths) wavelengths. The spectra were grouped into series taken under identical conditions except for the changes of a single variable (as the temperature of solution). The SVDLS mathematical and computational details have been reported previously.31, 32 Results Preparation of the Au-Cyt c Superstructure Biocomposite. Figure 1A shows the electronic absorbance spectra of Au-cyt c (dotted line) and free cyt c (solid line) in pH 7.2, 10 mM PBS. As shown, two spectra are nearly superposed, indicating that no denaturation of cyt c is induced upon exposure to the Au colloidal sol. Also, except the characteristic Q-band of cyt c at about 528 nm, we do not see the surface plasmon resonance of gold nanoparticles at about 520 nm from the spectrum of Au-cyt c. This indicates that cyt c is adsorbed on colloidal Au in the form of a multilayer assisted by the weak interactions of proteinprotein;16 that is, cyt c can self-associate into superstructure nucleated by colloidal Au as reported.15,16 AFM (Figure 1B) and TEM (Figure 1C) measurements also have been employed to investigate the formation of the superstructure biocomposite. As can be seen, colloidal Au (some small

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Figure 3. RR spectra of free cyt c (1 mg/mL) and Au-cyt c (1 mg/ mL) in the marker band region between 1300 and 1800 cm-1 obtained from pH 7.2, 10 mM PBS at room temperature (curves a and c) and 85 °C (curves b and d). The spectra were obtained with 514-nm excitation. Inset: the v2 mode of cyt c at room temperature (a) and 85 °C (b).

Figure 2. (A) Potential of reduction Ec versus T plots of free cyt c (solid line) and Au-cyt c (dotted line) at pretreated GCE in pH 7.2, 10 mM PBS. (B) Relative changes of absorbance difference (∆A800-695nm) of free cyt c (solid line) and Au-cyt c (dotted line) as a function of temperature in pH 7.2, 10 mM PBS. Insert: Electronic absorption spectra of 85 µM free cyt c (solid line) and 85 µM Au-cyt c (dotted line) at 85 °C in pH 7.2, 10 mM PBS.

spheres of white shown in AFM and dark shown in TEM) is surrounded by cyt c (grey shell), which means the Aucyt c superstructure biocomposite has been prepared successfully. Stability of Au-Cyt c Superstructure Biocomposite. The thermal stability of Au-cyt c can be gauged by electrochemistry and electronic absorption spectroscopy. Usually, native cyt c yields a one-electron, reversible redox waves with formal potential of 0.07 V in neutral solution of pH 7.0 at room temperature (vs Ag/AgCl in saturated KCl).33 Upon increasing the temperature, the reduction potential (Ec) shifts negatively compared with the formal potential of native cyt c. This is due to the heme group which is no longer well protected by the protein matrix and therefore difficult to be reduced.34-36 Figure 2A shows the temperature-dependent Ec values of free cyt c (solid line) and Au-cyt c (dotted line) at the pretreated GCE. In both cases, the reduction potential decreases linearly when increasing the solution temperature from 20 to 60 °C, but the slope of the Ec versus T plot of Au-cyt c (dotted line) is smaller than that of free cyt c (solid line). This indicates that the associated cyt c is significantly stabilized in Au-cyt c relative to free cyt c. The phenomenon can be verified by the different changes of temperature-induced electronic absorbance spectra of free cyt c and Au-cyt c. The UV-vis absorption spectrum of native cyt c displays the characteristic Soret band at 409 nm, Q-band at about 528 nm, and a very weak band at 695 nm. By increasing the temperature, the absorption band at 695

nm is eliminated due to the disruption of the Met 80-heme iron bond. The stability of native cyt c on heating can be monitored by a loss of the absorption band at 695 nm. Figure 2B shows the temperature-dependent absorbance changes (∆A800-695 nm) of free cyt c (solid line) and Au-cyt c (dotted line) in pH 7.2 PBS. Based on the inflection of the solid line, the midpoint of the thermal transition (Tm) for free cyt c is about 55 °C, whereas it shifts to 57.2 °C for Au-cyt c. Concomitant with the decrease of the 695 nm band in intensity, the Soret band at 409 nm shows a blue-shift with increasing the solution temperature to 85 °C. Apparently, it shifts down to 405 nm for free cyt c (inset, solid line), and only to 407 nm for Au-cyt c (inset, dotted line), suggesting that the different microenvironments of heme are induced from free cyt c and Au-cyt c upon heating. This phenomenon allows us to study the thermal stability mechanism of Au-cyt c by using RR and CD spectra since they are sensitive to the heme configuration and the protein conformation. RR and CD Spectroscopy Studies. Figure 3 shows the RR spectra of free cyt c (1 mg/mL) and Au-cyt c (1 mg/ mL) in 10 mM PBS (pH 7.2) at room temperature (curves a and c) and 85 °C (curves b and d), respectively. It can be seen that both free cyt c and Au-cyt c show main RR bands of V4, V11, V2, and V10 at 1370, 1564, 1585, and 1637 cm-1 at room temperature, except for the slight enhancement of the Raman signal of Au-cyt c (curve c) relative to that of free cyt c (curve a). In addition, the relative intensity of V10/V2 of Au-cyt c (curve c) is lower than that of free cyt c (curve a) due to the weak interactions of protein-protein among the associated cyt c.17 By increasing the solution temperature to 85 °C, V11 modes of both free cyt c and Au-cyt c disappear mostly with decreasing the intensity of the V10 band (curves b and d). Also for the V2 mode, it shows apparent broadening and downshifting accompanied with an appearance of a distinct shoulder at 1589 cm-1 for free cyt c (inset), whereas it is nearly unchanged for Au-cyt c (curve d). Such a

Cytochrome c Superstructure Biocomposite

Figure 4. (A) Near-UV CD spectra of 0.09 mM free cyt c at 25 °C (a), 59 °C (b), and 85 °C (c) in pH 7.2, 10 mM PBS. (B) Near-UV CD spectra of 0.1 mM Au-cyt c at 25 °C (a), 59 °C (b), and 85 °C (c) in pH 7.2, 10 mM PBS.

difference indicates that different configurations of heme are induced when the temperature is increased for free cyt c and Au-cyt c. These changes of the heme structure are accompanied with tertiary structure changes of cyt c, which can be revealed by CD spectra in the regions of the near-UV and the Soret bands. As shown in Figure 4, panels A and B, the near-UV CD spectra of free cyt c and Au-cyt c exhibit two sharp minima at 282 and 288 nm, respectively, in pH 7.2 PBS (curves a in panels A and B of Figure 4). An increase of the solution temperature to 59 °C leads to an apparent decrease of these two peaks in intensity for free cyt c (curve b of 4A), whereas they remain nearly unchanged for Au-cyt c (curve b of 4B). Further increasing the solution temperature to 85 °C results in a continuous decrease in intensities of these two peaks accompanied by 3 nm red-shift for free cyt c (curve c of Figure 4A) and a decrease of the peak at 282 nm for Au-cyt c (curve c of Figure 4B). These differences reflect different changes of tertiary structure of free cyt c and Au-cyt c. Figure 5 shows the temperature-dependent Soret CD spectra of free cyt c (A) and Au-cyt c (B) in pH 7.2 PBS. The Soret CD spectra of free cyt c and Au-cyt c display an S-shaped feature with a minimum and maximum at about 418 and 402 nm at room temperature, respectively (curves a in panels A and B of Figure 5). Upon increasing the solution temperature to 59 °C, the intensity of the negative band decreases about 51% and 43% for free cyt c (curve b of Figure 5A) and Au-cyt c (curve b of Figure 5B). When the temperature is further increased to 85 °C, the intensity of the positive band increases about 120% and

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Figure 5. (A) Soret CD spectra of 0.09 mM free cyt c at 25 °C (a), 59 °C (b), and 85 °C (c) in pH 7.2, 10 mM PBS. (B) Soret CD spectra of 0.1 mM Au-cyt c at 25 °C (a), 59 °C (b), and 85 °C (c) in pH 7.2, 10 mM PBS.

105% for free cyt c (curve c of Figure 5A) and Au-cyt c (curve c of Figure 5B). These differences indicate that, under the effect of Au colloid, the associated cyt c shows different changes in heme microenvironment upon heating. Figure 6 shows the temperature-dependent far-UV CD spectra of free cyt c (A) and Au-cyt c (B) in pH 7.2 PBS. It can be seen that the negative Cotton peaks of both free cyt c (A) and Au-cyt c (B) decrease gradually when increasing the solution temperature from 25 to 85 °C. Analysis of the far-UV CD spectra of Figure 6, panels A and B, using the SVDLS program shows that three secondary structures are changed for free cyt c and Au-cyt c, respectively, as shown in panels A′ and B′ of Figure 6. According to the Gauss-Markoff mode of protein secondary structure CD spectra and comparing with the CD spectra of 27 membrane proteins, curves a∼c in panels A′ and B′ of Figure 6 are attributed to R-helix, RT-helix, and random coil and R-helix, RT-helix, and β-sheet, respectively.37,38 The temperature-dependent fractional changes of each secondary structure obtained by SVDLS analysis are listed in Table 1. As shown in Table 1, the fraction of R-helix decreases, and the fractions of RT-helix and random coil increase for free cyt c when its solution is heated from 25 to 85 °C. This implies that the conformational transitions are mainly from R-helix to RT-helix and random coil, which results in an incompact conformation. As for Au-cyt c, although the fraction of R-helix also decreases upon increasing the solution temperature, its content is higher than that of free cyt c. Moreover, the decrease of R-helix is accompanied with the increase of RT-helix and β-sheet. This indicates that the conformational changes are from R-helix to RT-helix and

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Figure 6. (A) Temperature-linked far-UV CD spectra of 0.09 mM cyt c in 10 mM PBS solution, temperature values from (a-i) are 25, 34, 49, 54, 59, 64, 69, 74, and 85 °C. (A′) Spectra of three temperature-dependent conformations of cyt c obtained from panel A by SVDLS analysis; (a-c): R-helix, RT-helix, and random coil. (B) Temperature-linked far-UV CD spectra of 0.1 mM Au-cyt c in 10 mM PBS solution, temperature values from (a-k) are 25, 34, 39, 44, 49, 54, 59, 64, 69, 74, and 85 °C. (B′) Spectra of three temperature-dependent conformations of Au-cyt c obtained from panel B by SVDLS analysis; (a-c): R-helix, RT-helix, and β-sheet. Table 1. Fractions for the Conformations of Free Cyt c and Au-Cyt c at 25 °C and 85 °C Obtained by SVDLS Analysis cyt c R-helix RT -helix random coil β-sheet

Au-cyt c

25 °C

85 °C

25 °C

85 °C

0.60 0 0.10

0.15 0.30 0.30

0.62 0

0.18 0.30

0.03

0.20

n∆Ep > 200 mV, the electron-transfer rates, ks, of cyt c and Au-cyt c on a DNA film are estimated to be 1.2 and 2.3 s-1, respectively. These results indicate that Au colloid can affect the electrochemical behavior of cyt c as an efficient electron-conducting tunnel in the Au-cyt c biocomposite. This property enables the Au-cyt c biocomposite to become an attractive material platform for applications in biosensors. Discussion

β-sheet, which is different from that in free cyt c (to random coil). Thus, the conformational state of cyt c after Aunanoparticle-directed association is more compact than that of free cyt c at high temperature. Voltammetric Behavior of Au-Cyt c Adsorbed on DNA-Modified GCE. Figure 7A shows cyclic voltammograms of cyt c/DNA/GCE (solid line) and Au-cyt c/DNA/ GCE (dotted line) in 10 mM pH 7.2 PBS at 20 mV s-1. It can be seen that both modified electrodes lead to a pair of well-defined redox peaks. The formal potential and the peakto-peak separation of the redox reaction at cyt c/DNA/GCE are 0.04 and 0.036 V, respectively, whereas those at Aucyt c/DNA/GCE are 0.05 and 0.014 V, indicating that Aucyt c shows more reversible electrochemical behavior. When the scan rates are increased, the oxidation and reduction peaks shift to more positive and negative potentials for both modified electrodes. The anodic and cathodic peak potentials are linearly dependent on the logarithm of scan rates (ν) when n∆Ep > 200 mV, (Figure 7, panels B and C), which is in agreement with the Laviron theory.39 Based on the Laviron’s approach for diffusionless thin layer voltammetry with

As indicated by electrochemistry (Figure 2A) and spectroscopy (Figure 2B) studies, the Au-cyt c biocomposite shows more stability upon increasing the solution temperature. The midpoint of the thermal transition (Tm) shifts from 55 to 57.2 °C. This result is parallel to that reported by Rolison’s group.15,16 They studied the stabilization of Aucyt c by fluorescent monitoring of the degree of unfolding induced by guanidinium hydrochloride (GuHCl).15 They found that the midpoint for unfolding of cyt c shifted from 4.9 M GuHCl to 5.1 M. Although they have reported that the external protein could act as the “skin” to protect the internal protein at the extreme condition in such a proteinprotein superstructure,15 its mechanism has not been identified clearly. The Soret band of native cyt c at 409 nm originates from the π-π* transitions of the porphyrin ring. Increasing the solution temperature results in a small blue-shift (2 nm) of the Soret-band absorption of Au-cyt c (Figure 2B, inset, dotted line) relative to that of free cyt c (4 nm, Figure 2B, inset, solid line), suggesting that different states of heme are

Cytochrome c Superstructure Biocomposite

Figure 7. (A) Cyclic voltammograms of cytc/DNA/GCE (solid line) and Au-cyt c/DNA/GCE (dotted line) in 10 mM pH 7.2 PBS. Scan rate: 20 mV/s. (B) Laviron plot for cyt c/DNA/GCE in 10 mM pH 7.2 PBS. (C) Laviron plot for Au-cyt c/DNA/GCE in 10 mM pH 7.2 PBS.

induced. The temperature-induced 2 nm blue-shift in the Soret band of Au-cyt c is similar to that induced by addition of 2 mM sodium dodecyl sulfate (SDS) or 6 M GuHCl at pH 7.0, and it is also analogous to that induced by binding to phospholipid vesicles (DOPG) at a lipid/cyt c ratio of 50: 1.17 This implies that a similar configuration of heme may be induced. It can be further proved by the RR spectra. The characteristic bands of V4, V11, V2, and V10 at 1370, 1564, 1585, and 1637 cm-1 for free cyt c and Au-cyt c (Figure 3, curves a and c) reflect that cyt c is in a native state with six-coordinated low-spin (6cLS) configuration,17,40 in which cyt c is axially coordinated by His 18 and Met 80. The temperature-induced disappearance of V11 mode with a decrease in intensity of V10 band for Au-cyt c (Figure 3, curve d) is also similar to the phenomena when cyt c interacting with GuHCl, SDS, and DOPG liposomes.17 Such an excellent agreement of the UV-vis absorption and the RR spectra suggests the similar state of heme. In Au-cyt c, nonnative 6cLS configuration should be induced as those reported conditions, in which the axial ligands of cyt c are His 33 and His 18,17 whereas for free cyt c, the additional changes of V2 mode indicate that one or more other nonnative species are formed.17 However, we can only infer tentatively that it may be a six-coordinated high-spin configuration, in

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which His 18 and H2O act as axial ligands, or fivecoordinated high-spin configuration, in which only His 18 acts as an axial ligand.17 Furthermore, we are not sure which configuration it should be according to our results. However, we can still consider that the His 33 ligand is an energetically favored species in Au-cyt c biocomposite and such a bisHis configuration of heme can enhance the thermal stability of the associated cyt c. The near-UV CD spectrum of native cyt c characterized by two negative Cotton effects at 282 and 288 nm indicates a dense packing of tertiary structure in the vicinity of Trp59.17-19 Such dense packing of the tertiary structure is lost partly in free cyt c at 59 °C as indicated by the decrease of these two peaks in intensity (Figure 4A, dotted line), whereas it is not influenced in Au-cyt c (Figure 4B, dotted line) at the same temperature. When the temperature is increased up to 85 °C, such dense tertiary structure is lost mostly due to the large decrease of these two peaks in intensity and the red-shift in free cyt c (Figure 4A, dashed line), whereas in Au-cyt c, it is only disturbed slightly as revealed by the presence of the negative Cotton effect at 288 nm (Figure 4B, dashed line). Such a large difference suggests that the packing of the hydrophobic side chains around the Trp 59 plays a dominant role in stabilizing the native state of cyt c. The difference in the Soret CD spectra between free cyt c and Au-cyt c (Figure 5) reflects the role of the tertiary structure around the heme in stabilizing the native state of cyt c. The Soret CD spectrum originated from the coupling of the electronic transition dipole moments of the heme and nearby aromatic amino acids is considered as a fingerprint for the integrity of the heme pocket.17,18,23 The negative Cotton effect at 418 nm has been ascribed to the Phe-82 and Met-80-heme interaction, and the positive Cotton peak reflects the states of axial heme bonds.17,18,23 Increasing the solution temperature leads to an increase of the distance between the residues Phe 82 and Met 80-heme group.18 In Au-cyt c, the increase of the distance is smaller than that in free cyt c as assessed by the small decrease of the negative Cotton peak (Figure 5). So the interaction between the heme and the residues 70-85 polypeptide segment in Au-cyt c is stronger than that in free cyt c, which is also important in stabilizing the native state of cyt c. The study of temperature-induced far-UV CD spectra of cyt c (Figure 6A) and Au-cyt c (Figure 6B) indicates that a more compact conformation of the associated cyt c in Aucyt c biocomposite is induced relative to that of free cyt c at high temperature. Such a compact conformation leads to the increase of the hydrophobic nature of the associated cyt c,4 which decreases the degree of exposure of the protein to solution. The temperature effect on the internal microenvironment of protein is thus decreased. So the thermal stability of Au-cyt c can be enhanced inevitably. In the Au-cyt c superstructure model, the first layer of cyt c is adsorbed by electrostatic interactions between positively charged cyt c and negatively charged colloidal Au, in which the heme plane is slightly inclined relative to the surface of colloidal Au.41 Such an orientation will facilitate electrostatic contacts between two protein surfaces by coupling the areas with opposite charges.41 According to our

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results, hydrophobic interactions also play an important role. Both the electrostatic and hydrophobic interactions induce the organization of the second shell of proteins, resulting in further assembly that almost no unassociated cyt c is left in the buffer. Of course, a high radius of curvature formed by each shell of protein offers a special adsorption platform.15 Due to the electrostatic coupling of opposite charges among the proteins in such a superstructure, repulsions resulting from the same charges in protein can be reduced.23,42,43 Therefore, the conformational changes of cyt c induced by repulsions among the same charges in protein can be restrained. Thus, the associated cyt c becomes more stable at the extreme conditions. This also suggests that the selfassociation of protein molecules may contribute to the stability of protein. A similar conclusion has been drawn by Ping et al. when they studied the effect of macromolecular crowding on the stability of protein.24 Conclusions In conclusion, the thermal stability mechanism of the Aucyt c superstructure biocomposite formed by weak hydrophobic and electrostatic interactions of cyt c-cyt c and its voltammetric behavior at the DNA modified electrode were studied by UV-vis, RR, CD spectroscopy ,and electrochemistry. The main points of this study were summarized as follows: 1. The temperature-induced bis-His configuration of heme in the Au-cyt c superstructure biocomposite can increase the stability of the associated cyt c. 2. The high hydrophobic nature of the associated cyt c resulted from the compact secondary structure, and the packing of hydrophobic side chains around the Trp 59 and heme at high temperature provides the strongest contribution to the thermal stability of the Au-cyt c superstructure biocomposite. 3. The electrostatic coupling among shells of proteins with the opposite charges in Au-cyt c decreases the repulsions among the same charges in protein and thus increases the thermal stability of the associated cyt c. 4. The electron-transfer rate, ks, of Au-cyt c at DNA modified GCE is higher than that of free cyt c under identical conditions. Acknowledgment. This work was supported by the National Natural Science Foundation of China (Nos. 20275037, 20275036, 20210506). References and Notes (1) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Langmuir 2004, 20, 11594-11599. (2) Vertegel, A. A.; Siegel, R. W.; Dordick, J. S. Langmuir 2004, 20, 6800-6807. (3) Lundqvist, M.; Sethson, I.; Jonsson, B.-H. Langmuir 2004, 20, 10639-10647. (4) Jiang, X.; Jiang, J.; Jin, Y.; Wang, E.; Dong, S. Biomacromolecules 2005, 6, 46-53. (5) Lavan, D. A.; Lynn, D. M.; Langer, R. Nat. ReV. Drug DiscoVery 2002, 1, 77-84.

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