Biofunctionality of Calmodulin Immobilized on Gold Surface Studied

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Biofunctionality of Calmodulin Immobilized on Gold Surface Studied by Surface Enhanced Infrared Absorption Spectroscopy - Ca Induced Conformational Change and Binding to a Target Peptide 2+

Hidenori Noguchi, Tatsuhiko Adachi, Akiko Nakatomi, Michio Yazawa, and Kohei Uosaki J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b12724 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 8, 2016

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The Journal of Physical Chemistry

Biofunctionality of Calmodulin Immobilized on Gold Surface Studied by Surface Enhanced Infrared Absorption Spectroscopy - Ca2+ Induced Conformational Change and Binding to a Target Peptide Hidenori Noguchi,*,†,‡,§ Tatsuhiko Adachi,ǁ Akiko Nakatomi,# Michio Yazawa,# and Kohei Uosaki*,†,‡,§

† International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), Tsukuba 305-0044 Japan. ‡ Global Research Center for Environment and Energy based on Nanomaterials Science (GREEN), NIMS, Tsukuba, 305-0044, JAPAN. § Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, 0608628, JAPAN.

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ǁ Physical Chemistry Laboratory, Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. # Bioorganic Chemistry Laboratory, Division of Cellular Life Science, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan

Corresponding Authors *Hidenori Noguchi Tel: +81-29-860-4841 e-mail: [email protected] *Kohei Uosaki Tel: +81-29-860-4301 e-mail: [email protected]


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ABSTRACT Calcium ion (Ca2+) induced functional expression of calmodulin (CaM), which is a signal transmitter in living cell, is known to be associated with its conformation change. However, it is difficult to correlate the conformational change and functional expression induced by Ca2+ reversibly as it is not easy to change the Ca2+ concentration while keeping the CaM concentration in solution. This can be done if CaM is immobilized on a solid surface while maintaining its function intact. Here CaM is immobilized on a self assemble monolayer (SAM) of Ni-nitrilotriacetic acid (NTA) on a Au surface by histidine tag (His-tag) method. The conformational change of the immobilized CaM induced by the Ca2+ concentration change in solution was investigated using surface enhanced infrared absorption spectroscopy (SEIRAS). The Ca2+ induced conformational change of CaM immobilized on the Au surface was followed by monitoring IR intensities at 1550 and 1645 cm-1, which originated from the Ca2+ binding site (EF hand) in CaM and the central helix portion in CaM, respectively. The conformation of CaM was confirmed to be changed reversibly when the concentration of Ca2+ was increased and decreased between 10-5 and 10-3 M in a phosphate buffered D2O solution. The isotherm for immobilized CaM with respect to the Ca2+ concentration is in good agreement with that in solution, which confirms that the activity of CaM is maintained when immobilized on the surface. The interaction between CaM and a target peptide, mastoparan (MP), was also investigated using SEIRAS. Although MP was reversibly bound to and released from the CaM immobilized on the Au surface depending on the presence and absence of Ca2+, respectively, the dissociation constant of CaM immobilized on Au surface with MP was determined from the MP concentration dependence of attachment of MP as two orders of magnitude larger than that of CaM in homogeneous environment, possibly due to the difficulty for MP to access the binding site of CaM, which is immobilized on the surface with high order.



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1. Introduction Calcium ions (Ca2+) plays an important role in signal transduction pathways in cells and it is known that they act as second messengers, such as in neurotransmitters, in contraction of muscle, and also in biological clocks.1-2 These functions are the result of binding to specific proteins and calmodulin (CaM) is a ubiquitous and versatile Ca2+ binding protein. CaM is a small protein composed from 148 amino residues, which consist of two spherical domain connected by a long α-helix domain so called “central helix”. Each spherical domains contain two Ca2+ binding sites so-called “EF-hand”.3-4 When the intracellular Ca2+ level rises to 10-5 M, four Ca2+ ions bind to CaM, leads to a large conformational change of CaM and exposure of the hydrophobic central helix to the surrounding water.3-4 Finally, the Ca2+-CaM complexes to an enzyme, i.e. target protein, which initiates various signaling. One of the important Ca2+ signaling systems in the human body is synaptic activity in the brain, by which Ca2+ signals propagate into the nucleus by synapse-to-nucleus communications.5-6 A high content of CaM is confirmed and has been isolated from brain.5-6 Thus, the behavior of CaM and the Ca2+-CaM complex should be understood at a molecular level to reveal many unknown issues, not only in brain function but also in many Ca2+ signaling systems. CaM is a multifunctional protein responsible for the activation of various enzyme systems: therefore, it is important to understand its behavior in the presence and absence of Ca2+. Ca2+ induced conformational changes of CaM have been studied extensively using circular dichroism (CD),7-8 fluorescence,9 X-ray scattering10, and NMR.11 Characterization of the CaM structure and its functions with Ca2+ and target peptides have been studied mostly in homogeneous system.12-13 However, it is difficult to study the Ca2+ induced functional expression, in homogeneous systems, particularly the deactivation process of CaM caused by the reduction


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of Ca2+ concentration in solution, because once Ca2+ ions are added into the solution it is impossible to remove only Ca2+ from the system. This process could be investigated more easily by simply exchanging the solution if CaM could be immobilized on the surface with its native function intact. Many biomolecules including proteins have been reported to be immobilized on surfaces with controlled orientation while maintaining their structure and functionality14-16 by utilizing the formation of ternary metal-chellate complexes through the interaction of nitrotriacetic acid (NTA) and histidine-tagged (His-tag) biomolecules and/or exploition of the streptavidin/biotin interaction. Various surface analytical techniques such as surface plasmon resonance (SPR),17-18 quartz crystal microbalance (QCM),19 and total internal reflection fluorescence (TIRF) microscopy20 have been applied to clarify the relationship between the conformation and function of biomolecules at biointerfaces at the molecular level. However, these techniques cannot distinguish specifically adsorbed molecules from the non-specifically adsorbed one. Surface vibrational spectroscopy such as infrared absorption spectroscopy,21-23 Raman spectroscopy,24-25 and sum frequency generation (SFG) spectroscopy26 are the most suitable to obtain information on intra- and inter-molecular interactions between molecules and substrates. In this study, CaM was immobilized on a Au surface using the His-tag method, so that the orientation of CaM is highly controlled and the native conformation is maintained to keep the biofunctions at the surface. Surface enhanced infrared spectroscopy (SEIRAS) was employed to study the Ca2+ induced conformation change of CaM and the biofunctionality of CaM with a target peptide, mastoparan (MP), which is one of the frequently used model peptide to study binding interactions with CaM.



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2. Experimental Section 2.1 Synthesis and purification of His-tag CaM The cDNA fragment corresponding to the coding region of CaM was cloned from the scallop, Patinopecten yessoensis, testis cDNA library.27 After addition of two restriction sites for NspV and SacI at the 5’- and 3’-terminus, respectively, by polymerase chain reaction (PCR) amplification, the cDNA fragment was cloned into the NspV/SacI restriction site of plasmid pET30-b(+).The resulting plasmid, named pET-HisCaM, encodes the 27 residues of the tag sequence, containing a (His)6 segment at N-terminus, followed by the complete sequence of CaM (148 amino acid residues, 17 kDa).28 Nucleotide sequences were confirmed on an ABI 310 automated sequencer using the BigDye terminator cycle sequencing kit (Applied Biosystems). Escherichia coli strain BL21 (DE3) was transformed with pET-HisCaM, and the recombinant CaM protein (His-CaM) was over-expressed. The expressed His-CaM was purified according to a previously described method.29. 2.2 Immobilization of CaM on Au surface A thin Au film was formed on a flat surface of hemispherical shaped Si prism (Pier Optics) by an electro-less deposition technique.30 Au surface was cleaned by cycling the potential between -0.2 and 1.2 V at 100 mV/s in an Ar-saturated sulfuric acid solution more than 10 times using a three-electrode electrochemical cell with a Pt wire counter electrode and a Ag/AgCl (sat. KCl) reference electrode.30


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Scheme 1 shows the steps for attachment of His-tag CaM onto the Au surface by the affinity toward Ni-nitrilotriacetic acid (Ni-NTA). (i) The Au coated Si prism was immersed into a D2O solution of 1 mg/ml 3,3'-dithiobis[N-(5-amino-5-carboxypentyl) propionamide-N',N'diacetic acid] dihydrochloride (dithiobis-C2-NTA, Dojindo) to prepare an NTA terminated surface. (ii) The NTA terminated Au surface was immersed into a D2O solution of 50 mM NiCl2 (Wako) for 10 min to ligate Ni2+ ions by the three carboxylates and the tertiary amine of NTA. Incorporation of Ni2+ within the membrane was confirmed by X-ray photoelectron spectroscopy (XPS) measurements. Two D2O ligands are supposed to be bound to Ni center, because Ni complexation was performed in D2O solution and there exist two binding sites on Ni center for D2O. (iii) After rinsing with D2O, a D2O solution of 500 mM imidazole was added to the NiNTA modified Au surface to prepare an imidazole capped Ni-NTA surface. (iv) His-tag 10 µM CaM in phosphate buffered D2O solution (pH=7.4) was added to the surface to form the final CaM immobilized Au surface by the exchange reaction of imidazole with His-tag CaM.

Scheme 1. Surface modification of His-tag CaM on a Au surface.



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2.3 Surface enhanced infrared absorption spectroscopy (SEIRAS) measurements SEIRA spectra were recorded using a BioRad FTS30 spectrometer equipped with a HgCdTe (MCT) detector at a resolution of 2 cm-1 and light with p-polarization light. The Si prism was set into a spectrocell made by Kel-F and the incident angle of the IR beam into the prism was 60 degree. 80 or 512 interferograms were co-summated for each spectrum for in situ or ex situ measurements. To avoid the strong absorption peak from water bending and stretching mode, SEIRAS measurements were carried out in phosphate buffered D2O solution at room temperature (ca. 22 ºC). All the experiments were carried out at several times to check the reproducibility and only the reproducible data are shown. 3. Results and discussion 3.1 Chemical modification of Au with CaM Surface modification steps to build up a CaM immobilized surface were monitored using SEIRAS. Figure 1 shows the SEIRA spectra obtained at each of the surface modification steps shown in Scheme 1 for C=O (1475-1800 cm-1) and C-H (3000-3300 cm-1) stretching region. Fig. 1(a) shows the spectrum after Au surface was immersed in 1 mg/ml dithiobis-C2-NTA solution. Three peaks centered at 1477 cm-1, 1631 cm-1, and 1732 cm-1 were observed in the low wavenumber region. These peaks were assigned to C=O symmetric and antisymmetric stretching of carboxylate anion and C=O stretching of carboxyl group, respectively which suggest that the dithiobis-C2-NTA modified surface was terminated with both protonated and deprotonated carboxylate groups.31-33 No peaks were observed in C-H region. Fig. 1(b) shows the SEIRA spectrum after surface was treated with 50 mM NiCl2 solution. A broad positive peak centered around 1625 cm-1, which corresponds to the C=O antisymetric stretching of carboxylates group,


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and a negative peak at 1735 cm-1 corresponding to protonated carboxyl group complexed with Ni was observed. A broad positive peak at 1625 cm-1 is assigned to the C=O antisymetric stretching of Ni-compleaxed carboxylates group and a negative peak at 1735 cm-1 is due to the decrease of NTA carboxyl group during Ni-complex formation. These results suggest that the terminal carboxyl groups on dithiobis-C2-NTA SAM were changed into carboxylates by Ni2+ cation coordination. No peaks were observed in C-H region. Fig. 1(c) shows the SEIRA spectrum after surface was treated as 500 mM imidazole solution. No peaks were observed in C=O region, although C-H stretching peak of imidazole was observed at 3144 cm-1, which suggest that NiNTA was capped with imidasole. Fig. 1(d) shows the SEIRA spectrum after His-tag CaM was introduced. Two peaks at 1575 and 1646 cm-1 were observed which correspond to the amide II (C=N stretch coupled to N-H bending vibration) and I (C=O stretching vibration) bands of the CaM, respectively. No peaks were observed in C-H region.

Figure 1. SEIRA spectra after each modification step shown in Scheme 1. (a) dithiobis-C2-NTA modification (Reference spectrum: bare Au), (b) NiCl2 treatment (Reference spectrum: (a)), (c) imidazole treatment (reference spectrum: (b)), and (d) His-tag CaM modification (Reference spectrum :(c))



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The His-tag CaM immobilization processes were monitored by in situ time dependent SEIRAS measurements as sown in Figure 2. Two peaks that correspond to CaM were increased with time (Fig. 2(a)). The kinetics for the exchange reaction between imidazole and CaM is depicted in Fig. 2(b), where the intensity of the amide I band (1646 cm-1) is plotted against the reaction time. The intensity became saturated at ca. 60 min. Saturated CaM modified Au substrates were then used to study the biomolecular function of immobilized CaM on the Au substrate.

Figure 2. (a) Time dependent SEIRA spectra of the Au surface during the His-tag CaM modification in buffer solution containing 10 mM His-Tag CaM. (Reference spectrum: bare Au). (b) Time dependence of the peak height at 1643 cm-1.


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3.2 Probing the Ca2+ binding functionalities of surface immobilized CaM Figure 3 shows the SEIRA spectra for the Au surface modified with CaM as the Ca2+ concentration was increased from 10-7 to 10-3 M and then decrease again to 10-7 M. The reference spectrum was measured in Ca2+ fee phosphate buffer solution. Thus the positive peaks in the difference spectra correspond to an increase in intensity in the raw spectra, and vice versa. When Ca2+ was added to the buffer solution, a broad positive peak at 1646 cm-1 accompanied with two negative peaks at 1655 and 1680 cm-1 were observed in the amide I region. The negative and positive peak intensity increased with the Ca2+ concentration up to 10-3 M and then decreased with the Ca2+ concentration down to 10-7 M. Infrared studies on Ca2+ binding to CaM in homogeneous system have been reported by Trewhellar et al.12, Jackson et al.34, and Nara et al.35 Bands observed in the present region (1500-1750 cm-1) in Fig. 3 are correlated to the local environments of the carboxylate groups in the protein molecules. SEIRA spectra feature was almost the same as reported by Trewhella et al.12 According to their assignments, 1655 and 1680 cm-1 peaks are originated mainly from the carboxylate bond in α-helix and turns or bends structures, respectively, which exist in CaM.36 After CaM was bound with Ca2+, CaM will change their conformation and expose their hydrophobic central helix region to the solvent i.e., water.37 The hydrogen bond interaction between CaM and water will cause a low wavenumber shift of carboxylate bond peak, which appear as a broad positive peak at 1646 cm-1 after Ca2+ was added to the buffer solution. Such interactions with water would weaken these carboxylate bonds, thus lowering their vibrational frequencies.



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Figure 3. Ca2+ concentration dependent SEIRA spectra of CaM immobilized on the Au surface. (a) 10-7 M, (b) 10-6 M, (c) 10-5 M, (d) 10-4 M, (e) 10-3 M, (f) 10-4 M, (g) 10-5 M, (h) 10-6 M, and (i) 10-7 M. Experiments were conducted from (a) to (i). (Reference spectra: CaM modified Au surface measured in Ca2+-free solution.) Spectral change was also observed in amide II regions. A broad positive peak at 1550 cm1

and two negative peaks at 1570 and 1588 cm-1 were also observed. 1575 and 1588 cm-1 peaks

observed in amide II regions were close to the carboxylate bond of glutamic and aspartic acid side chains, respectively.12, 38 We think these two negative peaks which originally exist in Ca2+ free CaM shifted to low wavenumber and appeared as the positive peak at 1550 cm-1, which may assign to carboxylate groups which shifted to low wavenumber due to Ca2+ binding in the


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EF hands.12 Carboxylate groups can coordinate to Ca2+ and such interactions would also weaken these carboxylate bonds, thus lowering their vibrational frequencies. Figure 4 shows the change in absorbance at 1550 cm-1 against the log of the free Ca2+ concentration in buffer solution. The solid line in Fig. 4 represents the observed binding isotherm of Ca2+ reported by Crouch et al. in a homogeneous environment.13 The absorbance change against the Ca2+ ion concentration was in good agreement with the change in the number of Ca2+ ions in CaM. Clear reversibility of the ∆absorbance change against the Ca2+ concentration was also observed. From these results, it was concluded that the peak observed at 1550 cm-1 is related to the Ca2+ binding site, i.e. the EF hand, and CaM immobilized on the Au surface maintains its function to respond Ca2+ in homogeneous system.

Figure 4. Change in absorbance at 1550 cm-1 against the log of Ca2+ concentration in the buffer solution. The solid line is the binding isotherm for Ca2+ in a homogeneous environment reported by Crouch et al.13 3.3 Affinity of CaM immobilized on substrate to MP Figure 5 shows SEIRA spectra for the Au surface with and without modification by CaM measured in buffer solution containing Ca2+. A strong broad peak centered around 1653 cm-1 was observed when 120 µM MP was introduced to the Ca2+-activated CaM-modified surface (Fig.



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5(a)). According to the peak deconvolution, this broad peak has two components centered at 1653 and 1638 cm-1. The peak component at 1653 cm-1 originates from C=O hydrogen bonded with N-H in the α-helix structure and the 1638 cm-1 component originate from C=O hydrogen bonded with the surrounding solvent.39 The 1653 cm-1 component is almost twice as large as that at 1638 cm-1; therefore, the dominant conformation of MP bound to CaM is in the α-helix structure, as previously reported for the homogeneous system.40 When 120 µM MP was introduced to the surface without CaM modification, a broad peak centered around 1647 cm-1 was observed. From the spectrum deconvolution, this broad peak was also fitted by two components centered at 1653 and 1638 cm-1 (Fig. 5(b)). The peak areas of these two peaks were almost the same, which suggests that MP is nonspecifically adsorbed onto the substrate and interacts with the surrounded solvent.

Figure 5. SEIRA spectra of the Au surface (a) with and (b) without modification by CaM measured in a buffer solution containing 10-4 M Ca2+ and 120 µM MP. (Reference spectra: (a) CaM modified Au surface in a MP-free buffer solution containing 10-4 M of Ca2+. (b) Imidazole capped Ni-NTA surface in a MP-free buffer solution containing 10-4 M of Ca2+.


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Figure 6. SEIRA spectra of the Au surface (a) with and (b) without modification by CaM measured in a buffer solution containing 10-4 M Ca2+. (Reference spectra: (a) CaM modified Au surface in a MP-free buffer solution containing 10-4 M of Ca2+. (b) Imidazole capped Ni-NTA surface in a MP-free buffer solution containing 10-4 M of Ca2+.

One of the advantages with the immobilization of CaM on the surface is that the desorption process of target protein can be easily studied by the deactivation of CaM by simply reducing the concentration of Ca2+ in buffer solution. This procedure cannot be conducted in a homogeneous system. Figure 6 shows SEIRA spectra for the Au surface with and without modification by CaM measured in Ca2+-free buffer solution. When Ca2+ was eliminated from the buffer solution, the intensity of the peak at 1653 cm-1 was decreased in the case of the CaM modified surface, which suggests that MP was dissociated from CaM by deactivation (Fig. 6(a)). However, in the case of the surface without CaM modification, almost no spectra change was



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observed with Ca2+ elimination (Fig. 6(b)), which also supports the non-specific adsorption of MP onto the surface without CaM modification. To study the interaction between CaM and MP, the dependence of the absorbance change at 1653 cm-1 on the MP concentration, which corresponds to MP bound to CaM, was monitored against time by the addition of buffer solution containing MP onto the Ca2+-activated CaM modified surface, as shown in Figure 7. The spectrum obtained in phosphate buffer containing Ca2+ without MP was used as a reference. When a small amount of MP (300 nM) was added, no significant change in absorbance was observed, however, when more than 3 µM MP was added, the absorbance gradually increased and reached a constant value at each MP concentration, which suggests that all activated CaM was bounded with MP. Finally, when CaM was deactivated by replacing the solution with Ca2+-free buffer solution, a rapid decrease of absorbance to 0 was observed, which suggests that all MP bounded to CaM was then dissociated from CaM.

Figure 7. MP concentration dependence of absorbance change of 1653 cm-1. MP was added into the buffer solution with CaM activated by 10-4 M Ca2+.


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Figure 8. (a) Relationship between saturated absorbance observed in Fig. 7 after MP was introduced as a function of the MP concentration. (b) Langmuir plot of 1/absorbance vs. inverse of the MP concentration.

From these measurements, the dissociation constant for MP from CaM was determined from the adsorption isotherm using the saturated absorbance (Fig. 6) after MP was introduced to CaM immobilized on the surface, as shown in Figure 8(a). When the reciprocal 1/absorbance was plotted against the inverse of the MP concentration, a linear relationship was observed, as shown in Fig. 8(b) which suggests that the adsorption process of MP on CaM is a Langmuir type adsorption. The dissociation constant was determined from the slope to be ca. 30 µM. This dissociation constant was larger than that reported for a homogeneous solution at 300 nM41



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which indicates that it is more difficult for MP to bind with CaM immobilized on surface than with that present in solution. Figure 9 shows the model for MP-CaM complex formation on the Au surface. The large dissociation constant observed for the present CaM-modified surface could be due to (1) a high coverage of CaM on the surface blocking the binding sites for MP, or (2) His-tag modification on the N terminal of CaM may hinder its flexibility to change conformation after binding with MP.

Figure 9. Model for MP binding with CaM immobilized on the Au surface.

4. Conclusions In summary, CaM was immobilized on a Au surface by the His-tag method to investigate the biofunctionality of CaM in the presence and absence of Ca2+ using SEIRAS. We observed two different functions. One is a Ca2+ induced reversible conformational change of CaM, in the


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same manner as that in homogeneous solution and the other was the interaction between immobilized CaM and MP, although the dissociation constant determined from the MP concentration dependence was much smaller than that in a homogeneous environment. This result indicates that it is difficult for MP to bind with CaM immobilized on the Au surface due to the difficulty for MP to access the binding sites in well-ordered immobilized CaM on the surface. Non-specific adsorption onto the surface are still need to study carefully by changing the CaM coverage and surface modification with other molecules such as polyethylene glycol SAM to avoid non-specific adsorption of MP.

Acknowledgements The present work was partially supported by World Premier International Research Center Initiative on Materials Nanoarchitectonics (WPI-MANA) and the Development of Environmental Technology using Nanotechnology from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.



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Table of Contents



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Scheme 1. Surface modification of His-tag CaM on a Au surface. 209x154mm (200 x 200 DPI)


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Figure 1. SEIRA spectra after each modification step shown in Scheme 1. (a) dithiobis-C2-NTA modification (Reference spectrum: bare Au), (b) NiCl2 treatment (Reference spectrum: (a)), (c) imidazole treatment (reference spectrum: (b)), and (d) His-tag CaM modification (Reference spectrum :(c)) 170x186mm (200 x 200 DPI)



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Figure 2. (a) Time dependent SEIRA spectra of the Au surface during the His-tag CaM modification in buffer solution containing 10 mM His-Tag CaM. (Reference spectrum: bare Au). (b) Time dependence of the peak height at 1643 cm-1. 135x216mm (200 x 200 DPI)


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Figure 3. Ca2+ concentration dependent SEIRA spectra of CaM immobilized on the Au surface. (a) 10-7 M, (b) 10-6 M, (c) 10-5 M, (d) 10-4 M, (e) 10-3 M, (f) 10-4 M, (g) 10-5 M, (h) 10-6 M, and (i) 10-7 M. Experiments were conducted from (a) to (i). (Reference spectra: CaM modified Au surface measured in Ca2+-free solution.) 209x194mm (200 x 200 DPI)



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Figure 4. Change in absorbance at 1550 cm-1 against the log of Ca2+ concentration in the buffer solution. The solid line is the binding isotherm for Ca2+ in a homogeneous environment reported by Crouch et al.13 164x116mm (200 x 200 DPI)


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Figure 5. SEIRA spectra of the Au surface (a) with and (b) without modification by CaM measured in a buffer solution containing 10-4 M Ca2+ and 120 µM MP. (Reference spectra: (a) CaM modified Au surface in a MPfree buffer solution containing 10-4 M of Ca2+. (b) Imidazole capped Ni-NTA surface in a MP-free buffer solution containing 10-4 M of Ca2+. 172x204mm (200 x 200 DPI)



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Figure 6. SEIRA spectra of the Au surface (a) with and (b) without modification by CaM measured in a buffer solution containing 10-4 M Ca2+. (Reference spectra: (a) CaM modified Au surface in a MP-free buffer solution containing 10-4 M of Ca2+. (b) Imidazole capped Ni-NTA surface in a MP-free buffer solution containing 10-4 M of Ca2+. 164x182mm (200 x 200 DPI)


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Figure 7. MP concentration dependence of absorbance change of 1653 cm-1. MP was added into the buffer solution with CaM activated by 10-4 M Ca2+. 175x122mm (200 x 200 DPI)



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Figure 8. (a) Relationship between saturated absorbance observed in Fig. 7 after MP was introduced as a function of the MP concentration. (b) Langmuir plot of 1/absorbance vs. inverse of the MP concentration. 164x227mm (200 x 200 DPI)


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Figure 9. Model for MP binding with CaM immobilized on the Au surface. 109x146mm (200 x 200 DPI)



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49x48mm (200 x 200 DPI)


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Biofunctionality of Calmodulin Immobilized on Gold Surface Studied

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