CHEMPHYSCHEM ARTICLES DOI: 10.1002/cphc.201300863

Small-Angle Neutron Scattering Studies of Hemoglobin Confined Inside Silica Tubes of Varying Sizes Soumit S. Mandal,[a] Viviana Cristiglio,[b] Peter Lindner,[b] and Aninda J. Bhattacharyya*[a] In addition to the chemical nature of the surface, the dimensions of the confining host exert a significant influence on confined protein structures; this results in immense biological implications, especially those concerning the enzymatic activities of the protein. This study probes the structure of hemoglobin (Hb), a model protein, confined inside silica tubes with pore diameters that vary by one order of magnitude (  20–200 nm). The effect of confinement on the protein structure is probed by comparison with the structure of the protein in solution. Small-angle neutron scattering (SANS), which provides information on protein tertiary and quaternary structures, is employed to study the influence of the tube pore diameter on the structure and configuration of the confined protein in detail. Confinement significantly influences the structural stability of Hb

and the structure depends on the Si-tube pore diameter. The high radius of gyration (Rg) and polydispersity of Hb in the 20 nm diameter Si-tube indicates that Hb undergoes a significant amount of aggregation. However, for Si-tube diameters greater or equal to 100 nm, the Rg of Hb is found to be in very close proximity to that obtained from the protein data bank (PDB) reported structure (Rg of native Hb = 23.8 ). This strongly indicates that the protein has a preference for the more native-like non-aggregated state if confined inside tubes of diameter greater or equal to 100 nm. Further insight into the Hb structure is obtained from the distance distribution function, p(r), and ab initio models calculated from the SANS patterns. These also suggest that the Si-tube size is a key parameter for protein stability and structure.

1. Introduction Confinement of proteins inside various artificial porous substrates has become a subject of immense scientific importance as it is a realistic approach to mimic several in vivo biological processes. Confinement results not only in structural changes in the protein, but also influences its functional properties. Hence, confinement is also beneficial from the point of view of biotechnological applications. Structural changes in the confined protein are due to several physical and chemical factors related to the confining host. Porous substrates (e.g. Au, Ag, SiO2, TiO2) with proteins encapsulated inside them have been demonstrated to be highly effective in applications such as sensing, catalysis, and controlled delivery.[1–3] It has been observed that the surface chemistry of the host plays a significant role in altering the conformations as well as the dynamics of biologically relevant molecules. An important example is the case of enzyme delivery, in which the enzyme diffusion kinetics are significantly influenced by hydrophilic or hydrophobic groups on the host surface.[4]

[a] S. S. Mandal, Prof. A. J. Bhattacharyya Solid State and Structural Chemistry Unit Indian Institute of Science Bangalore 560012 (India) Fax: (+ 91) 80-23601310 E-mail: [email protected] [b] Dr. V. Cristiglio, Dr. P. Lindner Institut Laue-Langevin (ILL) 38042 Grenoble Cedex 9 (France) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cphc.201300863.

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It is a common phenomenon that proteins with a globular structure prefer to remain folded in compact configurations. Their folded configurations make these proteins biologically active. On the basis of the theoretical cage model proposed by Anfinsen,[5] it has been shown that confined denatured proteins fold at a much faster rate than proteins in bulk solution. Protein folding in a confined medium with attractive, repulsive, or inert walls has been extensively studied.[6, 7] Repulsive interactions may stabilize proteins to a greater extent than weak interactions (e.g. hydrogen bonding, van der Waals), which may destabilize proteins and lead to aggregation. However, strong interactions may cause partial or complete adsorption of the protein onto the confining host surface, which results in loss of its native structure. Within a cell, proteins participate in various biophysical processes in the presence of several types of macromolecules. Compact (folded) native states of proteins may be stabilized at intermediate concentrations but not at high concentrations. The folded states of the protein are stabilized by an efficient balance between repulsive and attractive interactions between the protein domains. In addition to the chemical nature of the host surface, the size and network of the pores in which the protein resides need serious consideration. It has been shown that if the pore size is very close to the critical size, which is determined by the smallest dimension of a protein’s folded state, protein stability is affected and the protein may not fold at all.[8, 9] With increasing protein size, the smallest dimension of a protein’s folded state also increases.[10–13] However, the folding rate may not match one-to-one with the changes in the host pore size. ChemPhysChem 2014, 15, 302 – 309

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CHEMPHYSCHEM ARTICLES The protein folding rates must have an optimum value if the pore size is much larger than the size of the folded state of the protein. If the pore size becomes comparable to the dimension of the folded state, the folding rate may decrease due to destabilization in the transition states related to the folding process. Most of the details discussed above are based on theoretical findings and experimental support is rare. There are few experimental reports that discuss the structural aspects of hemoglobin (Hb) on confinement inside various porous substrates.[14] In this paper, we experimentally investigate the effect of silica tube pore size on the protein structure. Smallangle neutron scattering (SANS) is used to directly probe differences in Hb that is confined inside Si-tubes of varying diameters. Small-angle scattering, in particular SANS, yields spatial information on the length scale from a few to hundreds of nanometers, and thus allows the characterization of changes that occur in the tertiary and quaternary structures of proteins. We have already demonstrated the beneficial effects of Hb confinement inside organic matrices, namely layered polymer capsules, by using synchrotron SAXS.[15, 16] The role of host size on the structure and function of confined Hb could not be studied in ref. [15] due to the non-trivialities in the synthesis of various sizes of polymer capsules. Ref. [16] serves as the basis for the undertaking of the present work, which intends to illustrate the full extent of the role of host pore size on protein structure and function. Modeling of the 1D scattering profiles obtained from SANS yields useful insight into the 3D protein structure. The radius of gyration (Rg) and polydispersity (p) in the size distribution were estimated following modeling of the SANS profiles. The SANS results combined with circular dichroism (CD) and UV/Vis spectroscopic investigations were carried out in order to obtain a comprehensive understanding of the effect of confinement inside Si-tubes.

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Figure 1. A) and B) SEM images of Si-tubes. C–D) TEM images of Si-tubes with pore diameter C) 20 nm, D) 100 nm, and E) 200 nm. F) Confocal laser scanning microscopy (CLSM) images of fluorescein isothiocyanate (FITC)tagged Hb confined inside Si-tubes.

Table 1. Concentration of confined Hb within Si-tubes of varying diameter.

2. Results and Discussion The as-synthesized Si-tubes were characterized using both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Figure 1). Figure 1 (C–E) show the Si-tubes with internal diameters of approximately 20–200 nm. The details of the tube morphology, in particular the various heterogeneities, have been discussed in refs. [17, 18]. The structural heterogeneities in the tube mainly arise from heterogeneities in the templates (Anodisc) used for Si-tube synthesis. The hydrophobic interactions between neighbouring Hb molecules due to the non-polar side chains of the amino acid residues are also very important. These interactions provide the driving force for the adsorption of a significant amount of Hb into the Si-tubes.[19] The amount of Hb encapsulated within the Situbes was estimated from UV/Vis spectroscopy. The values are given in Table 1. Si-tubes impregnated with Hb are abbreviated as Si-tube-Hb20 (Si-tube diameter = 20 nm), Si-tube-Hb100 (Sitube diameter = 100 nm), and Si-tube-Hb200 (Si-tube diameter = 200 nm).  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Sample

Concentration of confined Hb [mg mL1]

Si-tube-Hb20 Si-tube-Hb100 Si-tube-Hb200

3.48 3.65 3.84

CD Spectra The effect of the Si-tube pore diameter on the secondary structure of Hb was investigated using CD spectroscopy. Figure 2 shows the CD spectrum of bare Hb in solution and Hb confined inside Si-tubes of varying pore diameter. The two bands at 222 nm and 208 nm arise due to the peptide n–p* transition and exciton splitting of the lowest peptide p–p* transition, respectively. This result indicates that Hb exists predominantly in the ahelix conformation in solution. This is in close agreement with previous literature reports.[20] In addition, Hb predominantly retains its secondary structure under confinement inside the Situbes. To further probe the effect of the Si-tube pore diameter on the Hb structure, a least-squares fitting analysis of the secondary structure was performed in the wavelength range ChemPhysChem 2014, 15, 302 – 309

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Figure 2. CD spectra at 25 8C of Hb (-&-), Si-tube-Hb20 (-D-), Si-tube-Hb100 (-^-), and Si-tube-Hb200 (-*-) in 0.1 m PBS buffer (pH 7.0).

Table 2. Percentage of a-helix and b-sheet constituting the secondary structure of Hb. Sample

a -helix [%]

b-sheet [%]

Hb Si-tube-Hb20 Si-tube-Hb100 Si-tube-Hb200

50 40 48 45

8.19 17 12 14

(200–250) nm in order to account for the changes in the fractions of a-helix and b-strand. Table 2 shows the variation in fractions of the individual components obtained after fitting as a function of pore diameter. It is evident from the above analysis that confinement exerts a significant influence on the secondary structure of Hb in solution. As seen from the CD spectrum of Hb as well as the spectra of the Si-tube confined Hb samples, the intensity of the band at 222 nm was much more negative for Hb and Si-tube-Hb100 and Si-tube-Hb200 than that of Si-tube-Hb20. The variation in intensity is reflected in the fraction of a-helix and b-sheet. The fraction of b-sheet is approximately doubled in the case of Si-tube-Hb20 compared to bare Hb in solution, although in the other cases it is slightly less than double. This indicates that the secondary structure of Hb is significantly affected by confinement. SANS Analysis of Hb and Si-Tube-Hb Composite Samples: Effect of Pore Diameter Figure 3 shows the SANS profile of Hb in 0.1 m phosphate saline buffer (PBS) solution (pH 7). The SANS profile of Hb can be divided into three distinct regions, namely the low-q region up to 0.02 1, the intermediate region from 0.02 to 0.15 1 and the high-q region greater than 0.15 1. The global shape of the protein can be estimated from the scattering pattern in the intermediate-q region. In this region the scattering intensity decays rapidly with q. The scattering pattern can be modeled by assuming the molecular shape to be either a sphere or  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 3. SANS pattern for Hb in PBS solution. The solid black line indicates the fit assuming only the sphere model and the grey line indicates the fit assuming a Schultz distribution for the number distribution of the sphere sizes. A), B) and C) represent the different regions of the SANS profile.

an ellipsoid. Model fitting is more accurate than the Guinier analysis as it fits a larger q region to draw conclusions about the tertiary and quaternary molecular structure. In the high-q range the data points have lower statistical certainty and are affected by incoherent background correction, whereas at low q the data points are sensitive to the effect of the beam stopper and aggregation. In the low-q range, the experimental data for Hb show a strong increase in intensity that cannot be accounted for by any model. Although samples were freshly prepared and centrifuged prior to measurement, low-q upturn was significant and could not be reduced completely. The low-q upturn in scattering intensity has been reported for many protein systems and so far there is no conclusive explanation for this phenomenon.[21] It has been shown to depend on sample preparation, protein purity, and ageing.[21c] The upturn at low q could also be attributed to the existence of long-range[21a] or shortrange attractive interactions. Short-range attraction and longrange repulsion are known to be the major driving forces in the formation of equilibrium clusters in colloidal systems. These factors may also play an important role in protein solutions and might account for the observations at low q. Hydrophobic interactions in proteins are not unheard of and may lead to protein aggregation. This may contribute to the appearance of the upturn in the scattering profile in the low-q regime. We do not intend to speculate on or fit the upturn feature in the low-q region of the scattering profile as this is beyond the scope of this paper. The situation in the case of the Si-tube-Hb samples is different. The sharp rise in intensity observed in the low-q region, 0.007–0.02 1, can be attributed to the interfacial scattering from the interface of Si-tubes, which are approximately 20, 100, and 200 nm in diameter and polydisperse. The slope of the low-q region decreases as approximately q4, which indicates a smooth, sharp interface. Similar observations arising due to sharp interfaces have been reported in several neutroChemPhysChem 2014, 15, 302 – 309

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n[22a] and X-ray[22b,c] scattering experiments in dilute solutions and ultra filtrates. The interparticle separation of the particles constituting the Si-tube is below the minimum q accessible in SANS. A detailed study of the particles might be possible with ultra-small angle scattering methods. The SANS profile of Hb was first fitted with the sphere model as reported in the literature.[23] The deviation in the fitting (Figure 3) indicates that Hb molecules are no longer monodisperse with respect to their size. The SANS profile could be better fitted by introducing a polydispersity function to account for the non-uniform distribution in the size, shape, and mass of the spherical particles. The polydispersity is well accounted for by the Schultz sphere distribution formalism (Figures 3 and 4; see the Experimental Section for details). As

In order to compare the solution structure of Hb with the single crystal structure, the fitted profile was compared with the scattering profile generated from the PDB structure by using CRYSOL. The deviation of the scattering profile of Hb in solution from that of the PDB structure indicates that the protein structure in solution is different from the PDB structure. In addition, as discussed in the previous section, Hb has a polydisperse size distribution in solution, and this is another important reason for this deviation. It is known that the Hb molecule is composed of a tetramer of 2a and 2b subunits with a diameter of approximately 12 , each giving rise to an Rg value of 24 .[24] This Rg value is slightly affected by changes in its quaternary structure.[25] The Rg value was confirmed on the basis of the estimate made using the crystallographic PDB structure in the CRYSOL software. The discrepancy between the estimated and experimentally obtained Rg values could be attributed to the absence of monodispersity. The increase in Rg may be due to the partial unfolding of either of the subunits or to dimerization[26] in the Hb samples used in the experiments. Since the protein samples were obtained commercially, the samples may not be of the highest purity. However, the main thrust of this work was to investigate the effect of the host on the aggregated Hb structure, so these samples were used without further purification for other measurements. The high polydispersity (p = 0.6) also supports the presence of spherical particles of varying diameters. This becomes clearer in the subsequent sections of this paper, in which the pair distance distribution is discussed. The Hb in solution was compared with Hb confined inside the Si-tubes. Figure 4 shows the SANS profiles of Hb confined inside Si-tubes of varying diameters. In this case too, the SANS profiles can be well described using the Schultz distribution for the number distribution of Figure 4. SANS profiles for Hb and Si-tube-Hb in 0.1 m PBS. The Hb profile the sphere sizes. It can be seen in Table 3 that under confinewas fitted using a Schultz distribution for the number distribution of the ment the Rg decreases significantly except in the case of the sphere sizes. In the Si-tube-Hb system, the low-q data was fitted using a Porod function (q < 0.02, grey) and the higher-q region (q > 0.02, black) Si-tubes of 20 nm diameter. Both the Rg and the polydispersity was fitted using a Schultz distribution for the number distribution of the of Hb confined in Si-tube-Hb20 are very high compared to sphere sizes. For clarity, the curves have been shifted up using a suitable those of Hb inside Si-tube-Hb100 and Si-tube-Hb200. The Rg of factor. Hb inside Si-tube-Hb100 was closer to the values reported in the literature.[21] The variation in Rg and the polydispersity of stated earlier, Rg of Hb was estimated from the mean radius the Hb in Si-tube-Hb100 and Si-tube-Hb200 samples was at(Rm) of Hb and the polydispersity (p) factor obtained from the tributed to the environment created by the Si-tube around the fit. The value of Rg was estimated to be 31.2  with polydisHb molecules. The strong interaction between the hydrophilic groups on the Hb surface with the surface hydroxyl groups on persity, p = 0.6. The high values of Rg and p indicate the existhe Si-tube imposes constraints on the movement of the Hb tence of Hb (in buffer solution) in a highly polydisperse state molecules close to the surface inside the Si-tube. This prevents with respect to its size. The fitted parameters are shown in these Hb molecules (close to the host surface) from being in Table 3. close proximity to those molecules away from the surface. These results also suggest that Table 3. Fitted parameters for Hb in solution and confined inside Si-tubes of varying diameters. confinement in Si-tubes with diameters of 100 and 200 nm may Polydispersity(p) Rg (Guinier analysis) Rg (GNOM) Sample Rg (Schultz sphere) [] [] [] aid the reduction of the destabilization factors related to interHb 31.2  0.1 0.58  0.01 32.2  0.3 30.2  0.2 Si-tube-Hb20 33.8  0.2 0.59  0.05 32.6  0.7 33.3  0.6 molecular and molecule–solvent Si-tube-Hb100 23  0.1 0.45  0.02 23.5  0.2 23.1  0.1 interactions. In solution, an indiSi-tube-Hb200 26.5  0.3 0.55  0.06 26.3  0.3 25.29  0.1 vidual Hb molecule is under the  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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CHEMPHYSCHEM ARTICLES influence of the neighbouring Hb molecules. This significantly alters its configuration in solution. The situation in the case of Si-tube-Hb20 is not very different from that of the protein in solution. Due to the small diameter of the Si-tube, the proteins reside close to each other, which leads to the retention of its aggregated state. The Rg values (Table 3) also strongly suggest that the diameter of the Si-tube has a significant influence on the structure of Hb. We strongly believe that if the as-received Hb sample was purely monodisperse and non-aggregated in solution, it would retain a similar state regardless of the size of the confining Situbes. In the present study the protein is aggregated, and the size of the tube does matter. In the 100 and 200 nm diameter tubes, the transition from the aggregated to the non-aggregated state takes place due to the reasons explained earlier. However, the transformation from the aggregated to the non-aggregated state did not take place in the 20 nm diameter Sitube as in this case the detrimental intermolecular interactions could not be nullified. The Rg values estimated from model fitting were found to very close to those obtained from the Guinier approximation (Figure 5). Analysis of the Hb SANS profile in the Guinier

www.chemphyschem.org low-q region makes it difficult to determine the extent of aggregation in these samples. Dmax, the maximal diameter of the particle, is extracted from the pairwise distance distribution function profiles, p(r). The p(r) is determined from the Fourier transform of the scattering intensity profile. It is a representation of the distribution of radial distances throughout the macromolecule. For Hb in solution, Dmax was determined to be 88  and the Rg was estimated to be approximately 30 . From Figure 6, it can be observed

Figure 6. Distance distribution function, p(r) calculated for Hb (-&-), Si-tubeHb100 (-*-), and Si-tube-Hb200 (-~-) using the experimental SANS data. The distance distribution function was generated using the program GNOM.

Figure 5. Representative Guinier plots of SANS data. a) Hb, b) Si-tube-Hb20, c) Si-tube-Hb100, and d) Si-tube-Hb200. The curves have been shifted up for clarity.

region indicated that Hb has a slightly unfolded state in solution. This may be attributed to the starting material used for sample preparation, which might be in a slightly unfolded state even after centrifugation (10 000 rpm for 30 min) and separation. The ionic strength of the buffer solution and hence the presence of salts also screens the long range repulsive electrostatic interaction between proteins, which allows them to come closer together and thus form aggregates. The presence of aggregates in the solution means that the solution is no longer monodisperse. This would also explain the absence of an interaction peak in the data as there is no longer a welldefined average d-spacing between particles.[27] However, in the Si-tube-Hb samples, the contribution of the Si-tube in the  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

that the curve representing Hb has a broad peak extending from 20 to 40  and a small shoulder at 75  with comparatively lower intensity. The presence of the broad peak and a very high Dmax value of 88  indicate partial unfolding of Hb, which results in different distances between the macromolecule fragments. The curves for the Si-tube-Hb200 and Si-tubeHb100 samples were much smoother compared to the Hb solution sample. The Rg values estimated from the p(r) were found to be approximately 25.2 and 23.6  for the Si-tubes of 200 and 100 nm diameter, respectively. The Dmax was found to be 68 and 58  for the 200 and 100 nm samples, respectively. Smooth bell shaped plots were observed for the 100 and 200 nm Si-tubes. Confinement of Hb inside 200 and 100 nm Situbes led to a significant decrease in the Dmax value for Hb. In the case of Si-tube-Hb20, the curve was highly unsymmetrical (see Figure S2 in the Supporting Information). Due to the small size of the tube, a considerable fraction of the protein resides outside in very close proximity to the tube. The ensuing protein–protein and protein–tube interactions result in the formation of protein aggregates of widely varying sizes. The inhomogeneity in size and the distance between these aggregates makes the pairwise distance distribution highly uneven. To obtain further insight into the structure of Hb in solution and inside the Si-tubes, ab initio methods (based on the hardsphere approximation) have been developed for the reconstruction of low-resolution molecular models of protein moleChemPhysChem 2014, 15, 302 – 309

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www.chemphyschem.org propriate size may successfully nullify the detrimental intermolecular and molecule–solvent interactions that lead to protein aggregation. SANS can serve as a valuable tool in carrying out protein confinement studies. Using appropriate theoretical models, various important physical parameters related to proteins can be extracted from the SANS data. These can be very useful for both fundamental understanding and optimizations of various biotechnological applications.

Experimental Section Preparation of Silica Tubes

Figure 7. Ab initio shape reconstruction from SANS data collected for A) Hb in solution, B) Si-tube-Hb100, and C) Si-tube-Hb200.

cules using experimentally obtained SANS data. The program DAMMIN,[28] which calculates an arrangement of dummy atoms and minimizes their computed scattering profile against that of the experimental SANS data, was used. The final model represented in Figure 7 is the result obtained after averaging 15 model structures calculated using the program DAMAVER.[29] Top and side views of selected SANS-biased DAMMIN models of Hb and Hb confined inside Si-tubes are also shown in Figure 7. The model obtained for Hb in solution had an elongated shape with a longest dimension of approximately 88 . For Hb inside the 100 and 200 nm diameter Si-tubes, the generated models were nearly spherical or oval-shaped with dimensions of 58 and 68 . The differences between the Hb model generated here and the crystal structure of Hb may be due to the difference in experimental conditions involved. In the model Hb is in solution, whereas in the crystal state the structure depends on the conditions used for crystallization.

3. Conclusions We have successfully investigated the structure of Hb in solution and under confinement inside Si-tubes using SANS. We specifically focused on the effect of the Si-tube pore diameter on the structure of Hb. It was found that pore dimensions significantly influence the stability of Hb and hence its tendency towards aggregation. In comparison to its structure in solution, Hb preferentially remains in a more native-like (non-aggregated) state if confined inside Si-tubes of appropriate size. In exceptional cases, such as inside the Si-tube with a pore diameter of approximately 20 nm, Hb undergoes significant aggregation, which is evident from the high values of Rg. However, if the tube diameter is approximately 100 nm, the Rg of Hb is closer to the Rg obtained from the PDB structure, which indicates that the Si-tube with a diameter of 100 nm provides a more favorable environment than the 20 nm diameter Sitube. Thus, we can conclude that, in addition to the surface chemistry of the host, the host size (with respect to the size of the protein) is also a key parameter in protein confinement studies and related applications. It appears that a host of ap 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Si-tubes of varying diameter were synthesized using commercially available Anodisc templates with pore diameters of 50, 100, 200 ( 50, the thickness of the wall) nm. The Anodisc templates were immersed in SiCl4/CCl4 = 1:9 (10 vol %). The remnant SiCl4 was removed from the faces of the Anodisc templates by washing with CCl4. The templates were immersed in a fresh CCl4 solution for 30 min to remove the unreacted SiCl4 from the pores. Next, the Anodisc membranes were immersed in a (1:1 v/v) mixture of CCl4methanol solution for 2 min, then in ethanol for 5 min to displace the CCl4. The membranes were dried under an argon atmosphere then immersed in deionized water for 5 min and methanol for 2 min. The membranes were dried again under an argon atmosphere. This entire cycle was repeated 10 times. Finally, in order to separate the Si-tubes from the membrane, the Anodisc membranes were dissolved in an aqueous solution of 70 % H2SO4 at 70 8C. The Si-tubes were separated by centrifugation, then washed several times with millipore water and ethanol, and finally dried at 55– 70 8C to obtain dry Si-tubes.

Preparation of Silica Tube-Hemoglobin Composite Samples The Hb samples comprised a solution of human Hb (commercially acquired from Sigma Aldrich, 4 mg mL1) in a PBS solution (0.1 m, pH 7). This solution was centrifuged at 10 000 rpm at 4 8C for 30 min prior to measurement. For the preparation of the Si-tubeHb composite samples, the above supernatant was incubated with a solution of Si-tubes (varying diameters, 10 mg mL1) in PBS (0.1 m, pH 7) at 4 8C for 24 h. Following incubation, the free Hb was removed by centrifugation for 5 min at 2000–3000 rpm to separate the Si-tubes containing Hb. The concentration of the impregnated Hb within the Si-tubes was estimated from the difference in concentration of the parent solution used for incubation and that of the supernatant after centrifugation. The samples for neutron scattering measurements were prepared using the same protocol. PBS prepared in D2O was used for sample preparation. The use of D2O as a solvent instead of H2O provides a better contrast for hydrogenous proteins in neutron experiments. The concentrations of encapsulated Hb were almost the same as those mentioned in Table 1 under similar conditions.

Electron Microscopy TEM images were recorded on a FEI Technai F30 with an acceleration voltage of 200 kV. An ethanol dispersion of the Si-tubes was drop-casted on the Cu grid with a carbon-reinforced plastic film. The grid was dried overnight. SEM analysis of the sample drop-cast onto a silicon wafer was carried out using ESEM quanta using a voltage of 20 kV. ChemPhysChem 2014, 15, 302 – 309

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Confocal Laser Scanning Microscopy Confocal laser scanning microscopy (CLSM) imaging was carried out to confirm the location of Hb in the Si-tube-Hb composites. CLSM imaging was carried out using a Zeiss 510 Meta confocal microscope (60  objectives). The details of the sample preparation have been discussed in refs. [15, 16] and references therein. Briefly, Hb was tagged with a fluorescent dye, fluorescein isothiocyanate (FITC) prior to incubation with the Si-tube. After incubating for 24 h, the solution of the Si-tube with the tagged Hb inside was centrifuged and the Si-tube-Hb composite was separated. This composite was redispersed in PBS solution. Before imaging, the solution was dropped onto a microscope glass slide and covered with a cover slip. The edge of the cover slip was sealed with nail polish and the slide was analyzed by CLSM using an excitation wavelength of 488 nm.

UV/Vis Spectroscopy The confinement of Hb within the Si-tube was confirmed from the difference in concentration between the stock solution used for incubation and supernatant collected after centrifugation. The measurement was carried out using a Nanodrop UV/Vis spectrophotometer (ND-1000). The absorbance (at 280 nm) was converted to concentration using the molar extinction coefficient (e) value of 49 500 m1 cm1 (assuming the Hb to be met-Hb).

The radius of gyration (Rg) obtained using the model fitting was compared with that obtained from the Guinier plot (plot of lnI vs. q2). The pair-distance distribution functions were obtained by fitting the scattering data using the program GNOM.[31] This also provided an alternative method for the estimation of Rg. The maximum diameter of the particle (Dmax) was determined empirically by examining the quality of the fit to the experimental data for a range of Dmax values. The models of proteins were generated by an advanced modeling approach using the program DAMMIN,[28] an automated procedure based on simulated annealing, which uses compactness and connectivity constraints and optionally a priori structural information. To reduce the time required for the computations, the program uses spherical harmonics to calculate the model intensity. The input of experimental data occurred through a GNOM file, in which 16 independent calculations were carried out. Finally these structures were aligned using the program DAMAVER[29] to build the model structure of the protein. The program PYMOL was used for the primary pictorial representation of all models obtained. Surface representations of the models were applied to allow a closer comparison of the structural features of the various protein models.

SANS Analysis The coherent differential scattering cross-section per unit volume (dS/dW) for the monodisperse spheres can be expressed as shown in Equation 2:

Circular Dichroism Far-UV CD spectra were obtained using a JASCO-J-810 spectropolarimeter. A solution of Hb (10 mL) or Si-tube-Hb (10 mL) were mixed with fresh buffer solution (490 mL). These solutions were used to record the CD spectrum in quartz cuvettes with 1 mm path length. The least-squares fitting analysis of the secondary structures was performed over a wavelength of 200–240 nm with the program SELCON3.[30]

Small Angle Neutron Scattering Measurement and Data Treatment

  dSðQÞ ¼ nV 2 1p  1s 2 PðQÞSðQÞ þ Bkg dW

ð2Þ

where n denotes the number density, V is the particle volume, 1p and 1s are the scattering length densities of the particle (protein in this case) and solvent (buffer), respectively. P(Q) is the form factor of the particle (protein) and S(Q) is the interparticle structure factor arising as a result of the interaction between the particles. Bkg is a constant term representing the scattering due to the background. The value of P(Q) depends on the particle shape and size. For a spherical particle of radius R, its value is given by Equation 3: 

 3fsinðQRÞ  ðQRÞ cosðQRÞg 2 ðQRÞ3

The SANS experiments were performed on the D11 and D16 instruments at the Institut Laue-Langevin, Grenoble, France. The momentum transfer (q) and the scattering angle (2 q) are related to the neutron wavelength (l) according to Equation 1:

PðqÞ ¼

q ¼ ð4 p=lÞsinq

The S(Q) term provides information about the interactions and correlations between the particles. In dilute solutions the interparticle interactions become negligible and S(Q) = 1.

ð1Þ

On D11, the wavelength was set to 6 . The experiments were carried out with a sample-to-detector distance of 1.2 and 8 m to cover a q range of 0.007–0.5 1. The scattering intensities were normalized with a standard water measurement after the required transmission measurements had been carried out. On D11, the measurements were carried out in quartz cuvettes with 1 mm thickness. On D16, a neutron wavelength of 4.767  and a sampleto-detector distance of 1 m was used to cover a momentum transfer range of 0.06–1.8 1. The measurements were carried out in a cylindrical vanadium sample holder with an approximate diameter of 10 mm. This configuration was set on both instruments in order to give a considerable overlap. The scattering contributions of the empty sample holders and the buffer were subtracted from the samples. The scattering intensity was normalized with standard samples (Cadmium for D11, Vanadium for D16). The scattering pattern was analysed by fitting with a Schultz distribution of spheres.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ð3Þ

However, if the system deviates from monodispersity, a distribution needs to be included into the equation. For a polydisperse system, Equation 2 has to be modified to Equation 4: dSðQÞ ¼ dðQÞ

Z

dSðQ; RÞ f ðRÞdR þ Bkg dðQÞ

ð4Þ

where f(R) is the distribution function which takes into account the polydispersity factor. In the following discussion we use the Schultz distribution function.[32] The Schultz distribution function is given as Equation 5: f ðRÞ ¼

      Z þ 1 ðZþ1Þ Z Zþ1 1 R R exp  Rm Rm GðZ þ 1Þ

ChemPhysChem 2014, 15, 302 – 309

ð5Þ

308

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where Rm is the mean diameter, Z is the width of distribution and G represents the gamma distribution. The polydispersity factor (p) is related to the width of the distribution as shown in Equation 6: Z ¼ 1=ðp2  1Þ

ð6Þ

Acknowledgements

[15] [16] [17] [18] [19]

S.S.M. and A.J.B. would like to thank Bhabha Atomic Research Centre, Mumbai, India and Institut Laue-Langevin, Grenoble, France for providing the travel and instrumental support to carry out the SANS measurements.

[20] [21]

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Received: September 18, 2013 Revised: November 14, 2013 Published online on December 12, 2013

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