Structural Characterization of Myoglobin Molecules Adsorbed within

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Article Cite This: J. Phys. Chem. C 2018, 122, 15567−15574

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Structural Characterization of Myoglobin Molecules Adsorbed within Mesoporous Silicas Jun Kijima,† Yuuta Shibuya,†,⊥ Kazuya Katayama,† Tetsuji Itoh,‡ Hiroki Iwase,§ Yoshiaki Fukushima,§ Minoru Kubo,∥ and Akira Yamaguchi*,† †

Institute of Quantum Beam Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 310-8512, Japan Research Institute for Chemical Process Technology, National Institute of Advanced Industrial Science and Technology (AIST), Nigatake 4-2-1, Sendai 983-8551, Japan § Neutron Science and Technology Center, Comprehensive Research Organization for Science and Society (CROSS), Tokai, Ibaraki 319-1106, Japan ∥ RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan

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‡

S Supporting Information *

ABSTRACT: In the present study, we examined the secondary and tertiary structure of myoglobin (Mb) within folded sheets mesoporous material (FSM)- and Santa Barbara amorphous (SBA)-type mesoporous silicas. The Barrett− Joyner−Halenda pore diameters of SBA-type mesoporous silicas were 39, 70, and 75 Å, and that of FSM-type mesoporous silica was 40 Å. The secondary and tertiary structures of myoglobin were observed by Fourier transform infrared (FTIR) and small-angle neutron scattering (SANS), respectively. The FTIR and SANS results indicated preservation of the secondary and tertiary structures of myoglobin inside the pores of SBA-type mesoporous silicas. Adsorption of myoglobin within FSM-type mesoporous silica, however, resulted in perturbation of the tertiary structure, accompanied by partial unfolding of the secondary structure. Lower structural stability of myoglobin within the FSM-type mesoporous silica was also confirmed. These findings suggest that the Mb structure is more influenced by the inner pore surface characteristics than by geometrical pore size.

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INTRODUCTION Inorganic nanoporous materials hold promise as solid support for proteins because when adsorbed onto such supports, proteins can retain their functions under both physiological conditions and harsher reaction conditions.1−5 It is believed that the retention of protein function is due to stabilization of the native conformation of the proteins by spatial confinement and/or protein−surface interactions.1−5 The structural characterization of proteins is, therefore, an important step toward gaining a fundamental understanding of protein structure− function relationships in inorganic nanoporous support systems. Myoglobin (Mb) is a water-soluble globular protein.6 Although Mb held within nanoporous support can exhibit greater catalytic activity than free Mb in a bulk aqueous solution,7,8 it has been used as a model protein to study protein structure−function relationships in inorganic nanoporous support systems. The deformation behavior of Mb can be monitored by observing the Soret absorption band in the visible region.6,7 The amide I and II infrared bands are indicative of the secondary structure of Mb.8−10 Visible and infrared absorption spectroscopies have thus been used for structural characterization of Mb within a nanoporous material.6−10 For example, Itoh et al. examined the globular © 2018 American Chemical Society

structure of Mb within a folded sheets mesoporous material (FSM)-type mesoporous silica (MPS) support using the spectral shape of the Soret absorption band.7 Fourier transform infrared (FTIR) study by Sang et al. indicated that a slight change in the secondary structure of Mb was induced upon adsorption to Santa Barbara amorphous (SBA)-type MPS supports.8 Slight changes in the secondary structure were also examined using the UV circular dichroism spectrum for MB within a sol−gel silica glass and when in contact with silica nanoparticles.11,12 The pore size of an inorganic nanoporous material is one of the important factors affecting the catalytic activity of Mb within the pore. Sang et al. examined both the catalytic activities and secondary structures of Mb within SBA-type MPSs with different pore diameters (5.9, 8.5, and 11 nm).8 They found that Mb activity tended to decrease with increasing pore diameter of the MPS support. Analysis of the FTIR spectra of the Mb within MPSs suggested this be linked to deformation of the secondary structure of myoglobin. A protein’s function can be influenced not only by its secondary Received: May 8, 2018 Revised: June 16, 2018 Published: July 2, 2018 15567

DOI: 10.1021/acs.jpcc.8b04356 J. Phys. Chem. C 2018, 122, 15567−15574

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The Journal of Physical Chemistry C

we designate MPS as FSMxx or SBAxx, where xx indicates BJH pore diameter. The MPS powders were dried overnight at 200 °C prior to sample preparation. Structural Characterization of MPSs. The pore structures of MPSs were characterized by nitrogen adsorption and desorption isotherm and small-angle X-ray scattering (SAXS) measurements. The nitrogen adsorption and desorption isotherms were measured on a Micrometrics ASAP 2020 instrument. The specific surface area was calculated using the multiple-point Brunauer−Emmett−Teller (BET) method and the pore size distribution was calculated from the adsorption branch of the isotherm using the BJH method. The SAXS patterns of MPSs were recorded on a Rigaku Smartlab with Cu Kα radiation. The macroscopic structures of the MPS powders were observed by field-emission scanning electron microscopy (FE-SEM, Hitachi S-4800). Adsorption of Mb at MPS. In the SANS experiments, the dry MPS powders (30 mg) were rinsed with H2O/D2O mixture prior to Mb adsorption, following which they were added to the H2O/D2O mixture containing Mb. The total volumes of the H2O/D2O mixture and initial concentration of Mb were 3.0 mL and 2.5 mg mL−1, respectively. After being shaken for 20 h, the mixture was centrifuged at 14 000 rpm for 10 min. The supernatant was subjected to absorption spectrum measurement to estimate the amount of Mb adsorbed. The resulting Mb/MPS conjugates were carefully rinsed with the H2O/D2O mixture and then suspended in the H2O/D2O mixture for the SANS experiments. The pH of the solution was around 6.4. The adsorption isotherms of Mb were obtained by adding MPS powder to Mb aqueous (H2O) solutions. The amount of Mb adsorbed at MPS was estimated using the abovementioned procedure. The adsorption of Mb into the MPS pores was examined by differential scanning calorimetry (DSC) measurements. For the DSC experiments, Mb/MPS conjugates were prepared by adding MPS to an aqueous (H2O) solution of myoglobin. After rinsing with H2O, the wet Mb/MPS sample was placed in an aluminum sample pan and sealed with a crimper. The mass of the Mb/MPS conjugate was about 30 mg. DSC measurements were performed on a Rigaku (Tokyo, Japan) thermoPlus DSC-8230 instrument equipped with a cooling system using liquid nitrogen. The scanning speed was fixed at 2 K min−1. Optical Absorption Spectroscopy. The structure of Mb within MPS was characterized by UV−vis absorption and by attenuated total reflection Fourier transform IR (ATR-FTIR) spectroscopy. In the UV−vis absorption spectroscopy experiment, the suspension of Mb/MPS conjugate was poured into a quartz cell (1 cm × 1 cm) with a screw cap. The transmissionabsorption spectrum for the sample suspension was measured on a JASCO model V-570 spectrophotometer equipped with a cryostat (Unisoku Co. Ltd., CoolSpek USP-230). In the absorption spectrum experiment, an integrating sphere was set just after the output window of the cryostat, and the transmitted light collected by the integrating sphere was detected.23 ATR-FTIR spectra were obtained on a Bruker Vertex 70 FTIR Spectrometer at room temperature (ca. 25 °C). In the ATR-FTIR experiments, an aqueous (H2O) suspension containing Mb/MPS conjugate was placed on the surface of a Si ATR crystal with three effective internal reflections (Czitek, MicromATR).

structure but also by its tertiary structure. Direct observation of the tertiary structure of Mb should promote further discussion on the structure−function relationships of Mb within MPS. The size and shape of tertiary protein structures are also important factors affecting the adsorption behaviors of proteins. It is well known that a huge amount of Mb molecules can be loaded into MPS supports. Several comprehensive geometric pore-filling models for the tight packing of Mb molecules inside the MPS pores have been proposed.9,10,13 In the geometric calculation of the protein pore-filling model, the crystal structure of Mb has been used to analyze the molecular packing state inside the pores.9,10,13 Although the size and shape of the Mb tertiary structures are key parameters in the pore-filling model, they have not been experimentally determined. The possibility of an intermolecular association between the packed Mb molecules is also an important aspect that may shed light not only the packing properties but also on the catalytic activity of Mb molecules within MPS. Solvent contrast variation in small-angle neutron scattering (SANS) is a powerful technique for structural characterization in multicomponent systems.14,15 In a multicomponent system composed of silica, organic molecules, and mixed H2O/D2O solvent, the scattering length densities of individual components can be selectively matched to the solvent because the average scattering length densities of organic molecules and silica materials for neutrons are located between those of H2O and D2O.14,15 The scattering signal from silica phase or organic molecules thus can be separately observed by tuning the H2O/ D2O ratio. SANS with contrast variation has been applied for selective observation of molecular structures and molecular assemblies in various multicomponent systems, such as surfactant aggregates within MPS,16 proteins within silica sol−gel matrices,17,18 and mixtures of proteins, surfactants, and silica nanoparticles.19 However, it has not been applied to proteins within MPS, which has significant potential for use as solid support for proteins due to its uniform and tunable pore structure that is similar to protein dimensions.20−22 The purpose of this study is the application of the solvent contrast-variation method of SANS to the structural characterization of Mb molecules encapsulated within MPS supports. In the present study, FSM- and SBA-type mesoporous silicas with a series of pore sizes (Barrett−Joyner−Halenda (BJH) pore diameter, 39−75 Å) were prepared. The effects of the pore size and surface character on the Mb structure were then examined. The adsorption of Mb molecules inside the MPS pores was confirmed experimentally by adsorption assay and differential scanning calorimetry (DSC). The secondary structure of Mb within MPS was examined by IR absorption spectroscopy. Visible absorption spectroscopy was applied to monitor the heme environment and thermal stability of Mb. SANS experiments were performed to determine the tertiary structure of Mb within the MPS pores.

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EXPERIMENTAL SECTION Materials and Chemicals. Myoglobin (Mb) from equine heart and D2O were purchased from Sigma-Aldrich Japan (Tokyo, Japan). The molecular weight of Mb was 17 800 Da. Milli-Q water was used as H2O. FSM-type mesoporous silica powders were synthesized using stearyltrimethyl ammonium chloride and sodium silicate.20 SBA-type mesoporous silica powders were synthesized using Pluronic P123 or Brij S10 as a template surfactant.21−23 The silica source for the SBA-type mesoporous silica synthesis was tetraethoxysilane. Hereinafter, 15568

DOI: 10.1021/acs.jpcc.8b04356 J. Phys. Chem. C 2018, 122, 15567−15574

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The Journal of Physical Chemistry C SANS Experiments. The suspension of the MPS or Mb/ MPS conjugate was held in a custom-built quartz banjo cell (19 mm in inner diameter and 1 mm in thickness). SANS experiments were performed using the time-of-flight small- and wide-angle neutron scattering instrument TAIKAN at the Material and Life Science Experimental Facility (MLF) within the Japan Proton Accelerator Research Complex (J-PARC).24 Using white neutrons over a wide range of wavelengths (λ = 1−7.8 Å), the Q range covered was 0.005−17 Å−1, where the magnitude of the scattering vector Q is defined by Q = (4π/λ) sin(θ/2) (θ represents the scattering angle). SANS measurements were performed at room temperature (ca. 25 °C). All SANS data were normalized to an absolute intensity by the coherent scattering of a glassy carbon standard after the necessary data corrections, such as air scattering and cell scattering. The scattering intensities from the sample cell and the solvent were subtracted using the measured transmission and volume fraction of the solvent. Although the scattering intensities at low Q (Q < 0.01 Å−1) and high Q (Q > 0.4 Å−1) were still affected by this excess scattering, we used intensity data in the Q range from 0.01 to 0.4 Å−1. For contrast variation in SANS, we measured a series of Mb/ MPS samples dispersed in water with a D2O volume fraction, ϕD2O = 100, 80, 70, 62.1, and 40%. The contrast-matching point for the silica matrix of MPS was determined to be 62.1% from the contrast variation in SANS experiments. This contrast-matching point was almost the same as that in the literature.16,25 This contrast-matching point corresponds to the SLD of the silica matrix of 3.76 × 1010 cm−2. The average SLD of Mb used in this study was assumed to be 2.24 × 1010 cm−2.26

macroscopic particle morphology of FSM40 was not well defined, as shown in the SEM image (Figure 1B). The lower Porod slope for FSM40 is likely due to inhomogeneity of particle morphology and roughness on the surface or in the internal structure.25 Adsorption of Mb at MPS. From the adsorption isotherm experiments, it was confirmed that the maximum adsorption of Mb could be achieved upon addition of MPS (30 mg) in 3.0 mL of 2.5 mg mL−1 Mb aqueous solution (Figure S3). This experimental condition was applied to the preparation of Mb/ MPS conjugates used for the structural characterization of Mb within MPS. The amounts of Mb adsorbed at MPSs are summarized in Table 2. The adsorption behaviors will be discussed by considering the structure of myoglobin. In the present study, the volume fraction of pores occupied by Mb, f Mb, is defined by f Mb = VMb/Vpore by assuming the adsorption of Mb into the MPS pores. VMb is the total volume of Mb adsorbed at MPS and is described by VMb = AMb(4πNA/ 3Mw)R3, where R is the radius of globular Mb and AMb is the amount of Mb adsorbed at MPS. NA and Mw are Avogadro’s number and the molecular weight (17 800) of Mb, respectively. R was estimated to be 17.6 Å by the SANS experiment, as described below. The volume fraction of Mb within MPS (ϕMb) is defined as follows ϕMb = VMb/(Vpore + Vsilica)

where Vsilica is the total volume of the silica pore wall derived from the density of the silica pore wall. The density of the silica pore wall was estimated to be 2.38 g cm−3 from the contrast variation in SANS experiment described below. The estimated values of f Mb and ϕMb for Mb/MPS conjugates are summarized in Table 2. The adsorption of Mb into the MPS pores was characterized by DSC experiments. In the present study, we observed the lowering of melting/freezing temperatures of the pore water upon adsorption of Mb within MPSs. The DSC freezing curves are shown in Figures S4 and S5. The freezing temperature depressions upon adsorption of Mb were 0.63 K for Mb/ SBA39, 2.5 K for Mb/FSM40, 13 K for Mb/SBA70, and 9.5 K for Mb/SBA75 (Figures S4 and S5). Although the freezing temperature falls with increasing amount of Mb adsorbed (Figure S5), this lowering of freezing temperature can be ascribed to the adsorption of Mb molecules into the MPS pores. In the Gibbs−Thomson model, the freezing temperature is proportional to the inverse of the pore radius.27,28 Decreased average pore size accompanied by the pore adsorption of Mb is thus one plausible reason for this lowering of freezing temperature. Although the freezing/melting temperatures of hydration water of Mb are known to be lower than those for bulk water,29 the hydration water of Mb also appears to contribute to the lowering of freezing temperature. This adsorption of Mb into the MPS pores was also supported by the results of the contrast-variation SANS experiment. Characterization of Mb Structure by Optical Absorption Spectroscopy. The structural deformation of Mb within MPS was qualitatively characterized by measuring the Soret band of Mb in the visible absorption spectrum. As shown in Figure 2, a sharp Soret band peak was seen for all Mb/MPS conjugates at around 25 °C. On the other hand, the Soret band peaks became weaker and broader on raising the temperature to above 45 °C (Figure S6). These changes in the Soret band peak indicate the thermal deformation of Mb within MPS.6,12

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RESULTS AND DISCUSSION Structural Characterization of MPSs. The structural parameters of MPS powders obtained from the nitrogen adsorption isotherms and SAXS profiles are listed in Table 1. Table 1. Structural Parameters of Mesoporous Silica Powders SBA39 FSM40 SBA70 SBA75

DBJHa/Å

ABETb/cm2 g−1

Vporec/cm3 g−1

d100d/Å

39 40 70 75

1003 1211 664 796

1.10 1.96 0.85 0.93

56 51 98 96

(1)

a

BJH pore diameter. bBET surface area. cPore volume. dd100 spacing determined by SAXS.

The pore diameters of MPSs ranged from 39 to 75 Å (Figure S1). A hexagonal arrangement of one-dimensional pores was confirmed by the SAXS profiles of dry MPS powders (Figure S2). The hexagonal pore arrangements were also confirmed by SANS profiles obtained for a slurry of MPS in D2O (Figure 1A). The SBA-type MPSs had wheatlike macroscopic particle morphology with smooth and clear surfaces (Figure 1B). This particle morphology was also confirmed by the Porod slopes in the low Q region (0.01 Å−1 < Q < 0.18 Å−1); the Porod slopes for SBA39 and SBA75 were −3.2 and −4.0, respectively (Figure 1A). A Porod slope is generally −4 for particles with well-defined macroscopic morphology and with sharp surfaces.25 On the other hand, the Porod slope for FSM40 was −2.3, much lower than those for SBA-type MPSs. The 15569

DOI: 10.1021/acs.jpcc.8b04356 J. Phys. Chem. C 2018, 122, 15567−15574

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Figure 1. (A) SANS profiles of SBA39, FSM40, and SBA75 in D2O. (B) SEM images of SBA39, FSM40, SBA70, and SBA75. The solid lines in (A) are the Porod slopes obtained in the low Q range (0.01−0.018 Å−1).

Table 2. Parameters for Mb Adsorbed by MPSs and in Bulk Water Mb/SBA39 Mb/FSM40 Mb/SBA70 Mb/SBA75 bulk

AMba/mg mg−1

f Mbb

ϕMbc

ϕMb,Sd

DMbe/Å

0.049 0.39 0.18 0.20

0.035 0.15 0.16 0.16

0.025 0.12 0.11 0.12

0.035

35.2

0.15 0.12

34.7 34.6 35.2

a Amount of Mb adsorbed at MPS. bVolume fraction of pores occupied by Mb. cVolume fraction of Mb within Mb/MPS conjugate calculated using eq 1. dVolume fraction of Mb within Mb/MPS conjugate obtained by the best fit of SANS data. eDiameter of spherical Mb obtained by the best fit of SANS data.

Figure 3. Plots of normalized absorbance at 409 nm versus temperature (T). The solid lines are the results of best fit using the two-state denaturation mechanism.

Mb/MPS conjugates prepared with SBA-type MPSs exhibited relatively moderate changes in the Soret band absorbance with temperature. The plots of the Soret band absorbance against temperature were analyzed using a simplified two-state model, assuming zero heat capacity (Figure 3).30 Free energies of Mb denaturation at 25 °C, ΔGD, obtained by the fitting analysis, are 43 ± 4 kcal mol−1 for Mb/SBA39, 88 ± 20 kcal mol−1 for Mb/FSM40, 40 ± 9 kcal mol−1 for Mb/SBA70, and 42 ± 13 kcal mol−1 for Mb/SBA75. The 2-fold greater ΔGD for Mb/ FSM40 indicates that the stability of Mb within FSM40 is lower than when within SBA-type MPSs. The secondary structure of Mb within MPS was examined by ATR-FTIR measurements. Figure 4 shows typical FTIR spectra of Mb in bulk water and within MPSs. In each spectrum, no marked peaks due to an intermolecular β-sheet (1618 and 1683 cm−1)31 are seen. Intermolecular β-sheet bands are characteristic of intermolecular antiparallel β-sheet aggregation induced by thermal denaturation of Mb.31 It can, hence, be considered that monomeric Mb molecules are dispersed inside all of the MPS pores. In free Mb in bulk water, the peak position of amide I band is 1651 cm−1, and the peak intensity ratio of the amide I/II bands is 1.1. The spectral shapes for Mb within SBA-type MPSs are essentially same as those for free Mb: the peak position of the amide I band is 1651 cm−1 and the peak intensity ratio of the amide I/II bands is 1.1. It can thus be concluded that the secondary structure of Mb within the SBAtype MPSs is almost identical to that of folded Mb in bulk water.8 On the other hand, Mb within FSM-type MPS exhibited amide I peak at 1651 and 1640 cm−1. The peak at

Figure 2. Transmission-absorption spectra for Mb/MPS conjugates suspended in water and for Mb before/after thermal denaturation in bulk water. The absorption spectrum for the denatured Mb was obtained in water containing 3 M urea at 80 °C. Other spectra were obtained at 25 °C.

It can, therefore, be concluded that the heme environment of Mb is almost the same for all MPS systems at around 25 °C. Figure 3 shows the plots of Soret band absorbance against temperature. A significant decrease in the Soret band absorbance was seen for Mb/FSM40 above 320 K. In contrast, 15570

DOI: 10.1021/acs.jpcc.8b04356 J. Phys. Chem. C 2018, 122, 15567−15574

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decreasing the volume fractions of D2O and completely disappeared in the 62.1% D2O solution, the contrast-matching point for FSM40 (Figure S7). On the other hand, the SANS profile for 40% D2O, whose contrast was matched to Mb,26 is almost the same as that of MPS75 in 100% D2O (Figure 1A). The slight deviation in the high Q region is due to the influence of incoherent scattering of H2O. The scattering intensities of Mb/MPS75 in an H2O/D2O mixture can be described with partial scattering functions Sij(Q) by I(Q ) = ΔρS2 SSS(Q ) + 2ΔρS ΔρM SSM(Q ) + ΔρM2 SMM(Q ) (2)

where SSS and SMM are the self-terms of silica (MPS) and Mb, respectively.32 SSM is the cross-term between silica and Mb. Δρi (with i = S and M for silica and Mb, respectively) is the scattering contrast between component i and the solvent. The SANS data for Mb/MPS75 were decomposed into partial scattering functions using singular-value decomposition.32 The partial scattering functions estimated are shown in Figure 6. Figure 4. ATR-FTIR spectra for Mb/MPS conjugates suspended in water and for Mb in bulk water (20 mg mL−1). All spectra were obtained at room temperature (25 °C).

1640 cm−1 indicates an increased content of the unordered structure, accompanied by a decrease in the α-helix content.8,31 This change in the secondary structure is also indicated by the high peak intensity ratio (1.4) of the amide I/II bands.8 The results for UV−vis absorption spectra indicated that Mb molecules within FSM40 were not completely unfolded at 25 °C. It, therefore, can be concluded that adsorption into the pores of FSM40 causes Mb to partially unfold. Contrast Variation in SANS for Mb/MPS75. Figure 5 shows SANS profiles for Mb/MPS75 dispersed in H2O/D2O mixtures with different volume fractions of D2O (ϕD2O = 100, 80, 70, 62.1, and 40%). The SANS profiles of Mb/MPS75 in D2O show intense Bragg diffraction peaks due to the hexagonal pore arrangement. These Bragg diffraction peaks decreased on

Figure 6. Partial scattering functions obtained for Mb/SBA75 conjugate suspended in D2O/H2O mixture.

The profile of SSS agrees closely with the SANS profile of MPS75 in D2O (Figure 1A), and the profile of SMM reflects the structure and volume fraction of Mb within MPS75, as mentioned below. Of note is the negative value of SSM for the scattering functions. The negative sign suggests a spatial crosscorrelation between the silica matrix and Mb inside the pores of MPS75,32 that is, the Mb molecules are located in the pore interior. This result supports the lowering of freezing temperature due to the Mb adsorption into the MPS pores. Analysis of SANS Profiles of Mb/MPS Conjugates. The diameter of globular Mb used in this study was estimated by analyzing the SANS profile for Mb in 100% D2O solution. Figure 7A shows SANS profiles of diluted Mb solution (2.5 mg mL−1 in 100% D2O). The total scattering intensity I(Q) is taken as the sum of scattering from Mb molecules, IMb(Q), and background, Iinc, due to incoherent scattering from Mb protons and can be treated as a constant. On the basis of the Percus− Yevick (PY) hard-sphere model,33 the total scattering intensity is given by I(Q ) = P(Q )S(Q ) + Iinc

(3)

with S(Q) being the structure factor and P(Q) the form factor of Mb. P(Q) for the spherical shape is given by ÄÅ ÉÑ2 ÑÑ K ÅÅÅ 3V Δρ ÑÑ Å − P(Q ) = ÅÅ QR QR QR (sin( ) cos( )) ÑÑ ÑÑÖ V ÅÅÇ (QR )3 (4)

Figure 5. SANS profiles for Mb/SBA75 conjugates dispersed in H2O/ D2O mixtures with different volume fractions of D2O. 15571

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Figure 7. SANS profiles of (A) free Mb in bulk D2O and of (B)−(D) Mb/MPS conjugates prepared with SBA-type MPSs. The solid lines are the results of best fit using eq 3.

where R is the radius of spherical Mb, V is the volume of a single Mb molecule, Δρ is the scattering contrast between Mb and the medium surrounding Mb, and K is the scale (constant). The structure factor depends on the volume fraction (concentration) of Mb in the solution34 and can be regarded as 1 for a diluted Mb solution. SansView software was used for the following fitting analysis and simulation of SANS profiles (http://www.sasview.org/). The SANS profile for free Mb could be closely fitted to eq 3, and the fitting analysis provided a radius of 17.6 Å for Mb. This value agrees closely with 18 Å, determined by the SAXS experiment, for the same myoglobin provided by Sigma-Aldrich.35 None of the MPSs suspended in the contrast-matching solvent (62.4% D2O) showed the Bragg diffraction peaks in their SANS profiles (Figure S8). As shown in Figures 7B−D and 8, the Bragg diffraction peaks were also not recognized for all Mb/MPS conjugates suspended in the contrast-matching solvent. These results indicate that the SANS profiles for the Mb/MPS conjugates can be described by the self-term of Mb and incoherent scattering from protons of Mb, surface silanol groups, and H2O. 3 was, therefore, used to analyze the SANS profiles of the Mb/MPS conjugates. In the SANS profile for Mb/FSM40 conjugate (Figure 8), a monotonic increase in scattering intensity was identified in the low Q range (Q < 0.07 Å−1). Similarly, Mb/MPS conjugates composed of SBA-type MPSs also exhibit increased scattering intensity in the low Q range (Q < 0.03 Å−1; Figure S9). One plausible reason for the monotonic increase in intensity in the low Q range is neutron scattering from aggregated Mb molecules but Mb aggregation was not recognized in the ATR-IR spectra (Figure 4). We conclude that the neutron scattering in the low Q range is due to ambiguous particle

Figure 8. SANS profiles of Mb/FSM40 conjugates. The solid lines are the results of best fit using eq 3 with a fixed Mb radius (R). The fitting analysis was performed by fixing the radius of Mb ranging from 13 to 19 Å.

morphology and/or irregularities on the surfaces and the internal structures of MPS particles. In the contrast-matching condition, although each MPS particle possesses a large amount of Mb molecules within the particle interior (Table 2), the Mb molecules within the particle interior are likely to reflect these ambiguous MPS structures. This conclusion can be supported by the Porod slopes in the low Q range. The Porod slopes for Mb/SBA39, Mb/FSM40, Mb/SBA70, and Mb/SBA75 are −1.3, −1.9, −2.9, and −2.6, respectively (Figure S9). The lowest Porod slope is obtained for the Mb/ SBA39 conjugate with the lowest volume fraction of Mb (Table 2). The shape of the MPS particle morphology appears to become clearer by increasing the volume fraction of Mb. The neutron scattering due to the surface and internal 15572

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ratio of Q2/Q3 surface silanols. This difference in surface character may be due to the different surface charges of the template surfactant employed in the synthesis of MPS: FSMand SBA-type MPSs are synthesized using cationic and nonionic surfactants, respectively.36 The results in the present study also confirm that the surface characteristics of MPS are key influences on protein structure within MPS.

structure of MPS particle is likely to increase with adsorption of Mb within MPS. The SANS data in the middle Q range (0.05 Å−1 < Q < 0.4 −1 Å ) were used to analyze the Mb tertiary structure using eq 3. The fitting parameters are the radius and volume fraction of Mb, scale (K), and incoherent scattering from protons (Iinc). For Mb/MPS conjugates prepared by SBA-type MPSs, the SANS profiles could be closely fitted to eq 2, as shown in Figure 7B−D. The Mb diameter and volume fraction of Mb (ϕMb) within MPS, obtained by the fitting analysis, are listed in Table 2. The values of ϕMb are almost the same as those estimated by adsorption assay, indicating adequacy of the fitting analysis. Although the diameter of Mb within MPS is consistent with that of free Mb in bulk water, it can be concluded that Mb molecules retain their globular structure after their adsorption into pores of SBA-type MPSs. The preservation of the secondary structure of Mb before/after adsorption is confirmed by FTIR experiments (Figure 4). We, therefore, conclude that the Mb structure is not perturbed by adsorption within SBA-type MPSs with BJH pore diameters of 39−75 Å. The size matching between Mb (D = 35.2 Å) and pore (DBJH = 39 Å) does not affect the Mb structure. In contrast to SBA-type MPS systems, the SANS profile of Mb/FSM40 could not be closely fitted to the PY hard-sphere model. We, hence, fitted the experimental profile (0.09 Å−1 < Q < 0.4 Å−1) to eq 3 by fixing the radius of Mb. As shown in Figure 8, the fitting profiles obtained with a large Mb radius (17 and 19 Å) coincide with the experimental profile around 0.12 Å−1, whereas a deviation between the experimental and fitting profiles is apparent at above 0.18 Å−1. In contrast, the fitting profiles obtained with small Mb radii (13 and 15 Å) deviate from the experimental profile at around 0.12 Å−1. These results suggest that the size and shape of the Mb tertiary structure are not uniform inside the pores of FSM40. Lack of uniformity of the Mb tertiary structure is likely related to the partial unfolding of the Mb secondary structure. Our visible absorption spectroscopy experiments confirm that the globular Mb structure is not completely broken at 25 °C (Figure 3), suggesting that only slight perturbation of the globular Mb structure is induced by confinement inside the pores of FSMtype MPS. Although the BJH pore diameters of FSM40 and SBA39 are almost the same, changes in the secondary and tertiary structures of Mb are found only in the FSM40 system. This result suggests that the Mb structures are affected by the characteristics of the inner pore surface rather than by the geometrical pore size. Interfacial Mb/silica interaction is stronger in the FSM40 system than that in the SBA39 system because the greater amount of Mb can be loaded into the pores of FSM40. The volume fraction of pores occupied by Mb (f Mb) at the adsorption maximum is 4.8 times larger for FSM40 than that for SBA39 (Table 2). The stronger interfacial interaction is what appears to cause the changes in the Mb structure within FSM40. It is also likely to be responsible for the lower structural stability of Mb within FSM40 (Figure 3). Takahashi et al. reported that FSM-type MPS could adsorb larger amounts of horseradish peroxidase (HRP) than those of SBA-type MPS.36 In their study, the higher HRP adsorption ability of FSM-type MPS was explained by its different surface characteristics because only FSM-type MPS exhibited a significant dependence of solution pH on HRP adsorption. Their results suggest that FSM- and SBA-type MPSs have different Brønsted acidities of surface silanol groups and/or the

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CONCLUSIONS In the present study, we used visible and IR absorption spectroscopy, as well as SANS, to examine the structure of Mb confined within various MPSs. The results led us to conclude that Mb holds its globular structure after adsorption into the pores of SBA-type MPSs with BJH pore diameters ranging from 39 to 75 Å. Although FSM40 could adsorb large amounts of Mb due to strong interfacial Mb/silica interaction, the secondary and tertiary structures of Mb were perturbed inside its pores. The structural stability of Mb within FSM40 was lower than that within SBA-type MPSs. These findings suggest that the Mb structure is affected more by interfacial interactions than by geometrical pore size.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.8b04356. Pore distribution plots, SAXS data, adsorption isotherm data, DSC curves, additional absorption spectra for Mb/ MPS conjugates, and additional SANS data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-29228-8389. ORCID

Akira Yamaguchi: 0000-0003-3029-3775 Present Address ⊥

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan (Y.S.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by JSPS KAKENHI Grant nos. JP16H04160, 17K19022, and 15H03841. We acknowledge Dr. Kazuhiro Akutsu, CROSS, for the SANS measurements. The SANS experiment was performed with the approval of the Neutron Program Review Committee (Proposal nos. 2015A0086, 2016B0051, 2017A0078, and 2017B0130). This work benefited from the use of the SasView application, originally developed under NSF award DMR0520547. SasView contains code developed with funding from the European Union’s Horizon 2020 research and innovation program under the SINE2020 project, Grant agreement no. 654000.

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