pH-Dependent Structures of Ferritin and Apoferritin in Solution

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pH-Dependent Structures of Ferritin and Apoferritin in Solution: Disassembly and Reassembly Mihee Kim,† Yecheol Rho,† Kyeong Sik Jin,*,‡ Byungcheol Ahn,† Sungmin Jung,† Heesoo Kim,*,§ and Moonhor Ree*,†,‡ †

Department of Chemistry, Division of Advanced Material Science, Center for Electro-Photo Behaviors in Advanced Molecular Systems, BK School of Molecular Science, and Polymer Research Institute, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea ‡ Pohang Accelerator Laboratory, Pohang University of Science & Technology, Pohang 790-784, Republic of Korea § Department of Microbiology and Dongguk Medical Institute, Dongguk University College of Medicine, Gyeongju 780-714, Republic of Korea ABSTRACT: The pH-dependent structures of the ferritin shell (apoferritin, 24-mer) and the ferrihydrite core, under physiological conditions that permit enzymatic activity, were investigated by synchrotron small-angle X-ray scattering (SAXS). The solution structure of apoferritin was found to be nearly identical to the crystal structure. The shell thickness and hollow core volumes were estimated. The intact hollow spherical apoferritin was stable over a wide pH range, 3.40
10.0, and the ferrihydrite core was stable over the pH range 2.10
10.0. The apoferritin subunits underwent aggregation below pH 0.80, whereas the ferrihydrite cores aggregated below pH 2.10 as a result of the disassembly of the ferritin shell under the strongly acidic conditions. As the pH decreased from 3.40 to 0.80, apoferritin underwent stepwise disassembly by first forming a hollow sphere with two holes, then a headset-shaped structure, and, finally, rodlike oligomers. As the pH was increased from pH 1.96, the disassembled rodlike oligomers recovered only to the headset-shaped structure, and the disassembled headset-shaped intermediates recovered only to the hollow spherical structure with two hole defects. The apoferritin hole defects that formed during the disassembly process did not heal as the pH was increased to neutral or slightly basic conditions. The pH-induced apoferritin disassembly and reassembly processes were not fully reversible, although they were pseudoreversible over a limited pH range, between 10.0 and 2.66.

’ INTRODUCTION Ferritin is a major iron storage protein that can store up to 4500 iron atoms within the protein shell.1 Iron is sequestered as a hydrous ferric oxide phosphate mineral that is structurally similar to ferrihydrite (5Fe2O3 3 9H2O).2 The ferritin protein without bound iron is called apoferritin and is composed of 24 structurally homologous subunits.3
7 Two types of subunits are present, heavy (H) and light (L) subunits, classified according to the subunit molecular weight, 21 and 19 kDa, respectively.8 The ratio of the two types of subunits in apoferritin is species- and tissuespecific because each subunit carries out a different function.9 In horses, liver and heart apoferritins are composed of 40
50 wt % H subunits and 85
90 wt % H subunits, whereas spleen apoferritin contains 90 wt % L subunits.10 The H subunits play a major role in iron oxidation, and the L subunits are involved in the efficient nucleation and mineralization of iron.3,10 These subunits assemble as a hollow rhombic dodecahedron protein shell with 4-3-2 symmetry that forms intersubunit channels along the three- and four-fold axes to accommodate iron.11 The crystal structure of apoferritin was found to have a ferritin core (i.e., a ferrihydrite core),3
7 as determined by transmission r 2011 American Chemical Society

electron microscopy (TEM).12,13 The structures of apoferritin and the ferritin core in solution were investigated by small-angle X-ray scattering (SAXS). These studies provided only limited structural information, such as the form factor profiles, the shape of the spherical hollow, and the size.14 The stability of apoferritin in the presence of chemical and physical denaturants was tested using gel filtration, sedimentation
velocity analysis, circular dichroism, and fluorescence spectroscopy.15
19 Apoferritin was found to be highly resistant to chemical and physical denaturants. For example, horse spleen apoferritin denatured when heated above 80
C for 10 min15 or at pH values 13.0.17 These studies did not provide any direct structural insight into denaturation and the denatured state. The spherical hollow of apoferritin permits the protein’s use as a carrier for drug delivery, magnetic resonance imaging contrast agents, fluorescence dye markers, or other compounds. The hollow may also be used as a template for the fabrication of Received: January 5, 2011 Revised: March 18, 2011 Published: March 29, 2011 1629

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Biomacromolecules quantum dots and nanoparticles. For these reasons, apoferritin has attracted much attention from academia and industry. Recently, several research groups have investigated the use of apoferritin as a delivery carrier or template.20,21 These applications would be greatly assisted by an understanding of the solution structure of apoferritin, the structural changes induced in response to the solution environment, and the disassembly and reassembly processes of apoferritin. The molecular weight, secondary, and tertiary structural changes of apoferritin were examined as a function of disassembly by gel electrophoresis, ultracentrifugation, circular dichroism, and fluorescence spectroscopy.22
24 The studies suggest that apoferritin reversibly dissociates and associates, depending on the pH. However, these investigations provided only indirect evidence of structural changes during the disassembly and reassembly processes. Modeling of the full disassembly and reassembly processes, including the 3D intermediate structures, has not been possible. The structure of apoferritin under physiological conditions and its structural changes during disassembly and reassembly have not yet been unambiguously elucidated. In this study, we investigated the 3D structures of apoferritin and the ferrihydrite core under physiological conditions, and we examined their disassembly and reassembly processes using synchrotron SAXS measurements and quantitative data analysis. The stabilities of apoferritin and the ferrihydrite core were examined over a broad range of pH value in solution. Quantitative analysis of the structural details of the apoferritin intermediates associated with the disassembly and reassembly processes lends crucial insight into the assembly
disassembly mechanism of apoferritin.

’ EXPERIMENTAL SECTION Materials and Sample Preparation. Apoferritin from equine spleen in 0.15 M sodium chloride solution and ferritin from equine spleen in saline solution were purchased from Sigma Aldrich Company and used without further purification. Apoferritin and ferritin solutions were prepared over a concentration range of 1
10 mg/mL. In the case of ferritin, additional samples were prepared by using 80% sucrose solution by volume whose electron density is equal to that of the ferritin shell (namely, apoferritin), as described in the literature,5 to get information about the ferritin core (i.e., ferrihydrite core). For all of the protein solutions, their pH value was carefully adjusted with the addition of small amounts of sodium hydroxide or hydrochloric acid by a pH meter. Solution SAXS Measurements. SAXS measurements were carried out using the 4C1 SAXS beamline (BL)25,26 of the Pohang Accelerator Laboratory (that is so-called Pohang Light Source (PLS))27 at the Pohang University of Science and Technology, Korea. A light source from a bending magnet of the PLS storage ring was focused with a toroidal silicon mirror coated with platinum and monochromatized with a W/B4C double multilayer monochromator, giving an X-ray beam of wavelength 1.608 Å. The X-ray beam size at the sample stage was 1  1 mm2. A 2D charge-coupled detector (CCD) (Mar USA) was employed. Sample-to-detector distances of 0.5 and 3.0 m were used. The scattering angle was calibrated with silver behenate and poly(styrene-b-ethylene-b-butadiene-b-styrene) standards. We used solution sample cells with mica windows 10 μm in thickness, a volume of 50 μL, and X-ray beam path length of 0.7 mm. All scattering measurements were carried out at 25
C. The SAXS data were collected in 10 successive frames of 1 min each to monitor radiation damage. The absence of changes in the scattering patterns with time was confirmed, indicating that no radiation damage occurred during the scattering

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measurements. SAXS measurements were conducted on protein solutions over the concentration range 1
10 mg/mL to obtain good quality scattering data without any interference between the protein molecules (i.e., to eliminate any concentration effect), consequently finding that the highest quality scattering data can be obtainable with concentrations in the range 2
5 mg/mL. Each measured 2D SAXS pattern was circularly averaged from the beam center, normalized to the transmitted X-ray beam intensity (monitored with a scintillation counter placed behind the sample cell) and corrected for scattering arising from the buffer solution. Data Analysis. Experimental scattering data were analyzed with a scattering program GNOM28 to obtain the pair distance distribution function p(r) profiles. p(r) can be expressed as follows Z ¥ 1 pðrÞ ¼ 2 qrIðqÞ sinðqrÞ dq ð1Þ 2π 0 where q is the magnitude of the scattering vector defined by q = (4π/λ) sin θ, 2θ is the scattering angle, λ is the wavelength of the X-ray beam, and r is the distance between the paired scattering elements in the macromolecule. This approach provides an alternative method for the calculation of the radius of gyration (Rg,p(r)) and I(0) from the full scattering curve, and the maximum diameter of a given macromolecule (Dmax) can be found from the distance at which p(r) approaches zero. Rg, p(r) can be expressed by the following equation Z r 2 pðrÞ dr 2 ¼ Z Rg;pðrÞ ð2Þ 2 pðrÞ dr Because this expression makes use of the whole scattering curve, it is much less sensitive to factors (e.g., the presence of residual interparticle interactions or even of a small amount of aggregation) than the computationally more straightforward Guinier relation. Indeed, as with the Guinier relation, the effect of interparticle interaction is most restricted to the innermost part of the scattering pattern. Construction of 3D Structural Models. To reconstruct molecular shapes of the proteins in solution, we used the ab initio molecular shape determination programs GASBOR29 and DAMMIN.30 The reconstructed models were obtained without imposing any symmetry restrictions. The atomic coordinates of the crystallographic model of apoferritin (which was used in this study) were obtained from the Protein Data Bank (PDB code: 3F32).7 The SAXS curve from the atomic model was evaluated using the program CRYSOL.31 Superposition of the crystallographic atomic model to the structural models reconstructed from the experimental data was carried out by using the program SUPCOMB.32 Surface rendering was done by using the program Discovery Studio 2.0 (Accelrys).

’ RESULTS AND DISCUSSION Structural Stability of Apoferritin and Ferritin Core. Figure 1 shows the representative SAXS profiles of apoferritin solution measured at various pH values. Furthermore, the measured scattering data are compared with the theoretical SAXS profiles calculated for the crystal structure of apoferritin and its subunit (Figure 1). Here it is noted that the apoferritin crystal was prepared in a solution of pH 5.0, which was composed of 0.2 M sodium acetate, 0.2 to 1.2 M (NH4)2SO4, and 0.1 to 0.225 M CdSO4; the crystal was found to contain 201 water molecules.7 In addition to apoferritin, the ferritin core was investigated under various pH conditions. Representative scattering data are shown in Figure 2, which were measured for ferritin in 80% sucrose solution at various pH values. The used 1630

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Figure 1. Scattering profiles of apoferritin measured over a wide pH range of 0.80
10.0. The symbols indicate the experimental data, and the solid lines indicate the fits obtained using the GNOM program. The solid lines without symbols are the theoretical SAXS curves calculated from the crystal structure of apoferritin and its subunit crystal (PDB code 3F32). For clarity, each plot is shifted along the log I axis. Figure 3. Pair distance distribution functions for (a) apoferritin and (b) ferritin core in solution under several pH conditions, which were determined by an analysis of the experimental SAXS data using the GNOM program.

Figure 2. Scattering profiles of ferritin core measured in 80% sucrose solution over the pH range 0.80
10.0. The symbols indicate the experimental data, and the solid lines indicate the fits obtained using the GNOM program. For clarity, each plot is shifted along the log I axis.

sucrose solution has an electron density matched with that of the apoferritin, and thus the measured scattering data can give structural information about the ferritin core. We attempted to analyze further the SAXS profiles in detail. The pair distance distribution function p(r) is an expression in real space of the scattering profile so that it is an intuitively accessible function that helps in the visualization of ferritin shell and core under various pH conditions. This function further provides the radius of gyration Rg,p(r), which is based on the full scattering curve and gives the maximum dimension Dmax of apoferritin or ferritin core as the distance where the p(r) function approaches zero. Thus, p(r) functions were determined from the measured scattering profiles as well as the calculated scattering profiles of the apoferritin and subunit in crystal structures by indirect Fourier transform using the program GNOM.28 The obtained p(r) functions are displayed in Figure 3. The determined Rg,p(r) and Dmax values are listed in Table 1. As can be seen in Figure 1, the scattering profile of apoferritin measured at pH 7.30 reveals oscillation peaks, indicating that apoferritin has a spherical shape. This scattering profile resembles that calculated for the apoferritin crystal. Similar scattering profiles were observed for the apoferritin solutions with pH 3.40
10.0. This scattering characteristic is directly reflected in the p(r) function. Apoferritin reveals similar p(r) function over the range pH 1631

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Table 1. Structural Parameters Obtained from SAXS Measurements of Apoferritin and Ferritin Core in Various pH Solutions pH (solution)

apoferritin Rg,p(r) (nm)a

subunit crystal (i.e., monomer)

1.94 ((0.01)c

apoferritin crystal (24-mer)

5.28 ((0.06)

ferritin core pH (solution)

Dmax (nm)b

Rg,p(r) (nm)a

Dmax (nm)b

3.28 ((0.03)

10.2

6.10 12.0

10.0

5.48 ((0.01)

12.2

9.40

5.51 ((0.01)

12.3

10.0 9.00

3.29 ((0.02)

9.50

7.70

5.54 ((0.01)

12.2

8.00

3.23 ((0.02)

9.80

7.30

5.58 ((0.01)

12.3

7.20

3.27 ((0.02)

9.80

6.50

5.55 ((0.02)

12.2

6.00

3.25 ((0.01)

9.60

5.40 4.20

5.60 ((0.02) 5.54 ((0.02)

12.3 12.3

5.40 4.10

3.44 ((0.04) 3.54 ((0.03)

9.90 10.0

3.40

5.59 ((0.02)

12.3

3.10

3.29 ((0.02)

9.70

1.90

3.70 ((0.04)

11.5

2.10

3.06 ((0.02)

8.90

0.80

13.3d

0.80

a

14.1d

b

Rg,p(r) (radius of gyration) calculated from the p(r) function for the measured SAXS profile. Dmax (maximum dimension of particle) obtained from the p(r) function for the measured SAXS profile. c Standard deviation. d Rg obtained from the Guinier analysis of SAXS data.

3.40
10.0 (Figure 3a). The Rg,p(r) and Dmax values at pH 3.40
10.0 range 5.48
5.60 nm and 12.2
12.3 nm, respectively, which are comparable to those of apoferritin in crystal (Table 1). These results confirm that apoferritin is stable in the range of pH 3.40
10.0. The horse spleen apoferritin used in this study is mainly composed of L-chain subunit. Therefore, the observed stability of apoferritin at the wide pH range is attributed to the L-chain subunit’s stability. Such a high stability of L-chain subunit to the pH changes might be originated from the strong hydrogen bondings among inter- and intrasubunit and the salt bridge between Lys59 and Glu104, which were previously reported.19 However, in highly acidic solutions of below pH 3.40, apoferritin exhibited quite different scattering profiles. The apoferritin solution at pH 1.90 revealed no oscillation peaks (Figure 1). The p(r) function is also highly deviated from those at pH 3.40
10.0 and that of apoferritin crystal (Figure 3a). The obtained Rg,p(r) (3.70 nm) and Dmax (11.5 nm) are smaller than those of apoferritin at pH 3.40
10.0 and apoferritin crystal. The scattering profile and its p(r) function are somewhat similar to those calculated for the crystal structure of apoferritin subunit. In particular, the peak maximum of the p(r) function is close to that of the subunit crystal in rodlike structure. However, the Rg,p(r) and Dmax values are still almost two times larger than those of the subunit crystal, respectively. These results collectively indicate that under strong acidic condition such as pH 1.90 apoferritin underwent remarkable structural change from hollow sphere structure to disassembled oligomeric intermediates. Furthermore, at pH 0.80 apoferritin exhibits scattering profile significantly deviated even from that of the apoferritin subunit in crystal (Figure 1). The scattering profile in the Guinier plot was found to have no linearity (data not shown), informing us that apoferritin at pH 0.80 has no definite size and thus is polydisperse. By the Guinier analysis of the scattering profile, the largest radius of gyration (Rg) is estimated to be 13.3 nm, which is almost two times larger than that of apoferritin at pH 3.40
10.0. Hence, apoferritin at pH 0.80 is considered to be aggregated. Such aggregation was also detected in the gel filtration and sedimentation
velocity analysis conducted below pH 1.0.17 The aggregation might be originated from the denaturation of apoferritin that takes place below pH 1.0. The above scattering analysis results collectively give structural information on apoferritin in solution as follows. Apoferritin in

solution apparently retains its hollow spherical shape as the pH value is down to pH ∼3.0. However, below pH 3.0, apoferritin becomes unstable with decreasing pH value and then undergoes collapse and dissociation (i.e., disassembly). Under extremely strong acidic condition such as pH 0.80, the disassembled subunits further undergo aggregation, which might be driven by van der Waals and nonspecific hydrogen bonding interactions, ultimately causing inhomogenous and polydisperse apoferritin solution. SAXS data of ferritin core in solution and their p(r) function are shown in Figures 2 and 3b, respectively. Ferritin core at pH 7.20 reveals no oscillation peaks in the SAXS profile. Similar scattering profiles were observed over the pH range 3.10
10.0. These data are quite different from those of apoferritin under the corresponding pH conditions. The p(r) functions exhibit a single peak pattern (Rg,p(r) = 3.23 to 3.54 nm and Dmax = 9.50 to 10.2 nm), which is a typical characteristic of a compact globular form. In comparison, ferritin core at pH 2.10 apparently reveals scattering profile similar to those measured over the pH range 3.10
10.0. However, its p(r) function clearly shows somewhat difference from those at pH 3.10
10.0. Rg,p(r) and Dmax were determined to be 3.06 and 8.90 nm, respectively, which are a little bit smaller than those at pH 3.10
10.0. This relatively small reduction in the size of ferritin core is presumably attributed to several possible factors, such as partial dissolution of ferrihydrite core from the surface via chemical reactions with Hþ ion33 and escape of the free ferrous irons trapped in the core part with a limited volume, and release of the ferrous irons weakly bound to the core while the collapse of ferritin shell part occurs in solution under such an acidic condition. Furthermore, the ferritin core at pH 0.80 reveals scattering profile significantly different from those at pH 3.10
10.0 (Figure 2). In the Guinier plot, the scattering profile showed no linearity. The Guinier analysis found that ferritin core at pH 0.80 has a maximum Rg value of 14.1 nm, which is much larger than that of ferritin core itself and even larger than that of ferritin shell. The result indicates that at pH 0.80, the ferritin core underwent aggregation. The above scattering data analyses collectively inform us that the ferritin core apparently retains its shape over a wide pH range of 2.10
10.0 but undergoes aggregation below pH 2.10. In 1632

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Figure 5. Structural models reconstructed from the experimental SAXS data for ferritin core and apoferritin shell in neutral solution using the DAMMIN program: (a) ferritin core (i.e., ferrihydrite core) at pH 7.20; (b) the structural model of ferritin core was superimposed onto that of apoferritin in Figure 4a to provide information about degree of filling of ferrihydrite in the core of ferritin protein. Blue color and red color arrows mean horizontal cross-section and vertical cross-section, respectively. Surface rendering in the structural models was achieved using the Discovery Studio 2.0 program of Accelrys.

Figure 4. Structural models reconstructed from the experimental SAXS data for apoferritin in neutral solution: (a) structural model of apoferritin in solution of pH 7.30; (b) ribbon diagram for the crystal structure of apoferritin (PDB code 3F32); (c) ribbon diagram for the crystal structure of apoferritin in part b was superimposed on the reconstructed structural model of apoferritin at pH 7.30 using SUPCOMB to compare overall shape and dimension as well as to reconfirm the hollow sphere structure; (d) ribbon diagram for the crystal structure of apoferritin subunit, which is superimposed on the structural model reconstructed from the theoretical SAXS curve calculated from the subunit crystal using the CRYSOL program; (e) structural model cut into half of apoferritin structure in part a; and (f) structural model of the domain highlighted with pink color in part e, where ribbon diagram for the crystal structure of apoferritin subunits is superimposed on the structural model.

particular, the aggregation of ferritin cores at pH 0.80 seems to be a result of the disassembly of ferritin shell occurred under such the strong acidic condition. To corroborate the above p(r) function analysis results and to obtain a 3D representation of the structure of apoferritin and ferritin core under various pH conditions, model-independent structural models were reconstructed using the ab initio shape determination programs, GASBOR29 and DAMMIN.30 These reconstructions were performed inside the search volume of maximum dimension

Dmax calculated from the p(r) function determined for a set of measured scattering data by the GNOM program. The reconstructed models were obtained without imposing any restrictions on the symmetry and anisometry of the molecules. Figure 4a shows the structural model reconstructed for apoferritin in the solution of pH 7.30. The structure of apoferritin crystal, which was grown at pH 5.0,4,7 is presented in Figure 4b. The crystal structure is superimposed on the reconstructed 3D structure in Figure 4c. As can be seen in the Figure, the 3D structure reconstructed from the SAXS analysis is well-matched with the crystal structure, which shows highly ordered hollow spherical structure. As discussed in the Introduction, apoferritin is known to be composed of 24 subunits. From the single subunit structure,4,7 the SAXS profile was generated, as shown in Figure 1. From the SAXS profile and data analysis, a 3D structure was reconstructed. The reconstructed 3D structure is presented in Figure 4d and superimposed with the subunit structure, showing a good match. To obtain more detailed structural information and to reconfirm the hollow sphere structure of apoferritin, we did cut the reconstructed structural model of apoferritin into half in a twostep process. As can be seen in Figure 4e, the structural model cut in half along the horizontal axis of the reconstructed 3D structure clearly demonstrates a half-moon-shaped structure with empty core. A further structural model cut in half along the vertical axis of the half-moon-shaped structure shows a crescent-moonshaped structure (Figure 4f). From the reconstructed structural models, the shell thickness of apoferritin protein was estimated to be ∼2.3 ( 0.4 nm. Interestingly enough, the obtained shell thickness is in good agreement with that (2.5 to 3.0 nm) estimated previously from the crystal structure.4,7,34 The 3D structure reconstruction work was extended for ferritin core from the measured SAXS profiles. Figure 5a shows the 3D structure of ferritin core, which was reconstructed from the SAXS data measured under the neutral pH condition. As can be seen in the Figure, the overall shape of ferritin core apparently looks like a compact globular structure. This 3D structure is in reasonably good agreement with that previously reported from TEM analysis.12 To get information about the relative volume of ferrihydrite core with respect to the whole empty core space of 1633

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Table 2. Structural Parameters Obtained from SAXS Measurements of Apoferritin during pH-Induced Disassembly Process Apoferritin pH-induced process

pH (solution)

Rg,p(r) (nm)a

Dmax (nm)b

disassembly

3.40

5.59 ((0.02)c

12.3

3.24

5.31 ((0.02)

12.3

3.08

5.29 ((0.02)

12.3

2.95

5.23 ((0.02)

12.3

2.85 2.73

5.18 ((0.02) 5.19 ((0.02)

12.2 12.5

2.66

5.17 ((0.03)

12.6

2.40

4.82 ((0.05)

12.6

1.96

3.72 ((0.07)

11.4

1.90

3.70 ((0.04)

11.5

a

Rg,p(r) (radius of gyration) was calculated from the p(r) function by the program GNOM. b Dmax (maximum dimension of particle) was obtained from the p(r) function by the program GNOM. c Standard deviation.

Figure 6. Scattering profiles of apoferritin measured during disassembly process from pH 3.40 to 1.90. The symbols indicate the experimental data, and the solid lines indicate the fits obtained using the GNOM program. For clarity, each plot is shifted along the log I axis.

apoferritin, the 3D structure of ferrihydrite core was attempted to be superimposed to that of apoferritin (Figure 5b). The total volume of ferrihydrite is estimated to be ∼142.0 nm3 when the densely packed dummy atoms with a radius of 0.25 nm (which were used to reconstruct the 3D structural model of apoferritin shell from the SAXS data using the program DAMMIN) are adopted. The estimated volume of ferrihydrite corresponds to ∼60.9% of the hollow core volume of apoferritin (233.2 nm3). In fact, it was previously reported that individual native ferritin generally contains fewer than 2000 Fe atoms in the inner core, although the ferritin shell (i.e., apoferritin) has capacity to store ferrihydrite core composed of up to 4500 Fe atoms.21,35 Considering these facts, the above estimated volume of ferrihydrite core and its occupation ratio in the hollow core of apoferritin are quite reasonable. Disassembly and Reassembly Behavior of Apoferritin. As discussed in the previous section, apoferritin retains in solution the highly ordered hollow sphere structure over the wide pH range 3.40
10.0 but becomes unstable below pH 3.40. Thus, below pH 3.40, disassembly process of apoferritin in solution was investigated in detail by SAXS measurements and quantitative data analysis. The measured SAXS data are shown in Figure 6, and the scattering data analysis results are summarized in Table 2. Furthermore, we carried out 3D structural reconstructions from the scattering data by using the DAMMIN program, as described in the previous section. The reconstructed 3D structures are presented in Figure 7. As can be seen in Figure 6, the scattering of apoferritin at pH 3.24 apparently still resembles that measured at pH 3.40 or higher (Figure 1). However, the oscillation peaks in the scattering profile are damped down slightly compared with those of the scattering profile at pH 3.40. Such a damping of oscillation peaks

becomes more significant with decreasing pH in the solution. Such oscillation peaks ultimately disappear below pH 2.40. These results indicate that overall apoferritin reveals hollow spherical shape over the range of pH 3.40
2.40, but its structural details are somewhat changed depending on the pH condition; such structural change becomes more significant with decreasing pH value. Furthermore, below pH 2.40, apoferritin completely loses spherical structure, causing its structure to become very far from the spherical structure found at pH 3.40 or higher. These pH-induced structural changes are confirmed in the determined Rg,p(r) and Dmax data (Table 2) as well as in the reconstructed 3D structures (Figure 7). The 3D structure of apoferritin at pH 3.40 is identical to those under the neutral condition (pH 7.30) (Figures 4a and 7a), which shows an intact hollow sphere. Moreover, its dimension parameters are the same as those under the neutral condition. However, some structural changes are observed below pH 3.40. In the case of pH 3.24, overall apoferritin shows hollow spherical shape, but two holes are newly observed at the north and south poles of the spherical structure, respectively (Figure 7b). Each hole is estimated to have a diameter of ∼6.5 nm. This hole size is comparable to that of dimer, namely, two subunits. Thus, at pH 3.24, four subunits were disassembled away from the hollow spherical structure. The slight reduction observed in Rg,p(r) was attributed to the disassembled four subunits. Such holes become larger at pH 3.08, whose area is estimated to be ∼6.7  9.2 nm (Figure 7c). The hole size corresponds to that of three subunits. Therefore, six subunits were disassembled away from the hollow spherical structure. At pH 2.85, the area of the holes is determined to be ∼7.8  11.1 nm (Figure 7d), which corresponds to that of four subunits. As a result, at pH 2.85, eight subunits were disassembled away from the hollow spherical structure. These subunit disassemblies are correlated to the reductions in Rg,p(r) (Table 2). With further decreasing pH value, apoferritin loses more subunits, undergoing significantly structural changes. Apoferritin loses more subunits at pH 2.66, becoming a headset-shape structure (i.e., open-type of cage) (Figure 7e); at pH 2.40 apoferritin becomes more open headset-shape structure (Figure 7f). At pH 1.96, apoefrritin loses subunits significantly, becoming a rodlike structure (Figure 7g). This result confirms that at pH 1.96 apoferritin is 1634

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Figure 8. Scattering profiles of apoferritin measured during reassembly process from pH 3.24 to 9.48. The symbols indicate the experimental data, and the solid lines indicate the fits obtained using the GNOM program. For clarity, each plot is shifted along the log I axis.

Table 3. Structural Parameters Obtained from SAXS Measurements of Apoferritin during pH-Induced Reassembly Process Apoferritin pH-induced process

pH (solution)

reassembly from pH 3.24

3.24 3.60

5.31 ((0.02)c 5.29 ((0.02)

12.3 12.3

3.81

5.28 ((0.02)

12.5

7.25

5.24 ((0.01)

12.3

9.48

5.22 ((0.03)

12.4

2.66

5.11 ((0.02)

12.6

2.78

5.16 ((0.02)

12.5

2.91

5.21 ((0.02)

12.6

3.12 3.45

5.21 ((0.02) 5.22 ((0.02)

12.4 12.1

3.85

5.25 ((0.02)

12.6

7.34

5.29 ((0.02)

12.4

8.89

5.27 ((0.01)

12.1

1.96

3.72 ((0.07)

11.4

2.10

4.06 ((0.06)

11.6

2.53

4.63 ((0.04)

12.4

4.68 ((0.05)

12.4

reassembly from pH 2.66

Figure 7. Structural models of intermediates formed during the disassembly process of apoferritin, which were obtained from analysis of the scattering data in Figure 6 using the program DAMMIN. Surface rendering in the structural models was achieved using the Discovery Studio 2.0 program of Accelrys.

disassembled to rodlike oligomeric intermediates. Similar rodlike structure was found at pH 1.90 (data not shown). These drastic structure changes are evident in the Rg,p(r). With the above disassembly information, we attempted to examine reassembly process of apoferritin by increasing pH. Figure 8 presents the SAXS profiles of apoferritin measured with increasing pH value to 9.48 from 3.24. The scattering profile at pH 3.24 reveals oscillation peaks, which are attributed to spherical shape, as discussed above. Such a scattering profile was apparently found to retain during increasing pH of the solution to 9.48. The scattering data were further analyzed in the same manner, as

reassembly from pH 1.96

10.1

Rg,p(r) (nm)a

Dmax (nm)b

a

Rg,p(r) (radius of gyration) was calculated from the p(r) function by the program GNOM. b Dmax (maximum dimension of particle) was obtained from the p(r) function by the program GNOM. c Standard deviation.

described above. From the measured scattering data, the reconstructions of 3D structure were also conducted. As listed in Table 3, Rg,p(r) and Dmax are determined to be 5.22
5.31 and 12.3
12.5 nm, respectively, which are little varied with pH during 1635

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Figure 9. Scattering profiles of apoferritin measured during reassembly process from pH 2.66 to 8.89. The symbols indicate the experimental data, and the solid lines indicate the fits obtained using the GNOM program. For clarity, each plot is shifted along the log I axis.

Figure 10. Scattering profiles of apoferritin measured during reassembly process from pH 1.96 to 10.1. The symbols indicate the experimental data, and the solid lines indicate the fits obtained using the GNOM program. For clarity, each plot is shifted along the log I axis.

reassembly process. The 3D structure, which was reconstructed from the scattering data measured at pH 7.25, is identical to that at pH 3.24 (Figure 11a). The 3D structures, which were recovered from pH 3.08 and 2.85 by pH-induced reassembly processes, are identical to that at pH 3.24 (Figure 11b,c). These structures still have two holes as defects, which are a little bit far from the originally intact hollow sphere. These results collectively indicate that the two holes, which were once caused in the intact hollow spherical structure as defects during the pH-induced disassembly processes, could not be healed out by the pH-induced reassembly process. Figure 9 shows the SAXS profiles of apoferritin measured with increasing pH value to 8.89 from 2.66. As can be seen in the Figure, in the scattering profile, oscillation peaks (which are the scattering characteristics of spherical shape) become stronger in intensity as the pH of solution increases. These results suggest that the headsetshape of apoferritin at pH 2.66 is recovered back to spherical structure through reassembly process by increasing pH. The slightly low Rg,p(r) at pH 2.66 is again increased gradually with increasing pH, finally reaching 5.27 to 5.29 at pH 7.34 to 8.89 (Table 3). The recovered Rg,p(r) value is comparable to those at pH 3.08 and 3.24 in the disassembly process but still slightly lower than that (5.58 nm) of apoferritin under the neutral condition before disassembly process. Figure 11d shows a representative 3D structure, which was reconstructed from the scattering data measured at pH 7.34. The reconstructed 3D structure is apparently identical to that at pH 3.24 rather than that at pH 3.08 in the disassembly process. These analysis results inform that the headset-shape structure of apoferritin, which was once formed at pH 2.66 by disassembly process, is recovered to a hollow spherical structure having two holes as defects but could not be recovered completely to the original intact hollow spherical structure under the neutral condition. Figure 10 shows the SAXS profiles of apoferritin measured with increasing pH value to 10.1 from 1.96. With increasing pH,

oscillation peaks are developed very weakly but still very far from those observed under the neutral condition before disassembly process. The scattering data were analyzed and 3D structures were reconstructed. The results are shown in Table 3 and Figure 11e. The Rg,p(r) at pH 1.96 is 3.72 nm and somewhat increased with increasing pH, finally reaching 4.68 nm at pH 10.1, whereas the Dmax is increased from 11.4 nm at pH 1.96 to 12.4 nm at pH 10.1. The Rg,p(r) value is smaller than that of apoferritin under the neutral condition. As can be seen in Figure 11e, a headset-shaped structure is found at pH 7.70, which is similar to that observed at pH 2.66 in the disassembly process but very far from that under the neutral condition before disassembly process. These results conclude that the rodlike structure of apoferritin (which was once formed in disassembly process) is recovered to a headset-shaped structure with severe structural defects but never recovered to the original intact hollow spherical structure under the neutral condition. One may concern that the disassembly
reassembly process discussed above involves multicomponents in the solution, depending on pH values. For all measured scattering data of our study, their Guinier analysis showed only one slope. This result might be due to the following. The solution at a given pH value is composed of intermediates (which have a structure that is stabilized in the pH condition) and some fragments (which came off from apoferritin). The size of the intermediates is always greater than those of the fragments. Therefore, the larger size of the intermediates contributes substantially to the X-ray scattering, compared with the smaller sizes of the fragments. Therefore, the measured scattering data can monitor the structure of apoferritin and its intermediates during the disassembly
reassembly process. With the above structural results, we tried to figure out in detail structural components of the intermediates formed during disassembly and reassembly processes. We extracted various oligomeric fragments from the crystal structure of apoferritin consisting of 24 1636

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Figure 12. Crystal structures cut into fragments of apoferritin superimposed onto the structural models reconstructed from the experimental SAXS data measured during the disassembly and reassembly process, which were obtained using the programs SUPCOMB and Discovery Studio 2.0.

Figure 11. Structural models of the products recovered by subsequent reassembly processes of the intermediates formed in the disassembly of apoferritin, which were obtained from analysis of the measured scattering data using the program DAMMIN. Surface rendering in the structural models was achieved using the Discovery Studio 2.0 program of Accelrys.

subunits and then computed their SAXS profile and p(r) function. The computed SAXS profiles and p(r) functions were compared with those measured during pH-induced disassembly and reassembly process. The representative results are shown in Figures 12 and 13 and Table 4.

Figure 12 shows some of the oligomeric fragments extracted from the apoferritin crystal structure, which are superimposed onto the 3D structures reconstructed from the measured SAXS data by using the program SUPCOMB and Discovery Studio 2.0. The structure of a fragment consisting of 20 subunits (i.e., 20mer) is reasonably well-matched with the hollow sphere structure with two holes measured at pH 3.24 in the disassembly process (Figure 12a). The SAXS profile calculated from the fragment and its p(r) function are also reasonably matched with those measured at pH 3.24 in the disassembly process, respectively (Figure 13). These results confirm that the hollow sphere structure found at pH 3.24 is composed of 20 subunits and has two holes as defects. Therefore, the results further inform that the two holes formed as defects are attributed to the disassembly of two subunits (i.e., dimer), respectively. The headset-shaped structure measured at pH 2.66 in the disassembly process is well-matched with that of 12-meric fragment (Figure 12b). The scattering profile and p(r) function calculated for the fragment are also reasonably matched with those measured at pH 2.66 (Figure 13). These results support that the headset structure of apoferritin found at pH 2.66 is composed of 12 subunits. Furthermore, the rodlike structure measured at pH 1.96 is in good agreement with that of trimeric fragment (Figure 12c). The scattering profile calculated for the fragment is fairly wellmatched with that measured at pH 1.96 (Figure 13). Its p(r) 1637

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Table 4. Structural Parameters for the Extracted Oligomeric Crystal Structures and Apoferritin at Specific pH Values during Disassembly and Reassembly Process Apoferritin pH (solution)

a

Rg,p(r) (nm)

crystal structure b

Dmax (nm)

oligomer

a

Rg,p(r) (nm)

Dmax (nm)b

disassembly: 3.24

5.31 ((0.02)c

12.3

20-mer

5.34 ((0.02)

12.6

2.66

5.17 ((0.03)

12.6

12-mer

5.22 ((0.01)

12.6

3.72 ((0.07) 5.29 ((0.02)

11.4 12.4

3-mer 20-mer

3.05 ((0.01) 5.34 ((0.02)

9.90 12.6

1.96 reassembly (from pH2.66): 7.34

a Rg,p(r) (radius of gyration) was calculated from the p(r) function by the program GNOM. b Dmax (maximum dimension of particle) was obtained from the p(r) function by the program GNOM. c Standard deviation.

Figure 13. (a) Theoretical SAXS curves and (b) p(r) functions of the oligomeric fragments extracted from the crystal structure of apoferritin using the programs CRYSOL and GNOM. The symbols are the experimental data, and the solid lines are the fits using the GNOM program. For clarity, each plot is shifted along the log I axis.

function is close to the measured one but shows some deviations. The Dmax and Rg,p(r) values of the trimeric fragment are slightly smaller than those of the measured one, respectively (Table 4). The trimeric fragment is derived from the intact apoferritin by deleting the other 21 subunits and thus the dimer and monomer

in the trimeric fragment contact each other with an appropriate angle (Figure 12c), as observed in the intact hollow spherical apoferritin in 24-mer. In comparison, the trimer that resulted from the disassembly process may not hold such a specific angle between the dimer and monomer because it has a certain degree of mobility in solution. Therefore, the slightly larger Dmax and Rg,p(r) values of the measured one might come from loosing of such the specific contact angle between the dimer and monomer of the disassembled trimer in solution. In the case of the reassembly process of apoferritin from pH 2.66, the fragment consisting of 20 subunits (i.e., 20-mer) reasonably well fits the 3D structure determined at pH 7.34 (Figure 12d). The structure is the same as that observed at pH 3.24 in the disassembly process. The SAXS profile calculated from the fragment and its p(r) function are also reasonably matched with those measured at pH 7.34, respectively (Figure 13). These results collectively indicate that the headset structure of apoferritin formed in the disassembly process can be recovered to hollow spherical structure in the subsequent reassembly process, but the recovered sphere structure still has two holes as defects, which is a little bit far from the originally intact hollow sphere. As discussed in the Introduction, apoferritin is known to be composed of structurally homologous 24 subunits (i.e., 24-mer), making two-, three-, and four-fold symmetry.3
7,11 Six four-fold symmetries constitute hydrophobic channels with a size of 1.2 nm long and 0.3 to 0.4 nm wide, whereas eight three-fold symmetries constitute hydrophilic channels with a size of 0.3 to 0.4 nm long and wide.6 Each axis is connected with subunits by intersubunit hydrogen bonding and electrostatic interactions. The number of interaction sites is the most abundant in the twofold axis;6 therefore, the two-fold axis is the stable site. In contrast, the three-fold axis is known to be the least interaction site;6 therefore, the three-fold axis is the weakest site. Taking the above facts into account, the observed disassembly process results inform us that apoferritin in an intact hollow sphere structure initiates pH-induced disassembly process at the vicinity of three-fold channels (which are located in the opposite direction each other) and further undergoes the disassembly process in a stepwise manner through the formation of hollow sphere with two holes and headset-shaped structure as structural intermediates, ultimately dissociating to rodlike oligomers. Therefore, the formations of such intermediates are pivotal steps in the pH-induced disassembly process. In the reassembly process, the trimers in rodlike structure (which were formed as the most stable form through complete dissociation of apoferritin under highly acidic condition, pH 1.0 to 2.0) can gather together regardless of their orientation by increasing pH and undergo self-assembly, ultimately recovering 1638

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Biomacromolecules back to headset-shaped structures rather than the original intact hollow spherical structure. However the resulting headset structure is still very far from the originally intact hollow spherical structure of apoferritin under the neutral condition before disassembly process. In the case of the headset-shaped intermediates (which were formed by the disassembly process at pH 2.66), they can be recovered back to hollow spherical structure by increasing pH; but the recovered hollow sphere still has two holes as defects, which is a little bit far from the original intact hollow spherical structure. Overall, in the subsequently pHinduced reassembly process, structural recovery is highly dependent on the intermediates formed in the disassembly process. Here two questions arise from the results observed in the reassembly process. The first arising question is why the headsetstructured oligomer (which was reassembled from the rodlike oligomers) cannot undergo further to hollow spherical structure, compared with the headset-shaped oligomer formed in the disassembly process that can structurally recovered to hollow spherical structure. Such the limitation in the structural recovery from the rodlike trimer intermediate might be attributed to the improper contact angle in the components of the intermediate as well as in the resulting headset structure. As discussed above, the rodlike trimer intermediate in solution does not hold the specific contact angle between the dimer and monomer components, which is observed in the intact hollow spherical apoferritin but still has an adoptability for trimers or monomers that can be added to the intermediate. As a result, the reassembled headset structure may no longer hold a certain degree of specific contact angle to adopt trimers or monomers for continuing reassembly process further. The second question is why the hollow sphere with two holes as defects (which was formed over the pH range 3.24 to 2.85 in the disassembly process) could never be recovered back to the originally intact hollow sphere. At this moment, this observation is not yet understood. However, the observed result suggests that appropriate specific intersubunit interactions are required for arranging 24 subunits to make intact hollow spherical structure. This may need additional support from surroundings (for example, polyribosome, specific enzymes, etc.) which are available only in vivo. Perhaps, such the hollow spherical apoferritin with two holes as defects is a better template candidate for the fabrication of various nanodots because such the two holes can be utilized as open doors for transportation of materials (atoms, ions, molecules, drugs, etc.) into or out of the hollow core, compared with the intact hollow spherical apoferritin without defects.

’ CONCLUSIONS We investigated the structures of apoferritin and the ferritin core in a neutral solution by SAXS measurements. Under neutral conditions, apoferritin assumed an intact hollow spherical structure that was identical to the structure determined in the crystalline state. The structure included 24 homologous subunits (to form a 24-mer). The intact hollow spherical structure (Rg,p(r) = 5.58 nm and Dmax = 12.3 nm) of apoferritin was stable over the pH range 3.40
10.0. The apoferritin shell thickness was 2.3 nm, and the hollow core volume was 233.2 nm3. Below pH 3.40, apoferritin became unstable and underwent stepwise disassembly through several structural intermediates: a hollow spherical structure with two holes, a headset-shaped structure, and, ultimately, rodlike oligomers (mainly trimers) or monomers. Below pH 0.80, the disassembled subunits aggregated, which was attributed to denaturation.

ARTICLE

Structural recovery of the intermediates during the pH-induced reassembly process depended on the history of the disassembly process: (i) Structural recovery of the rodlike oligomers was limited to the headset-shaped oligomers. (ii) Structural recovery of the headset-shaped oligomers was limited to the hollow spherical structures with two hole defects (20-mer). (iii) The hollow sphere with two hole defects never recovered back to the originally intact hollow sphere. Because the two holes acted as channels for mass transport, the hollow spherical apoferritin with two holes may be useful as a template for nanodot fabrication. The ferrihydrite core of ferritin under neutral conditions assumed a compact globular structure (Rg,p(r) = 3.27 nm and Dmax = 9.80 nm). The ferrihydrite core was estimated to have a volume of 142.0 nm3, which corresponds to ∼60.9% of the hollow core volume of apoferritin. The ferrihydrite core (i.e., the ferritin core) was stable over the pH range 2.10
10.0. Below pH 2.10, the ferritin core underwent aggregation as a result of the disassembly of the ferritin shell under such strongly acidic conditions.

’ AUTHOR INFORMATION Corresponding Author

*Tel: þ82-54-279-2120 (M.R.), þ82-54-770-2417 (H.K.), þ8254-279-1573 (K.S.J.). Fax: þ82-54-279-3399 (M.R.), þ82-54770-2447 (H.K.). E-mail: [email protected] (M.R.), hskim@ dongguk.ac.kr (H.K.), [email protected] (K.S.J.).

’ ACKNOWLEDGMENT This study was supported by the National Research Foundation (NRF) of Korea (Basic Research Grant No. 2010-0023396 and Center for Electro-Photo Behaviors in Advanced Molecular Systems (2010-0001784)) and the Ministry of Education, Science and Technology (MEST) (Korea Brian 21 Program and World Class University Program (R31-2008-000-10059-0)). The synchrotron X-ray scattering measurements at the Pohang Accelerator Laboratory were supported by MEST and POSCO Company and POSTECH Foundation. ’ REFERENCES (1) Ford, G. C.; Harrison, P. M.; Rice, D. W.; Smith, J. M.; Treffry, A.; White, J. L.; Yariv, J. Philos. Trans. R. Soc., B 1984, 304, 551. (2) Mann, S.; Bannister, J. V.; Williams, R. J. P. J. Mol. Biol. 1986, 188, 225. (3) Lawson, D. M.; Artymiuk, P. J.; Yewdall, S. J.; Livingstone, J. C.; Treffry, A.; Luzzago, A.; Levi, S.; Arosio, P.; Cesareni, G.; Thomas, C. D.; Shaw, W.; Harrison, P. M. Nature 1991, 349, 541. (4) Harrison, P. M. J. Mol. Biol. 1959, 1, 69. (5) Fischbach, F. A.; Anderegg, J. W. J. Mol. Biol. 1965, 14, 458. (6) Granier, T.; Gallois, B.; Dautant, A.; Langlois D’Estaintot, B.; Pr
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