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May 3, 2016 - was reported by Sano and Shiba,5 who originally discovered 12- .... the protein surface and partly from bulk water, in which the water p...
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Structure and Dynamic Properties of a Ti-Binding Peptide Bound to TiO2 Nanoparticles As Accessed by 1H NMR Spectroscopy Yu Suzuki,*,† Heisaburo Shindo,‡ and Tetsuo Asakura‡ †

Tenure-Track Program for Innovative Research, University of Fukui, 3-9-1, Bunkyo, Fukui-shi, Fukui 910-8507, Japan Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo 184-8588, Japan

‡

S Supporting Information *

ABSTRACT: Saturation transfer difference (STD) NMR spectroscopy is a powerful method for detecting and characterizing ligand−receptor interactions. In this study, the STD method was used to characterize the interactions of a Tibinding peptide (TBP:RKLPDA) with TiO2 nanoparticles. The water peak in the NMR spectrum was selectively saturated, and the STD amplitudes for TBP were observed in the presence of TiO2, demonstrating that the side chains of the N-terminal residues Arg1 and Lys2 exhibit the strongest saturation transfer effect from water molecules; i.e., the two Nterminal residues are in contact with the TiO2 surface. The relaxation rate in the rotating frame, R1ρ, was observed to be high at the N-terminal residues; R1ρ decelerated toward the Cterminus, indicating that the N-terminal residues serve as anchors on the TiO2 surface and that the TBP motion bound to TiO2 particles is modeled as a wobble-in-cone with a fairly flexible C-terminus. The dissociation constant Kd of the TBP−TiO2 nanoparticle complex was 4.9 ± 1.8 mM, as estimated from the STD experiments and R1ρ measurements. The combination of these results and the negative zeta potential of the TiO2 surface validate that both the positively charged guanidyl group of Arg1 and amino group of Lys2 play key roles in interaction with the TiO2 surface by electrostatic force.

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INTRODUCTION The field of nanoscale engineering of organic/inorganic hybrid materials continues to grow and includes a broad range of applications, such as molecular biomimetics, implant surface engineering, and the construction of nanodevices.1−3 The adhesion and selectivity at the interface of biomacromolecules and inorganic materials are critical to the construction of wellorganized hybrid materials. To address this point, researchers have used peptide motifs that undergo specific interactions with inorganic materials. Such motifs have often been observed in natural proteins and have been artificially selected from a mixture of peptides with random sequences via the so-called artificial evolution systems first established in the early 1990s.4 One such binding peptide, titanium-binding peptide (TBP), was reported by Sano and Shiba,5 who originally discovered 12residue peptides using a phage display method. One of the peptides binds the surface of metals with high selectivity, as monitored by phage binding assay.6 They observed that the Nterminal sequence in the peptide, R1K2L3P4D5A6, the so-called TBP, was sufficient for Ti binding. TBP has subsequently been used in nanobiotechnological applications such as functionalizing the surfaces of medical titanium materials7−9 and multilayer constructions using its binding and mineralization properties.10,11 The mechanisms by which TBP binds to the TiO2 surface have been suggested by several groups. Sano and Shiba suggested that a positive charge on the side chain of Arg1 and a © 2016 American Chemical Society

negative charge on the side chain of Asp5 electrostatically interact with −Ti−O− and −Ti−OH2+ on the TiO2 surface, respectively. Hayashi et al.12 used atomic force microscopy (AFM) to study the interaction of ferritins fused with TBP and that of its mutants with Ti nanoparticles. Replacement of Arg1, Lys2, and Pro4 in TBP with Ala resulted in substantially weaker binding to TiO2; they therefore concluded that the local structure of TBP governs the arrangement of charged residues and affects the binding strength. Fukuta et al.13 also investigated the binding behavior of ferritins with Ala-substituted TBPs using AFM and revealed that Arg1 or Lys2 anchors ferritin to the TiO2 surface through electrostatic interaction; moreover, they observed that the adsorption force of Lys2 is stronger than that of Arg1. Skelton et al.14 reported that TBP initially recognizes the water layers on the nanoparticle surface via a pair of oppositely charged groups, the combination of Arg1 and Asp5, or Lys2 and Asp5, as studied by molecular dynamics (MD) simulations. Schneider et al.15 also used MD simulations to deduce that Arg1 is mainly responsible for TBP anchoring to the TiO2 surface and that Lys2 and Asp5 contribute to the adsorption equilibrium on the Ti surface. As previously mentioned, subtle discrepancies and ambiguities exist among Received: March 31, 2016 Revised: May 2, 2016 Published: May 3, 2016 4600

DOI: 10.1021/acs.jpcb.6b03260 J. Phys. Chem. B 2016, 120, 4600−4607

Article

The Journal of Physical Chemistry B

a train of Gaussian-shaped pulses with a saturation length of 60 ms and 71.5 dB attenuation; WATERGATE was used for water suppression. The number of selective pulses determining the presaturation periods ranged from 10 to 320, leading to a total length for the saturation pulse train of 0.6−19.2 s to obtain the STD buildup curves. The total number of scans was 256 when the spectral width for the 1D STD NMR spectra was 15 ppm. On-resonance irradiation of the adsorbed water molecules was performed at a chemical shift of 4.7 ppm, and off-resonance irradiation was applied at −10 ppm. The NMR data were processed using the Delta program on the ECA600II instrument. Analyses of STD and T1ρ. Several methods of STD experiment for identifying or characterizing ligands that bind to a target protein have been proposed. One of the STD experiments is often used to measure the degree of magnetization transfer to ligand from the protein, in which the entire protein is saturated via spin-diffusion by strong irradiation at any protons within the protein.19−21 A variation of STD, the socalled WaterLOGSY experiment,22,23 is used to measure magnetization transfer to ligand from the waters hydrated on the protein surface and partly from bulk water, in which the water peak is selectively saturated. STD intensity can also be used to elucidate the interaction of TBP with TiO2 nanoparticles, as described later. The STD factor Astd is defined in general by eq 1:20,24

the preceding studies; also, the binding mechanism for this system has not been fully elucidated. In this study, we investigated the structure and dynamic nature of TBP bound to TiO2 nanoparticles in solution using solution NMR spectroscopy. First, we measured the relaxation time T1ρ to evaluate the dynamic nature of TBP interacting with the TiO2 surface. Second, we conducted saturation transfer difference (STD) NMR measurements to identify the TBP sites that interact with the TiO2 surface and to obtain the dissociation constants. Third, we performed mutagenesis experiments to elucidate the roles of individual residues of TBP in its interaction with TiO2 nanoparticles. Fourth, we conducted nuclear Overhauser effect spectroscopy (NOESY) experiments to determine the three-dimensional structure of TBP bound to the TiO2 surface. Finally, on the basis of these NMR results, combined with the surface potential of the TiO2 nanoparticles, we propose a model for the binding of TBP onto the TiO2 surface.

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MATERIALS AND METHODS Sample Preparation. Peptides with purity greater than 98% were purchased from Funakoshi Co. Ltd., Japan. Their amino acid sequences are RKLPDA (TBP) and analogues AKLPDA (R1A), KKLPDA (R1K), RKLADA (P4A), and RKLPAA (D5A), where the abbreviations in parentheses are the abbreviations of the peptides used in this study. The TiO2 nanoparticles (Aeroxide TiO2 P25, obtained from Evonik Industries) had an average particle size of 21 nm and surface area of 50 ± 15 m2/g. The nanoparticle dispersion in water was prepared as follows. The TiO2 nanoparticles were suspended in water and sonicated twice for 10 min with a 50% duty cycle. The nanoparticle suspension was mixed with the peptide solutions in the appropriate proportions, and the pH of the resulting suspensions was adjusted to 7.0. The final concentrations of the nanoparticles and peptides were 1 mg/mL and 1−6 mM in 20 mM phosphate buffer pH 7.0 with 10% D2O, respectively. NMR Measurements. All 1H NMR experiments were performed on a JEOL Resonance ECA600II spectrometer operating at a 1H frequency of 600.17 MHz at 298 K. Resonance assignments for the 1H signals were accomplished by total correlation spectroscopy (TOCSY), NOESY, and rotating-frame Overhauser effect spectroscopy (ROESY) experiments with WATERGATE for solvent suppression. TOCSY spectra, with a MLEV-17 mixing sequence, were recorded with a mixing time of 50 ms, 15 ppm spectral width in both the t1 and t2 dimensions, 256 and 1024 complex points in the t1 and t2 dimensions, respectively, and 32 scans. NOESY spectra were recorded with a mixing time of 150 ms, 15 ppm spectral width in both the t1 and t2 dimensions, 256 and 1024 complex points in the t1 and t2 dimensions, respectively, and 32 scans. ROESY spectra were recorded using the same parameters as those in the NOESY experiments, except for a mixing time of 250 ms. The NMR data were processed using NMRPipe,16 and the two-dimensional spectra were analyzed using SPARKY.17 Distance constraints obtained from the NOESY and ROESY spectra were used as input for the DYANA program18 to determine the three-dimensional structure. The T1 and T1ρ relaxation times were measured by the inversion recovery method and spin-lock method, respectively. Segments of peptides in direct contact on the TiO2 surface were identified by saturation transfer difference NMR19,20 using

A std = (I0 − Isat)/I0 = Istd /I0

(1)

where I0 is the intensity of a given signal in the off-resonance or reference NMR spectrum, Isat is the intensity of a signal in the on-resonance NMR spectrum, and (I0 − Isat) represents the intensity of the STD spectrum; thus, Astd expresses the intensity of a saturated spectrum as a fraction of the intensity of an unsaturated reference spectrum. The saturation of ligand protons in the bound state has been reported to be counteracted by their longitudinal relaxation times T1 in the unbound state.24 Therefore, the difference in T1 values among ligand protons must be taken into account. Short saturation times should be more accurate for STD measurements because the T1 effect should be weaker. However, the sensitivity of STD under short saturation times is greatly reduced, and short saturation times are thus associated with larger measurement errors. Consequently, quantitative evaluation of STD values is expected to be difficult under such conditions. Researchers have therefore proposed using an STD buildup curve to prevent possible misinterpretations.21,24 To eliminate the T1 bias at long saturation times, we calculated the slope of the STD buildup curve at saturation time zero by fitting the saturation curve using eq 2:21 A std = A max (1 − exp(−ksattsat))

(2)

where Astd is the STD factor of a given proton at saturation time tsat, Amax is the maximal STD factor when long saturation times are used, and ksat is the saturation rate constant. Multiplication of ksat by Amax yields the initial slope of the buildup curve at zero saturation time, which corresponds to the STD intensity without T1 bias, denoted as STD0.21 Evaluation of the binding affinity of TBP to the TiO2 particles is essential. Angulo et al. reported a method for obtaining the dissociation constant Kd from STD measurement data.25,26 Using the binding system with a known Kd value, they showed that the value obtained from the initial slope of the 4601

DOI: 10.1021/acs.jpcb.6b03260 J. Phys. Chem. B 2016, 120, 4600−4607

Article

The Journal of Physical Chemistry B

Figure 1. (a) Overlaid 1D 1H NMR spectra of TBP alone (black) and the TBP−TiO2 sample (red) in solution and their signal assignments; (b) 1D STD NMR spectrum of TBP in the TBP−TiO2 sample; (c) 1D STD NMR spectrum of TBP alone in solution.

These results indicate that TBP interacts with TiO2 nanoparticles and undergoes a fairly rapid exchange between association and dissociation onto the TiO2 surface. To elucidate the dynamic nature of the bound state of TBP on TiO2, we measured the T1 and T1ρ relaxation times for the TBP and TBP−TiO2 samples. T1ρ is especially important because it is sensitive to slow motions. Relaxation rates R1 and R1ρ (the reciprocals of the relaxation times) of the Hβ protons for each residue are plotted along with the residue number in Figure 2. The Hβ of Leu3 was excluded because the resonance severely overlapped other peaks at approximately 1.5 ppm in Figure 1a. As expected, the R1 values for Hβ of each residue are approximately the same between TBP alone and TBP−TiO2

STD buildup curve is much more accurate than that obtained from a single STD experiment with a given saturation time. STD0 = STDmax [L]/(Kd + [L])

(3)

.where STD0 is the initial slope of the STD buildup curve as a function of ligand concentration [L], and STDmax is the maximum STD0. The longitudinal relaxation rate in the rotating frame R1ρ (the reciprocal of T1ρ) is known to be sensitive to slow molecular motions and is thus a useful parameter for evaluating association−dissociation reactions between ligand molecules and proteins. Thus, the binding affinity of TBP to the TiO2 particles was obtained from the ligand concentration dependence of R1ρ. Fielding has reviewed various NMR methods for determining the dissociation constant Kd.27 In the case of relaxation-rate data, the following equation is given: [P]ΔR max /ΔR = [L] + Kd

(4)

where [P] and [L] are the concentrations of binding sites on the TiO2 particles and on TBP, respectively, ΔR is the difference between R1ρ values in the presence and absence of TiO2 as a function of the TBP concentration [L], and ΔRmax is the difference between the R1ρ of TBP free in solution and that of TBP bound to TiO2. The dissociation constant is easily calculated from linear plots of 1/ΔR vs [L].

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RESULTS AND DISCUSSION R1ρ and Motional Mode of TBP Bound to TiO2. The overlaid 1H one-dimensional (1D) spectra of TBP alone (black) and TBP−TiO2 (red) are shown in Figure 1a with the signal assignments made through analysis of the NOESY and TOCSY spectra. Line broadening was observed for most of the TBP peaks in the presence of TiO2 nanoparticles, and chemical shift changes were small but clearly observed for peaks Hα, Hβ, and Hδ of residue Arg1 and most likely for peak Hε of Lys2.

Figure 2. Relaxation rates R1 and R1ρ plots of Hβ (not available for Leu) of each residue for TBP in the absence of TiO2 (●) and in the presence of TiO2 (▲), respectively. 4602

DOI: 10.1021/acs.jpcb.6b03260 J. Phys. Chem. B 2016, 120, 4600−4607

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The Journal of Physical Chemistry B samples because a small fraction of TBP in the bound state relative to TBP in bulk has a very slow relaxation rate, resulting in only a small contribution to the observed R1. By contrast, the R1ρ values of TBP−TiO2 are much faster than those of TBP alone, suggesting that TBP in the bound state on the TiO2 surface substantially contributes to the observed R1ρ. Interestingly, the large difference in R1ρ between the presence and absence of TiO2 nanoparticles observed for the N-terminal residues gradually decreases toward the C-terminus, indicating that the N-terminal residues are more restricted in motion than the C-terminal region. Therefore, the N-terminal residues of TBP likely serve as an interfacial anchor on the TiO2 surfaces and the motion of TBP bound to TiO2 surfaces would be modeled as a wobble-in-cone with fairly flexible chains. STD Measurements and Its Mechanism. In the STD experiments, water molecules were selectively saturated by strong irradiation at the frequency corresponding to the water peak and a difference spectrum between on-resonance saturation at the frequency corresponding to the water peak and off-resonance saturation at −10 ppm was recorded. The STD spectrum for the TBP−TiO2 sample is shown in Figure 1b. The STD peaks were observed only in the presence of TiO2, not in the absence of TiO2 (Figure 1c). These results provide clear evidence for the occurrence of saturation transfer from water molecules to the TBP molecules bound to the TiO2 surface. What is the mechanism of the STD observed here for the TBP−TiO2 system? The STD and WaterLOGSY techniques have been widely used for screening and characterizing ligand− protein interaction systems.19−23 It should be noted that intensity profiles of the STD spectrum observed here were nearly identical to those of the WaterLOGSY (see Figure S1 in the Supporting Information), suggesting that the mechanism of the STD observed for the TBP/TiO2 sample is essentially equivalent to that of WaterLOGSY. By analogy to the crossrelaxation of the ligand bound to proteins from bulk water molecules observed in WaterLOGSY experiments,22,23 and intermolecular NOEs observed between protein protons and the hydration water molecules,23 the saturation transfer to a given proton occurs (1) directly from water molecules in the bound layers on the TiO2 surface, (2) from labile protons (exchanged with water protons), (3) from labile protons remote from a given proton but propagated to it via spin diffusion, and (4) from Hα protons with a chemical shift near the selectively saturated water peak. Mechanism 4 is excluded by the fact that the STD observed for the TBP−TiO2 sample in D2O solution was negligible (Figure S2). Moreover, the spindiffusion mechanism (3) is unlikely because of relatively weak affinity, i.e., an association−dissociation exchange of TBP to TiO2 particles that is faster than the spin-diffusion rates. Mechanisms 1 and 2 have long been debated in the hydration of biomolecules (see the review article by Otting28 and references therein) and also in the ligand−protein interaction systems22,23 in aqueous solution. NOE (cross-relaxation) from water molecules hydrated on the protein surface to the protons of ligands and/or protein has often been observed in NOESY experiments.28,29 However, in the case where a given proton in the ligand has close contact with a labile proton in a protein or in the ligand itself, the dominant process will be saturation transfer from this labile proton, which has been exchanged with water protons in bulk, to the protons of the ligand.28,29 STD factors Astd for well-separated peaks are plotted against the saturation time (0.6−19.2 s) in Figure 3a. The saturation

Figure 3. (a) STD amplification factors plotted against saturation time for well-separated peaks and (b) the structure of TBP and the value (%) of STD0 relative to that of Arg Hδ, which was set as 100%.

time constant ksat and maximum Amax in eq 2 were obtained by least-squares curve-fitting of the STD buildup curves in Figure 3a; the results are listed for representative protons in Table 1, Table 1. Maximum STD Amplitude Amax, Saturation Rate ksat, and Initial Slope STD0 Obtained via Least-Squares Fitting of the STD Buildup Curvesa residue

observed protons

Amax

Ksat (s−1)

STD0 (×10−2 s−1)

STD0 (%)

Astd(7.2 s) (%)

Arg1 Arg1 Lys2 Lys2 Lys2 Leu3 Pro4 Asp5 Ala6

Hβ Hδ Hβ Hγ Hε Hδ Hβ Hβ Hβ

0.19 0.24 0.16 0.17 0.21 0.04 0.07 0.05 0.03

0.35 0.31 0.34 0.3 0.32 0.28 0.28 0.33 0.26

6.7 7.5 5.4 5 6.8 1.2 1.9 1.6 0.7

89 100 72 67 91 16 25 21 9

77 100 62 71 91 17 25 23 11

a

STD amplitudes STD0 (%) are normalized, with the highest value for Arg1 Hδ being set to 100.

together with the initial slope STD0. The STD0 values were relativized for comparison, where the largest STD value of Arg1 Hδ was set to 100%. Figure 3b displays the relative STD0 values in percent. The STD0 values for Arg1 Hδ and Lys2 Hε are high: 100 and 91%, respectively. With respect to the mechanism of STD, we focus on parameter STD0 of the side chain protons of Arg1 and Lys2. In the case of the Lys2 residue, the relative STD0 values for CH2 at the Lys2 side chain (Hβ, Hγ, and Hε) were 72, 67, and 91, 4603

DOI: 10.1021/acs.jpcb.6b03260 J. Phys. Chem. B 2016, 120, 4600−4607

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The Journal of Physical Chemistry B

R1A after standing overnight, whereas they remained in suspension in the presence of the other three analogues under the same conditions (data not shown). These results suggest that the positively charged N-terminal residues of TBP analogues are essential for protecting the suspended TiO2 nanoparticles against aggregation, indicating that three TBP analogues exhibit sufficient binding ability despite the presence of R1A on the particles’ surface. In Table 1, the relative STD0 values are compared with relative STD factors Astd(7.2) at a long saturation time of 7.2 s. A comparison of these two parameters reveals that their values are comparable. We concluded that STD is only slightly affected by the T1 bias in the TBP−TiO2 system. Thus, Astd(7.2) values are sufficient for quantitative evaluation of STD effects, as discussed in the following section. The binding abilities of R1K, P4A, and D5A to the TiO2 surface were evaluated by STD measurements. The relative STD factor Astd(7.2) in percent was used to evaluate the binding ability of TBP analogues. Here, the Astd(7.2) value of Arg1 Hδ of TBP was set to 100%. The relative Astd(7.2) values of TBP vs those of the TBP analogues are represented by bar graphs in Figure 4. In the case of R1K, Lys1 and Lys2 Hβ

respectively. The high relative STD0 value observed for Lys Hε is likely due to the close proximity of this proton to the Lys2 side-chain amino group, whose protons are rapidly exchangeable with water, suggesting that saturation transfer from the labile amino protons to Lys Hε protons would be dominant compared to direct saturation transfer from water molecules.29 By contrast, the relative STD0 values for the Hβ and Hγ protons of Lys2 are small because these protons are distant from the amino protons. Given that the saturation transfer rate is proportional to the reciprocal of the sixth power of distance between protons, direct saturation transfer to these protons would be dominant from water molecules in the bound layers of the TiO2 surface. If this is the case, the water molecules would be considerably tightly bound to the TiO2 surface with a residence time of longer than 1 ns, as estimated from the NOESY experiments on hydration water molecules trapped inside a cavity of proteins.28 A similar situation occurs for the side chain protons of Arg1; in this case, saturation transfer to protons Hδ from labile protons of guanidyl groups would be dominant. The Hβ protons located far from these labile protons would exhibit only a small saturation transfer effect, if any, from these exchangeable protons; thus, they are most appropriate for comparing direct saturation transfer from water molecules in the bound layers on the TiO2 surface. Nonetheless, distinguishing and experimentally quantifying STD contributions from water molecules in the bound layers on the TiO2 surface and labile protons are difficult. Further quantitative studies of these two STD mechanisms in the TBP−TiO2 system are currently underway in our laboratory. General Features of STD for TBP and Its Analogues. The STD intensity generated by saturation of water peaks could be used to evaluate the relative proximity of the protons of TBP to water molecules in the bound layers on TiO2 surface. To this end, Hβ protons are most appropriate for comparing the STD strength of residues along the peptide chain, as previously described. Arg1 Hβ and Lys2 Hβ have large relative STD0 values of 89 and 72%, respectively, whereas Pro4 Hβ and Asp5 Hβ have much smaller relative STD0 values, ranging from 16 to 25%. The lowest relative value is observed to be only 9% for the Ala6 Hβ of the C-terminal residue. These results suggest that the side chains of N-terminal residues Arg1 and Lys2 closely contact on the TiO2 surface, whereas C-terminal residue Ala6 is most distant from the surface. This result is consistent with the results of the T1ρ measurements, suggesting again that, in the bound state of TBP on the TiO2 surface, the N-terminal residues initially bind to the TiO2 surface, whereas the Cterminal regions remain relatively free. In cases where STD factors are compared among different ligand systems, the STD factors should be multiplied by the ligand excess to normalize the differences in receptor molecule concentrations.20 In this study, however, Istd/I0that is, Astd can be used as the STD factor because the concentrations of TiO2 and peptides were the same in all analogous TBP samples. As previously described, the side chains of the N-terminal residues of Arg1 and Lys2 play an essential role in the interaction between TBP and the TiO2 surface. To clarify the roles of individual residues of TBP, we performed site-directed mutation analysis using TBP analogues: AKLPDA (R1A), KKLPDA (R1K), RKLADA (P4A), and RKLPAA (D5A). Initially, the mixtures of TiO2−TBP analogues were subjected to precipitation tests. The TiO2 nanoparticles suspended in aqueous solution precipitated in the presence of

Figure 4. Bar graphs of the relative Astd(7.2) value for Hβ protons of each residue along the TBP chain (black bars) and for TBP analogues R1K, P4A, and D5A (white bars). Here, the Astd(7.2) value for Hβ protons of Arg1 is set to 100%.

proton peaks were superimposed in the spectrum so that the observed Astd(7.2) is considered to be the average value. This value is slightly smaller than that of Lys2 Hβ in TBP and approximately 27% smaller than that of Arg1 Hβ in TBP. Moreover, the Astd(7.2) for residues Leu3−Ala6 of the R1K analogue range from approximately two-thirds to one-third of those observed for the corresponding residues of TBP, indicating that the interaction of R1K with the TiO2 surface is slightly weaker than that of TBP. The higher basicity of the guanidyl group of the Arg side chain compared to that of the amino group of the Lys side chain may result in a stronger interaction for TBP than for R1K with the negatively charged surface of the TiO2 particles. Additionally, hydrogen donor groups (NH and NH2) in the Arg side chain may contribute to hydrogen-bond formation with oxygen atoms on the TiO2 surface. The Astd(7.2) value for P4A decreased by more than 30% for Arg1 Hβ and Lys2 Hβ compared to those of the corresponding 4604

DOI: 10.1021/acs.jpcb.6b03260 J. Phys. Chem. B 2016, 120, 4600−4607

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The Journal of Physical Chemistry B protons of TBP, suggesting that Pro4 somewhat strengthens the interaction of Arg1 Hβ and Lys2 Hβ as well as the Cterminal residues. The presence of Pro induces the rigidity of the peptide because an −N−Cα− bond in a ring cannot rotate freely. This rigidity may favor the interaction between TBP and the TiO2 surface. The Astd values for D5A are slightly higher than those observed for TBP. In fact, the replacement of Asp5 by Ala may reduce electrostatic repulsion with the negatively charged TiO2 surface, resulting in an increase in interaction of the peptide− TiO2 complex. Binding Affinity of TBP to TiO2 Nanoparticles. Evaluation of the binding strength of TBP to TiO2 particles is essential for characterizing such ligand−nanoparticle interaction systems. Angulo et al. reported a method for obtaining the dissociation constant Kd from STD measurement data.25 Using a binding system with a known Kd value, they showed that the value obtained from the initial slope of the STD buildup curve is much more accurate than that obtained from a single STD experiment with a given saturation time. We plotted the STD0 values for the Hβ of each residue except Leu as a function of the TBP concentration. The plot fitted with the Langmuir equation, as given by eq 3, is shown in Figure 5 for Arg1 Hβ; plots for other residues are presented in the Supporting Information, Figure S3.

Figure 6. 1/ΔR1ρ plotted against TBP concentration at a constant concentration of TiO2 particles for Arg1 Hβ. The fitting with eq 4 yields the dissociation constant Kd (6.7 ± 0.5 mM) which is almost identical to the value obtained in Figure 5.

reliable and are often used to obtain the binding affinities of ligand−receptor systems. Within the experimental errors, the Kd values obtained from these two methods are identical for the Hβ protons of a corresponding residue; however, the Kd values are high by a few multiples for the N-terminal residues, compared with that for the C-terminal residues, although we expected that the dissociation constants at the C-terminal residues should be identical to those at the N-terminal residues. The reasons for this discrepancy have not yet been determined. Nevertheless, importantly, these Kd values from T1ρ experiments are nearly identical to those obtained from STD data, as previously described. The apparent dissociation constants obtained in this study are greater by approximately 2 orders of magnitude compared to those previously reported in the literature, 13.2 ± 4.0 μM.6 This discrepancy in Kd may originate from the substantial difference in size of the TiO2 particles used (average diameters of 21 nm vs