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The incorporation of GALA peptide into a protein cage for an acid-inducible molecular switch
Biomaterials 31 (2010) 5191e5198
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The incorporation of GALA peptide into a protein cage for an acid-inducible molecular switch Seung-Hye Choi, Kuiwon Choi, Ick Chan Kwon, Hyung Jun Ahn* Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-Gu, Seoul 136-791, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2010 Accepted 9 March 2010 Available online 31 March 2010
Caged proteins have been utilized as a biological container in a wide range of applications from material science to biomedicine, and GALA peptide has been known to undergo coil-to-helix transition upon the increased acidity. In this study, GALA synthetic peptide is incorporated to cage protein by genetic modification. Our engineered caged scaffold retains intact at the physiological pH but dissociate completely at pH 6.0, and the dissociated subunits are re-assembled simply by neutralization to biological pH. This acid-induced dissociation has the potential as molecular switch in vivo as well as in vitro so that the acid-sensitive caged proteins are applicable to drug delivery system for acidic target sites such as tumor. Since our design depends on the conformational transition of GALA peptide, not on removal of characteristic interface observed only in viral capsid-like protein, non-viral caged proteins can also be engineered to have molecular switching function. Therefore, this design for acid-sensitive scaffold would broaden the width of applications in nanotechnology including biomimetic material synthesis and biomedicine. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Cage protein GALA peptide Self-assembly Molecular switch Disassembly
1. Introduction Caged proteins are the spherical macromolecules that are precisely self-assembled from a limited number of subunits. This assembly is modulated by the extensive subunitesubunit interactions between the adjacent subunits. Container-like cage proteins have played a role as the nanoscale platform in the biomimetic nanoparticle synthesis [1] and have provided a wide range of possible applications in biomedicine, including medical imaging [2], and drug delivery [3]. Since the caged proteins have three distinct interfaces (the interior, exterior, and interface between the subunits), chemical or genetic modifications at these interfaces have imparted designed functionality to the cage [2]. However, despite recent advances in protein synthetic and computational methods, it has been still a considerable challenge to redesign cage proteins for the functionality. Several caged proteins have been reported that their assembly could be modulated by pH variation [4e8]. For example, CCMV (Cowpea Chlorotic Mottle Virus) and Norwalk capsids assemble at low pH and disassemble at high pH [4e6]. Ferritin proteins remain intact over a wide range of pH [9], and reversibly disassembles under the acidic guanidine hydrochloride (GdnHCl) or strongly
* Corresponding author. Tel.: þ82 2 958 5938; fax: þ82 2 958 5909. E-mail address: [email protected] (H.J. Ahn). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.03.016
acidic solutions [7,8,10]. However, any natural caged proteins have not been reported to assemble at the physiological pH but reversibly dissociate pH 6.0. Others have speculated that redesigning subunitesubunit interactions in the caged protein complex could be a potential strategy for controlling its architecture and assembly [11]. In several viral capsids, the assembly profile was altered by removing the N-terminus of the subunits, resulting in the virus particle with a smaller volume [12,13]. Similarly by deletion of an embracing N-terminus arm, Wang and co-workers reported the first example that one caged protein, dihydrolipoamide acetyltransferase (E2) could be engineered to correctly assemble at physiological pH but irreversibly disassemble into aggregation at pH 5.0 [14,15]. However, this approach has the limitation that cage protein without such N-terminally embracing arm cannot be engineered. The N-terminal control of assembly, which modulates the scaffold in viral capsid-like cages, is not the common feature in the ferritin-like [9,16] and closed shell cage-like cages [17e19]. For example, ferritin and Dps (DNA-binding protein from the starved cells) caged proteins are composed of the compact subunits without an embracing structural motif, so the deletion method is not applicable to these non-viral capsid-like cages for acid-induced molecular switch. In the applications of drug and gene delivery, the GALA peptide has been designed to interact with membranes in a pH-sensitive manner [20,21]. This peptide is composed of the artificial synthetic
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amino acid repeat of a Glu-Ala-Leu-Ala [20], and one sequence example of GALA peptide is WEAALAEALAEALAEHLAEALAEALEALA, of which the sequence length can vary to longer or shorter than 30 amino acids. The most remarkable feature of this peptide is its reversible conversion (Fig. 1) from a random coiled structure to an a-helix when the pH is reduced from 7.0 to 5.0 [20,21]. This unusual behavior originates from the ordered repetition of the apolar and polar residues. At the neutral pH, electrostatic repulsions between the carboxylic acid moieties of the glutamic acids are expected to destabilize the a-helix, whereas at pH 5.0, the neutralization of these groups should promote a-helix formation, resulting in the localization of the hydrophobic leucine residues on one side of the a-helix and the pH-titratable hydrophilic glutamic residues on the other side. Moreover, as the glutamic acids are protonated, the hydrophobicity of the glutamic acid side chain increases [20]. The pH value at the midpoint of maximum change of the helical content in GALA peptide was reported to be 6.0, and pKa value of glutamic acid in polyglutamic acid was close to 6.0 [21,22]. Circular dichroism (CD) measurements also demonstrated the increase of an a-helical conformation in GALA peptide as the pH was decreased from 7.5 to 5.0 [21]. These data indicated that GALA peptide could undergo coilto-helix transition near pH 6.0. In this study, we designed cage protein genetically to utilize GALA peptide as acid-inducible molecular switch (Fig. 1). We selected human ferritin light chain for a cage model. This cage protein differed in viral capsid-like proteins in that there was no N-terminal embracing arm (Fig. 2A and B) and therefore, we could not use deletion method. Ferritin is typically spherical capsid with 432 point symmetry [23]. Cage structure of ferritin has 24 subunits with each subunits composed of four-helix bundle (which included A, B, C, and D helix) and E-helix (linked at the C-terminal end) (Fig. 2A). A fifth short a-helix is tilted towards the bundle axis and masks the one end of the cylindrical bundle. The recombinant human ferritin heavy chain has been subjected to numerous mutational studies to investigate in vivo reassembly. Although in vitro stability was markedly decreased, assembly was not prevented by the deletion of the first 13 N-terminal residues (D1e13), of the last 22 C-terminal residues (D161e182) [24], or by various substitutions in the four-helix bundle, in the hydrophilic channels, or in the inner and outer surfaces of the cage [25]. Cage proteins with acid-inducible molecular switch are potentially applicable to drug delivery systems [2,26]. For example, capsulated drug within cage protein can avoid side effects and physicochemical instability in vivo. And drug release is expected to be accomplished by dissociation of cage after arrival at acidic target sites [26e29].
In the present studies, the engineered proteins were expressed in Escherichia coli expression systems and purified after genetic modification according to our strategy. The resulting proteins were characterized by size exclusion chromatography, dynamic light scattering, SDS polyacrylamide gel electrophoresis, and Circular Dichroism. To utilize the coil-to-helix transition of GALA by acid, various lengths of GALA peptides were incorporated into the scaffold and investigated. 2. Materials and methods 2.1. Design and construction of ferritin-GALAn hybrid protein The human ferritin light chain gene was amplified by the polymerase chain reaction using human cDNA library as template. Three different length of GALA peptides were chosen to be inserted to E-helix truncated ferritin, and depending on the composition of amino acids, three ferritin-GALA hybrids, ferritin-GALA2, ferritinGALA4, and ferritin-GALA6 were named fGALA2, fGALA4, fGALA6, respectively. The forward oligonucleotide primers for fGALAn (n ¼ 2, 4, and 6, respectively) constructs were 50 -G GAA TTC CAT ATG AGC TCC CAG ATT CGT CAG-30 and shared for each of fGALAn hybrids. The reverse oligonucleotide primers for fGALA2, fGALA4, and fGALA6 constructs were 50 -CCG CCG CTC GAG TTA AGC TTC AGC TAA AGC TTC AGC TAA AGC CTC CGG GCC-30 , 50 -CCG CCG CTC GAG TTA TTC AGC TAA AGC TTC AGC TAA ATG TTC AGC TAA AGC TTC AGC TAA AGC CTC CGG GCC-30 , 50 -CCG CCG CTC GAG TTA TTC AGC TAA AGC TTC AGC TAA AGC TTC AGC TAA AGC TTC AGC TAA ATG TTC AGC TAA AGC TTC AGC TAA AGC CTC CGG GCC-30 respectively, where the bases in bond represent the NdeI and XhoI restriction enzyme cleavage sites. Amplified wild-type ferritin DNA was templated to remove the whole C-terminal fragment region ranging between the E-helix domain (161Gly-170Leu) and C-terminus (175Asp). And three different length of GALA genes were substituted for those eliminated C-terminal fragment, resulting in fGALAn hybrid construct. The amplified DNA was inserted into the NdeI/XhoI e digested expression vector pET-28a(þ) by T4 DNA ligase. This vector construction adds a 20-residue tag (MGSSHHHHHHSSGLVPRGSH) to the aminoterminus of the gene product in order to facilitate protein purification. Each of constructed expression vectors were sequenced and confirmed. 2.2. Protein expression and purification The proteins were over-expressed in E. coli BL21(DE3) cells. Cells were grown at 310 K to an OD600 of 0.5 in LB medium containing 50 mg/mL kanamycin and protein expression was induced by 1.0 mM isopropyl-b-D-thiogalactopyranoside (IPTG). Cell growth continued at 293 K for 18 h after IPTG induction and cells were harvested by centrifugation at 4200g (6000 rev min1; Sorval GSA rotor) for 10 min at 277 K. The cell pellet was resuspended in ice-cold lysis buffer (50 mM TriseHCl pH 8.0, 100 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride) and homogenized with an ultrasonic processor. The crude lysate was centrifuged at 70 400g (30 000 rev min1; Beckman 45Ti rotor) for 1 h at 277 K and the recombinant protein in the supernatant fraction was purified in two chromatographic steps. The first step utilized the hexahistidine tags by metal-chelate chromatography on Ni-NTA resin (Qiagen). Next, size exclusion chromatography (SEC) was performed on a Superdex 200 10/ 300 GL column (GE Healthcare) previously equilibrated with buffer containing 50 mM TriseHCl (pH 8.0), 100 mM sodium chloride, and 1 mM mercaptoethanol. The homogeneity of the purified protein was assessed by SDS-PAGE. The protein solution was concentrated using an YM10 ultrafiltration membrane (Amicon). The protein concentration was estimated using Bradford assay method with bovine serum albumin as a standard. 2.3. Analysis of oligomeric state of proteins
Fig. 1. Schematic diagram of acid-inducible disassembly of engineered protein cage by GALA peptide. GALA peptides (yellow) are introduced to pore sites on cage scaffold without loss of assembly. Coil-to-helix transition of GALA (magenta) is triggered by acid and induces the conformational change around artificial pores, leading to dissociation of caged particles. Our engineered protein scaffold retains intact at physiological pH but reversibly dissociate at pH 6.0. The dissociated subunits re-associate simply by returning to physiological pH. Each subunits of caged protein is represented by hexagons, and yellow and magenta colours on protein subunits denote the random coiled and helical structures in GALA, respectively.
After purification, each of all proteins in this study was incubated for 1 h at room temperature in the pH range 3.0e8.0, and then analyzed by size exclusion chromatography (Superdex 200 10/300 GL column). Oligomeric states were judged from the elution volume (Ve) and bands on denatured SDS-PAGE. Regardless of buffer condition, all equilibrated buffer contained 100 mM sodium chloride and 1 mM mercaptoethanol, and injection volume on SEC did not exceed about 2 mL. Before analyzing proteins in this research, standard protein curve was prepared so that 24-mer of wild-type ferritin (Mr 440 kDa), BSA (Mr 67 kDa), and Cytochrome C (Mr 14 kDa) were eluted at Ve ¼ 9e10 mL, 15e16 mL, and 20e21 mL, respectively. Protein elution profiles on SEC were monitored by measuring the absorbance at 280 nm. When needed, the protein samples were concentrated on Centricon 30 membrane (Amicon). 2.4. Dynamic light scattering (DLS) and circular dichroism (CD) DLS experiments were performed using a model DynaPro-801 instrument from Protein Solutions (Lakewood, New Jersey). The data were measured at 297 K with the protein at 1 mg/mL concentration in a variety of buffer conditions containing
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100 mM sodium chloride and 1 mM mercaptoethanol. CD scans were performed on a Jasco J-715 spectropolarimeter equipped with a Jasco peltier temperature controller. We used far-UV circular dichroism to characterize the protein folding/ unfolding and thermal stability. Samples at concentration of about 0.075 mg/mL in 50 mM potassium phosphate (pH 6.0 and 7.0, respectively) and 100 mM NaCl were scanned between 190 and 260 nm at 25 C at a scanning speed of 10 nm/min in 0.1 cm path length quartz cells. To evaluate thermal stability, we monitored the ellipticity at 222 nm from 5 C to 100 C at a heating rate of 0.5 C/min. 2.5. Electrophoresis and dialysis Denaturing electrophoresis was carried out on 15% poly-acrylamide gels, and proteins were stained with Coomassie Blue or silver stain (Bio-Rad). Samples were diluted with an 5-fold concentrated sampling buffer (0.25 M Tris/HCl buffer, pH 6.8), which contained SDS, b-mercaptoethanol, and glycerol. After destaining, if needed, densitometry was performed on a Computing Densitometer (Molecular Dynamics). We changed pH by buffer exchange with dialysis membrane tube (Mr. cutoff 3000 Da, Millipore) at room temperature for at least 4 h.
3. Results and discussion 3.1. Design of protein scaffold for acid-sensitive disassembly Structural analysis of ferritin in atomic levels revealed that two kinds of pores existed around the 3- and 4-fold axis, respectively (Fig. 2B). Around the 4-fold axis, six pores, which were composed of the adjacent four E-helices, were investigated for possible incorporation of GALA peptide (Fig. 2B). To substitute the E-helix with GALA peptide (Fig. 2B and C), we linked GALA peptide to the end of DE loop (160Ala) by genetic modification (Fig. 3A and B). SDS-PAGE analysis revealed that alteration of the native pores by deletion of the E-helices did not affect the expression and solubility of the subunits (Fig. 3C). Since the sequences of DE loop were not modified and sequentially followed by GALA, we expected that GALA could form the artificial pores on behalf of the E-helices (Fig. 3C). To examine if the sequence length of GALA peptide affected on acid-sensitive disassembly, three kinds of ferritin-GALA hybrids with variation of Glu-Ala-Leu-Ala amino acid repeat were prepared by the same redesign (fGALA2, fGALA4, and fGALA6 respectively) (Fig. 3A). Each constructs showed the high expression and solubility levels, as did the native ferritin (Fig. 3D), implying that genetic modification for incorporation of GALA peptide did not hinder the correct folding of subunits. Because lysis buffer maintained pH 8.0, a random coiled structure of GALA peptide at least did not affect the folding process of the subunits. 3.2. Correct assembly of native ferritin and ferritin-DE mutant at a wide range of pH 4.0e8.0 Generally, ferritin caged proteins have been reported to dissociate only under the strongly acidic condition and reversibly re-associate at or above neutral pH [30e32]. Horse spleen apoferritin dissociates under the extreme pH conditions below 2.8 and reversibly re-associates at pH value above 3.0 [30,31]. Rhodobacter capsulatus bacterioferritin is 100% 24-mer over the pH range 3.0e10.0 and dissociates at pH below 2.0 as well [32]. These assembly behaviors have been utilized in the medical imaging so that GdIII chelates, GdHPDO3A could be entrapped within ferritin
Fig. 2. Ribbon diagram of monomer subunit and native pore structure in ferritin caged protein, and schematic diagram of fGALA hybrid around symmetry axis. (A) Subunit is composed of four-helix bundle and fifth E-helix. Each of five helices are displayed with different colours. (B) Individual subunits of 24-mer caged particle are represented with
different colours around 3- and 4-fold symmetry axis, respectively (left and right). Pore structures around 4-fold symmetry axis were magnified by dashed circle after rotated by 90 along the horizontal axis. For clarity, one monomer in the relation of the 4-fold symmetry is abbreviated and three E-helices are coloured by green. (C) Incorporation of GALA peptide into protein cage. The view point is the same as Fig. B. Random coiled structure of GALA peptide in pH 7.4 (yellow) does not hinder assembly of protein cage, but transition to helical structure at pH 6.0 yields molecular repulsion (magenta) between GALA peptide in narrow artificial pores. This figure is prepared with PyMOL (DeLano, 2002).
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Fig. 3. Various mutants of ferritin caged proteins and secondary structure analysis. (A) Summary of various ferritin mutants and GALA peptide sequences. GALA sequences are denoted by one-letter code. (B) Secondary structure analysis of human ferritin light chain reveals five helices on sequences. The cylinders correspond to helices and deleted E-helix region is highlighted with a rectangular. (C) Denatured SDS-PAGE analysis for E-helix-deleted ferritin shows the high level of expression and solubility in E. coli system. () and (þ) represent before and after IPTG induction, respectively. (D) Solubility test of fGALA hybrids in E. coli expression system. fG2, 4, and 6 represent each of fGALA2, 4, 6 hybrids.
interior by dissociation at pH 2.0 and subsequently re-association at pH 7.0 [10]. However, we are not aware of any ferritin proteins that remain assembled at the neutral pH but disassemble at pH 6.0. At first, we examined the pH-dependent disassembly of the native human ferritin light chain using size exclusion chromatography (SEC) and denatured SDS-PAGE. Each protein samples were dialyzed at room temperature against the different pH values ranging from 3.0 to 8.0, and then concentrated to 1 mg/mL by ultrafiltration membrane. To make the same environmental condition as protein samples, the SEC column was pre-equilibrated with corresponding buffer and 2 mL of samples was loaded on every injection. Below pH 4.0, most ferritin existed as unassembled building blocks as judged from the elution volume, Ve ¼ w20 mL after size exclusion chromatography, and any cage structures were not observed (Fig. 4). However, in the pH range of 4.0e8.0, the peak appeared in the earlier elution volume (Ve ¼ w9 mL) and corresponded to the size of 24-mer ferritin (Fig. 4), indicating the native ferritin subunits self-assembled and retained its cage structure in these pH range. This result demonstrated that disassembly in human ferritin light chain was triggered only by harsh acidic condition (below pH 4.0), as were other ferritin proteins. Since we tried to build the artificial pores by alteration of the native pores, we needed to examine if the native pore structures modulate assembly of cage. Therefore, pH-dependent profile of the truncated ferritin (ferritin-DE) was investigated with variation of pH. The elution peaks resembled those of the native ferritin (Fig. 4) at a wide range of pH values, as ferritin-DE assembled intact at pH 4.0e8.0 and dissociated below pH 4.0. This suggested that we could alter the pore-forming structural motif without loss of assembly. Especially, because the E-helices were positioned in the hollow sphere of the cage, the outer diameter of ferritin-DE was expected not to differ in that of the native ferritin, and actually there was no
difference in the elution volume of 24-mer between native and ferritin-DE. The hydrodynamic diameters measured by DLS for the native and ferritin-DE were 12.4 0.4 nm and 12.9 0.8 nm at the pH 7.0, respectively, and there was no noticeable difference in size between the native and ferritin-DE. But in the pH value below 4.0, DLS analysis could not measure the exact size due to the high polydispersity (more than 90%), which seemed to be another evidence of disassembly. 3.3. Effect of GALA peptides on cage assembly in pH 7.0 and 8.0 Because our engineered protein should retain intact cage assembly at the physiological pH, we investigated the effect of GALA peptides on assembly of the cage at or above neutral pH. Each
Fig. 4. Size exclusion (SEC) chromatography profile of native ferritin and mutant ferritin-DE. Straight lines correspond to SEC profiles at various pH range (4.0, 5.0, 6.0, 7.0, and 8.0), and dashed lines do at pH 2.5 and 3.0. There was no difference in elution profiles between native and ferritin-DE.
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Fig. 5. SEC profiles of fGALA4, 6 hybrids. (A) Oligomeric states of fGALA4, 6 hybrids are analyzed on SEC column at various pH solutions. Population of each species is analyzed on denatured SDS-PAGE. Circles, squares, and triangles denote the different oligomeric states of protein particles, respectively. In the view of acid-dependant profile, only fGALA2 hybrid coincided with the native ferritin rather than other two hybrids. (B) Superimposed SEC profiles of each fGALA4, 6 hybrid at various pH conditions. Whenever protein samples were injected, their volume and concentration were adjusted to the equal value for comparison between profiles. (C) Decreased peak areas corresponding to 24-mer in Fig. B are plotted at various pH conditions to predict the transition pH range of caged particles. The calculated area at 24-mer peaks is postulated to be proportional to the amount of assembled cage.
of three different fGALAn (n ¼ 2, 4, and 6) hybrids were dialyzed against both pH 7.0 and 8.0, and subsequently each samples were concentrated to 1 mg/mL. 2 mL of samples were injected on the pre-equilibrated SEC column with both pH 7.0 and 8.0 buffer, and collected fractions from the individual peaks were analyzed by denatured SDS-PAGE. At both pH values 7.0 and 8.0, all fGALAn hybrids retained their cage structures as judged from the elution volume (Ve) (Fig. 5A). Especially, the elution peak Ve ¼ w9 mL coincided with that of the native ferritin at pH 4.0e8.0 (Fig. 4), so it corresponded to the size of 24-mer. Dynamic light scattering analysis also indicated all fGALAn hybrids to have molecular weight of w500 kDa (12.5 0.3 nm in diameter) with a polydispersity range of 20.0e24.5%. Because the calculated monomer weight including the N-terminal tag is about 22 kDa, it corresponded to 24-mer cage particle. From these results, we could prove that all fGALAn hybrids existed as 24-mer at pH 7.0 and 8.0, and the artificial pores at least did not disrupt the cage structure in these pH range. And the length of GALA in the artificial pores was not likely to affect the assembly, either. 3.4. Acid-sensitive dissociation of protein cage in pH 6.0 To demonstrate the acid-sensitive disassembly predicted by our designing, each fGALAn sample was prepared at various acidic conditions by dialysis as described above and then, analyzed on the SEC columns and denatured SDS-PAGE. In contrast to at pH 7.0 and 8.0, as pH decreased from 7.0 to 6.0, two fGALA4 and fGALA6
hybrids, not fGALA2, yielded the shifted elution peaks on SEC column, implying the conformational transition from 24-mer to small building blocks (Fig. 5A and B). In this study, two fGALA4 and fGALA6 hybrids were named fGALA4, 6 hybrids to distinguish this behavior from that of fGALA2 hybrid because we did not notice any difference between two fGALA4 and fGALA6 hybrids. Two fGALA4, 6 hybrids revealed the apparent dissociation under weak acidic conditions as following. At pH 6.8, the peak corresponding to Ve ¼ w20 mL in fGALA4, 6 hybrids, increased as much as the height of 24-mer peak decreased, and this revealed the partial dissociation was triggered. Interestingly, at more acidic condition, pH 6.5, another peaks of Ve ¼ w15e17 mL were subsequently eluted with considerably high population after the 24-mer peak (Fig. 5A). These fractions corresponded to the intermediate oligomers because they eluted faster than Ve ¼ w20 mL but slower than Ve ¼ w9 mL. Since the heights of 24-mer and smallest building block decreased at the same time, these fractions were regarded as mixture of partially dissociated particles or subassembly of the smallest building blocks (Fig. 5B). And the polydispersity value of DLS analysis at pH 6.5 increased more than 90.0% so that it implied the species of cage particles was highly heterogeneous. Remarkably, below and at pH 6.0, the caged particles of fGALA4, 6 hybrids dissociated completely so that only single peak was observed at Ve ¼ w17 mL (Fig. 5A and B). According to comparison with our standard markers, elution of this single peak was slower than BSA (Mr 67 kDa) but faster than cytochrome C (Mr 14 kDa), so
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we could demonstrate that cage particles dissociated completely at pH 6.0 and below pH 6.0. DLS analysis also revealed that the molecular weight was w70 kDa with 4.2 0.2 nm in diameter (whereas the calculated molecular weight of cage was about 530 kDa), supporting disassembly of cage particles. When compared with the native ferritin in Fig. 4, our engineered ferritin cage dissociated completely at pH 6.0, whereas the native ferritin did below pH 4.0. Since most natural ferritin proteins dissociate in harsh acidic condition such as at pH 3.0 [7e9], this is the first example of complete dissociation of ferritin cage at pH 6.0 without loss of assembly at neutral pH, and these results showed our design could impart the acid-sensitive disassembly to the engineered cage protein. Since the individual peak areas on SEC profiles were proportional to the population of the corresponding species, we tried to estimate a transition pH value by comparison between SEC profiles. The decreased ratio of cage assembly was determined by calculation of 24-mer peak areas at each of pH values (Fig. 5B and C). Although the aggregation tendencies of concentrated samples increased at acidic conditions, such aggregates were removed by the high speed ultracentrifugation before injection and only the soluble fractions were analyzed on SEC column. Whenever analyzing the proteins samples on SEC column, since we adjusted the sample concentration and the injection volume to the equal values (1 mg/mL and 2 mL, respectively) so that it was possible to compare the elution profiles. A sigmoidal curve fitted to this data, reveals that two fGALA4, 6 hybrids undergo a transition from an assemble state to a disassemble state in the pH range of 6.0e7.0, and w80% dissociation of cage particles was observed near pH 6.5 for two fGALA4, 6 hybrids (Fig. 5C). On the other hand, fGALA2 did not dissociate its cage in the pH range of 4.0e8.0, but disassembled completely to be small building blocks only below pH 4.0 (data not shown). This acidity-dependant disassembly of fGALA2 resembled that of the native ferritin (Fig. 4). Relative to two fGALA4, 6 hybrids, the artificial pores in fGALA2 was built by only two repetition of Glu-Ala-Leu-Ala amino acids, and this implied that the length of GALA was too short to have any stable a-helical structure or to yield molecular interaction between the adjacent GALA peptides.
3.6. Unfolding of subunits coupled to dissociation of caged particles Since the ferritin proteins are composed only of the a-helices, they have the characteristic profile of minima at 208 and 222 nm on circular dichroism spectra. In the stability study of R. capsulatus bacterioferritin, the CD and tryptophan fluorescence emission spectra demonstrated that any secondary or quaternary structural change were not observed in the pH range 3.0e8.0, but the a-helix content at or below pH 2.0 decreased substantially [32]. Another CD spectra of the recombinant horse ferritin light chain reported a decrease in the a-helix content at acidic conditions (at or below pH 2.0) as well [31]. These reports explain unfolding of the subunits is coupled to dissociation of the native ferritin at strongly acidic conditions although a great deal of ordered structures are still present in the subunits and unfolding mechanism still remains veiled [31,32]. Similarly, in the native ferritin of our scaffold, we have also observed the representative CD spectra of a-helical proteins at pH 6.0 and 7.0 (Fig. 6A), and a-helix content also decreased substantially below pH 4.0 (data not shown).
3.5. Re-assembly capability The design strategy proposed by Wang and coworker reported that their engineered cage protein could not re-assemble after dissociation because disassembly of protein cage lead to aggregation [14,15]. However, the re-assembly capability is important in application to drug delivery system because drug or cargo should be capsulated by the dissociated subunit during reassembly process. If dissociation and re-association process of the caged particles are irreversible, it would be impossible to capsulate the cargo inside cage particles. Hence, we tried to investigate if the disassembled subunits could re-assemble into caged particle simply by buffer change. The corresponding peak of dissociated subunits was pooled after the sample was injected on SEC column at pH 6.0. To neutralize the sample, the pooled fraction was dialyzed against the buffer containing 50 mM TriseHCl (pH 7.0) and 100 mM NaCl, and then concentrated to 1 mg/mL. When the sample was injected again on SEC column equilibrated at pH 7.0, we could observe the same 24-mer peak as Fig. 5A, indicating that acid-sensitive dissociation was reversible and the dissociated subunits could re-associate into caged particles by pH neutralization. Therefore, this result shows the first example of engineered caged scaffold to be reversibly re-assembled by neutralization.
Fig. 6. Far-UV circular dichroism spectra for native and fGALA4, 6 hybrids. (A) CD spectra of the native ferritin at pH 6.0 and 7.0. Since ferritin subunits are composed of only a-helices, caged scaffold shows two minima at 208 and 222 nm. (B) CD spectra of fGALA4, 6 hybrids at pH 6.0 and 7.0. Loss of 208 nm minimum and a shift in 222 nm minimum at pH 6.0 indicate unfolding events.
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Fig. 7. SEC profiles of fGALA4, 6 hybrids at various protein concentrations. Equal volume of protein samples with variation of its concentration (0.075, 0.15, 0.3, and 0.6 mg/mL, respectively) are injected on SEC column at pH 7.0. These profiles reveal that assembly of caged particles is independent of protein concentration.
Interestingly, as we expected, our engineered cage protein revealed the different CD profile at pH 6.0. Before triggering dissociation, two fGALA4, 6 hybrids also had the similar CD profile to that of the stable caged particles at pH 7.0 (Fig. 6B). However, when disassembly process was accomplished at pH 6.0, a remarkable decrease of a-helix content at 208 nm minimum and a shift in 222 nm minimum were observed, representing variation of secondary and quaternary structure (Fig. 6B). Especially, robust variation of minima at 208 nm accounted for unfolding process of subunits. Since this variation of CD spectra meant unfolding of subunits, and elution profile on SEC column explained dissociation of cage, we could conclude that unfolding of the subunits in two fGALA4, 6 hybrids were coupled to dissociation of caged particles. The native ferritin did not show any CD spectral variation in pH 6.0 so that the unfolding was anticipated to be triggered by coil-tohelix transition of GALA. 3.7. Effects of protein concentration and ionic strength One variant with the substitution Lys169 / Arg in the recombinant human ferritin heavy chain has been reported to exist as dimer in low protein concentration, but to be readily reassembled after concentration by membrane ultrafiltration [33]. To examine if our fGALA4, 6 hybrids form caged particles only in the high concentrated state, we have compared the oligomeric states at the different protein concentrations of 0.075, 0.15, 0.3, and 0.6 mg/ mL, respectively. Due to the relatively low solubility of the samples, it was impossible to concentrate more than 1 mg/mL. At pH 7.0, although protein concentrations were increased up to eight times higher for each of two fGALA4, 6 hybrids, only the caged particles were observed on the profiles of SEC column (Fig. 7), and assembly of two fGALA4, 6 hybrids was not affected by the protein concentration. In addition, when the acidity increased, acid-sensitive disassembly of two fGALA4, 6 hybrids did not depend on the protein concentration either (data not shown). If unfolding of the subunits is modulated by electrostatic forces, variation of ionic strength would affect the acid-sensitive disassembly of cage particles. To investigate the influence of ionic strength on acid-sensitive disassembly, we increased the NaCl concentration from 100 mM to 500 mM or decreased from 100 mM to zero, but did not observe any different behavior on SEC profiles (data not shown). Hence, we could conclude that assembly process of our protein scaffold was not affected by either protein or salt concentration. 3.8. Thermal stability of fGALA4,
6
hybrids
Because incorporation of artificial pores had possibility to lower the thermostability of our scaffold, we monitored the ellipticity at 222 nm as the protein samples were heated and calculated
Fig. 8. Temperature stability profiles of native ferritin and fGALA4, 6 hybrids. The ellipticity value at 222 nm is monitored to evaluate the degree of unfolding by heating. (A) The native ferritin does not show any transition event over a wide range of temperature both at pH 6.0 and 7.0. The slow increase of molar ellipticity during heating accounts for the high degree of stability in native ferritin. (B) In contrast to the native ferritin, fGALA4, 6 hybrid reveals unfolding with a sigmoidal profile (Tm 78.0 1.0 C) at pH 7.0. As pH is lowered more to pH 6.0, additional transition point is observed at 39.0 0.5 C. At pH 6.0, any transition event is not observed.
apparent midpoint unfolding temperatures (Tm). The native ferritin proteins remain intact over a wide range of temperature and this high thermostability has enabled ferritin to be utilized widely as nanocontainer in material synthesis [9]. In the native ferritin of our study, we could not measure any apparent melting temperatures at pH 6.0 and 7.0 (at which cage assembly retained intact) because the ellipticity values increased slowly and gradually (Fig. 8A). Relatively small variation of ellipticity in a wide range of temperature explained high thermostability of the native protein. In contrast, our acid-sensitive scaffold revealed the apparent sigmoidal curve with a Tm 78.0 1.0 C at pH 7.0 (Fig. 8B) and these transition inflections could account for unfolding of scaffold by heating. Although the different profile between the native and our scaffold was anticipated to be due to incorporation of the artificial pores into scaffold, the acid-sensitive scaffold was still thermostable at pH 7.0. Remarkably, at pH 6.5, which corresponded to the early transition state of disassembly, acid-sensitive scaffold had two melting temperature at 39.0 0.5 C and 75.0 1.0 C (Fig. 8B). According to the analysis of SEC profiles on above, the acid-sensitive scaffold existed as mixture of several oligomeric states in the transition pH range so we interpreted this unfolding profile as superimposition of several profiles, which corresponded to each oligomeric species. The first
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transition temperature, Tm 39.0 0.5 C, was likely to imply unfolding of the intermediate oligomers, which were less stable than assembled cage particles. Moreover, this profile suggested potentially another proof that the coil-to-helix transition of GALA could destabilize the assembly of caged particles. At pH 6.0, which corresponded to complete dissociation of our scaffold, any apparent transition between folding and unfolding was not observed (as we expected), because there were not any stable secondary and quaternary structures (Fig. 8B). After unfolding of the subunits and subsequent dissociation of cage particles were triggered, dissociated form had the lowered thermostability than caged scaffold. However, our acid-sensitive scaffold was sufficiently stable in a wide range of temperature before acidity-inducible disassembly was initiated. 4. Conclusions In summary, we demonstrated the engineered cage protein can dissociate completely at pH 6.0 but reversibly re-assemble at pH 7.0. In this study, ferritin scaffold was genetically modified by our strategy so that artificial GALA peptides were incorporated into caged protein. Coil-to-helix transition of GALA peptide inducible by acid was suggested to impart acid-sensitive disassembly function to cage protein. Our design strategy overcomes the limitation that depended on the specific structural motif of viral capsids. The disassembly and re-assembly process of caged protein were sensitive to weak acid like pH 6.0 so that engineered cage protein would be potentially applicable as acid-inducible molecular switch. Acknowledgements This research was supported by the Pioneer Research Center Program (2009-0081523) and Global Research Laboratory Project of MEST. Appendix Figures with essential color discrimination. Many of the figures in this article have parts that are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/j.biomaterials.2010.03.016. References [1] Kramer RM, Li C, Carter DC, Stone MO, Naik RR. Engineered protein cages for nanomaterial synthesis. J Am Chem Soc 2004;126:13282e6. [2] Uchida M, Klem MT, Allen M, Suci P, Flenniken M, Gillitzer E, et al. Biological containers: protein cages as multifunctional nanoplatforms. Adv Mater 2007;19:1025e42. [3] Flenniken ML, Willits DA, Harmsen AL, Liepold LO, Harmsen AG, Young MJ, et al. Melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture. Chem Biol 2006;13:161e70. [4] Johnson JE, Speir JA. Quasi-equivalent viruses: a paradigm for protein assemblies. J Mol Biol 1997;269(5):665e75. [5] Bancroft JB, Hills GJ, Markham R. Study of the self-assembly process in a small spherical virus. Formation of organized structures from protein subunits in vitro. Virology 1967;31(2):354e79. [6] Ausar SF, Foubert TR, Hudson MH, Vedvick TS, Middaugh CR. Conformational stability and disassembly of Norwalk virus like particles: effect of pH and temperature. J Biol Chem 2006;281(28):19478e88.
[7] Stefanini S, Finazzi-Agro A, Chiancone E, Antonini E. Binding of hydrophobic compounds to apoferritin subunits. Effects on the polymerization state. FEBS Lett 1979;100:296e300. [8] Gerl M, Jaenicke R. Assembly of apoferritin from horse spleen: comparison of the protein in its native and reassembled state. Biol Chem Hoppe-Seyler 1987;368:387e96. [9] Harrison PM, Arosio P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta 1996;1275:161e203. [10] Aime S, Frullano L, Geninatti Crich S. Compartmentalization of a gadolinium complex in the apoferritin cavity: a route to obtain high relaxivity contrast agents for magnetic resonance imaging. Angew Chem Int Ed Engl 2002;41 (6):1017e9. [11] Varpness Z, Peters JW, Young M, Douglas T. Biomimetic synthesis of a H2 catalyst using a protein cage architecture. Nano Lett 2005;5:2306e9. [12] Tang TH, Johnson JM, Dryden KA, Young MJ, Zlotnick A, Johnson JE. The role of subunit hinges and molecular “switches” in the control of viral capsid polymorphism. J Struct Biol 2006;154:59e67. [13] Larson SB, Lucas RW, McPherson A. Crystallographic structure of the T ¼ 1 particle of brome mosaic virus. J Mol Biol 2005;346:815e31. [14] Dalmau M, Lim S, Wang SW. Design of a pH-dependent molecular switch in a caged protein platform. Nano Lett 2009;9:160e6. [15] Dalmau M, Lim S, Wang SW. pH-triggered disassembly in a caged protein complex. Biomacromolecules 2009;10(12):3199e206. [16] Bozzi M, Mignogna G, Stefanini S, Barra D, Longhi C, Valenti P, et al. A novel non-heme iron-binding ferritin related to the DNA-binding proteins of the Dps family in Listeria innocua. J Biol Chem 1997;272(60):3259e65. [17] Koeck PJ, Kagawa HK, Ellis MJ, Hebert H, Trent JD. Two-dimensional crystals of reconstituted beta-subunits of the chaperonin TF55 from Sulfolobus shibatae. Biochim Biophys Acta 1998;1429(1):40e4. [18] Trent JD, Kagawa HK, Yaoi T, Olle E, Zaluzec NJ. Chaperonin filaments: the archaeal cytoskeleton? Proc Natl Acad Sci U S A 1997;94(10):5383e8. [19] Steinbüchel A, Marchessault RH. Biopolymers for medical and pharmaceutical applications, vol. 2. Wiley; 2005. p. 951e972. [20] Subbarao Jr NK, Parente RA, Szoka Jr FC, Nadasdi L, Pongracz K. pH-dependent bilayer destabilization by an amphipathic peptide. Biochemistry 1987;26:2964e72. [21] Li W, Nicol F, Szoka Jr FC. GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery. Adv Drug Deliv Rev 2004;56:967e85. [22] Haas DH, Murphy RM. Design of a pH-sensitive pore-forming peptide with improved performance. J Pept Res 2004;63:9e16. [23] Wang Z, Li C, Eijenburg M, Soistman E, Ruble J, Wright B, et al. Structure of human ferritin L chain. Acta Crystallogr 2006;D62:800e6. [24] Levi S, Luzzago A, Cesareni G, Cozzi A, Franceschinelli F, Albertini A, et al. Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants. J Biol Chem 1988;263:18086e92. [25] Santambrogio P, Levi S, Arosio P, Palagi L, Vecchio G, Lawson DM, et al. Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins. J Biol Chem 1992;267:14077e83. [26] Taillefer J, Brasseur N, van Lier JE, Lenaerts V, Le Garrec D, Leroux JC. In vitro and in vivo evaluation of pH-responsive polymeric micelles in a photodynamic cancer therapy model. J Pharm Pharmacol 2001;53:155e66. [27] Ojugo AS, McSheehy PM, McIntyre DJ, McCoy C, Stubbs M, Leach MO, et al. Measurement of the extracellular pH of solid tumours in mice by magnetic resonance spectroscopy: a comparison of exogenous (19)F and (31)P probes. NMR Biomed 1999;12(8):495e504. [28] Lee ES, Shin HJ, Na K, Bae YH. Poly(l-histidine)ePEG block copolymer micelles and pH-induced destabilization. J Control Release 2003;90:363e74. [29] Kim GM, Bae YH, Jo WH. pH-induced micelle formation of poly(histidine-cophenylalanine)-block-poly(ethylene glycol) in aqueous media. Macromol Biosci 2005;5:1118e24. [30] Crichton RR, Bryce CFA. Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH. Biochem J 1973;133:289e99. [31] Yoshizawa K, Mishima Y, Park SY, Heddle JG, Tame JRH, Iwahori K, et al. Effect of N-terminal residues on the structural stability of recombinant horse L-chain apoferritin in an acidic environment. J Biochem 2007;142:707e13. [32] Kilic MA, Spiro S, Moore GR. Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein. Protein Sci 2003;12:1663e74. [33] Santambrogio P, Pinto P, Levi S, Cozzi A, Rovida E, Albertini A, et al. Effects of modifications near the 2-, 3- and 4-fold symmetry axes on human ferritin renaturation. Biochem J 1997;322:461e8.
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The incorporation of GALA peptide into a protein cage for an acid-inducible molecular switch Seung-Hye Choi, Kuiwon Choi, Ick Chan Kwon, Hyung Jun Ahn* Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-Gu, Seoul 136-791, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2010 Accepted 9 March 2010 Available online 31 March 2010
Caged proteins have been utilized as a biological container in a wide range of applications from material science to biomedicine, and GALA peptide has been known to undergo coil-to-helix transition upon the increased acidity. In this study, GALA synthetic peptide is incorporated to cage protein by genetic modification. Our engineered caged scaffold retains intact at the physiological pH but dissociate completely at pH 6.0, and the dissociated subunits are re-assembled simply by neutralization to biological pH. This acid-induced dissociation has the potential as molecular switch in vivo as well as in vitro so that the acid-sensitive caged proteins are applicable to drug delivery system for acidic target sites such as tumor. Since our design depends on the conformational transition of GALA peptide, not on removal of characteristic interface observed only in viral capsid-like protein, non-viral caged proteins can also be engineered to have molecular switching function. Therefore, this design for acid-sensitive scaffold would broaden the width of applications in nanotechnology including biomimetic material synthesis and biomedicine. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Cage protein GALA peptide Self-assembly Molecular switch Disassembly
1. Introduction Caged proteins are the spherical macromolecules that are precisely self-assembled from a limited number of subunits. This assembly is modulated by the extensive subunitesubunit interactions between the adjacent subunits. Container-like cage proteins have played a role as the nanoscale platform in the biomimetic nanoparticle synthesis [1] and have provided a wide range of possible applications in biomedicine, including medical imaging [2], and drug delivery [3]. Since the caged proteins have three distinct interfaces (the interior, exterior, and interface between the subunits), chemical or genetic modifications at these interfaces have imparted designed functionality to the cage [2]. However, despite recent advances in protein synthetic and computational methods, it has been still a considerable challenge to redesign cage proteins for the functionality. Several caged proteins have been reported that their assembly could be modulated by pH variation [4e8]. For example, CCMV (Cowpea Chlorotic Mottle Virus) and Norwalk capsids assemble at low pH and disassemble at high pH [4e6]. Ferritin proteins remain intact over a wide range of pH [9], and reversibly disassembles under the acidic guanidine hydrochloride (GdnHCl) or strongly
* Corresponding author. Tel.: þ82 2 958 5938; fax: þ82 2 958 5909. E-mail address: [email protected] (H.J. Ahn). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.03.016
acidic solutions [7,8,10]. However, any natural caged proteins have not been reported to assemble at the physiological pH but reversibly dissociate pH 6.0. Others have speculated that redesigning subunitesubunit interactions in the caged protein complex could be a potential strategy for controlling its architecture and assembly [11]. In several viral capsids, the assembly profile was altered by removing the N-terminus of the subunits, resulting in the virus particle with a smaller volume [12,13]. Similarly by deletion of an embracing N-terminus arm, Wang and co-workers reported the first example that one caged protein, dihydrolipoamide acetyltransferase (E2) could be engineered to correctly assemble at physiological pH but irreversibly disassemble into aggregation at pH 5.0 [14,15]. However, this approach has the limitation that cage protein without such N-terminally embracing arm cannot be engineered. The N-terminal control of assembly, which modulates the scaffold in viral capsid-like cages, is not the common feature in the ferritin-like [9,16] and closed shell cage-like cages [17e19]. For example, ferritin and Dps (DNA-binding protein from the starved cells) caged proteins are composed of the compact subunits without an embracing structural motif, so the deletion method is not applicable to these non-viral capsid-like cages for acid-induced molecular switch. In the applications of drug and gene delivery, the GALA peptide has been designed to interact with membranes in a pH-sensitive manner [20,21]. This peptide is composed of the artificial synthetic
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amino acid repeat of a Glu-Ala-Leu-Ala [20], and one sequence example of GALA peptide is WEAALAEALAEALAEHLAEALAEALEALA, of which the sequence length can vary to longer or shorter than 30 amino acids. The most remarkable feature of this peptide is its reversible conversion (Fig. 1) from a random coiled structure to an a-helix when the pH is reduced from 7.0 to 5.0 [20,21]. This unusual behavior originates from the ordered repetition of the apolar and polar residues. At the neutral pH, electrostatic repulsions between the carboxylic acid moieties of the glutamic acids are expected to destabilize the a-helix, whereas at pH 5.0, the neutralization of these groups should promote a-helix formation, resulting in the localization of the hydrophobic leucine residues on one side of the a-helix and the pH-titratable hydrophilic glutamic residues on the other side. Moreover, as the glutamic acids are protonated, the hydrophobicity of the glutamic acid side chain increases [20]. The pH value at the midpoint of maximum change of the helical content in GALA peptide was reported to be 6.0, and pKa value of glutamic acid in polyglutamic acid was close to 6.0 [21,22]. Circular dichroism (CD) measurements also demonstrated the increase of an a-helical conformation in GALA peptide as the pH was decreased from 7.5 to 5.0 [21]. These data indicated that GALA peptide could undergo coilto-helix transition near pH 6.0. In this study, we designed cage protein genetically to utilize GALA peptide as acid-inducible molecular switch (Fig. 1). We selected human ferritin light chain for a cage model. This cage protein differed in viral capsid-like proteins in that there was no N-terminal embracing arm (Fig. 2A and B) and therefore, we could not use deletion method. Ferritin is typically spherical capsid with 432 point symmetry [23]. Cage structure of ferritin has 24 subunits with each subunits composed of four-helix bundle (which included A, B, C, and D helix) and E-helix (linked at the C-terminal end) (Fig. 2A). A fifth short a-helix is tilted towards the bundle axis and masks the one end of the cylindrical bundle. The recombinant human ferritin heavy chain has been subjected to numerous mutational studies to investigate in vivo reassembly. Although in vitro stability was markedly decreased, assembly was not prevented by the deletion of the first 13 N-terminal residues (D1e13), of the last 22 C-terminal residues (D161e182) [24], or by various substitutions in the four-helix bundle, in the hydrophilic channels, or in the inner and outer surfaces of the cage [25]. Cage proteins with acid-inducible molecular switch are potentially applicable to drug delivery systems [2,26]. For example, capsulated drug within cage protein can avoid side effects and physicochemical instability in vivo. And drug release is expected to be accomplished by dissociation of cage after arrival at acidic target sites [26e29].
In the present studies, the engineered proteins were expressed in Escherichia coli expression systems and purified after genetic modification according to our strategy. The resulting proteins were characterized by size exclusion chromatography, dynamic light scattering, SDS polyacrylamide gel electrophoresis, and Circular Dichroism. To utilize the coil-to-helix transition of GALA by acid, various lengths of GALA peptides were incorporated into the scaffold and investigated. 2. Materials and methods 2.1. Design and construction of ferritin-GALAn hybrid protein The human ferritin light chain gene was amplified by the polymerase chain reaction using human cDNA library as template. Three different length of GALA peptides were chosen to be inserted to E-helix truncated ferritin, and depending on the composition of amino acids, three ferritin-GALA hybrids, ferritin-GALA2, ferritinGALA4, and ferritin-GALA6 were named fGALA2, fGALA4, fGALA6, respectively. The forward oligonucleotide primers for fGALAn (n ¼ 2, 4, and 6, respectively) constructs were 50 -G GAA TTC CAT ATG AGC TCC CAG ATT CGT CAG-30 and shared for each of fGALAn hybrids. The reverse oligonucleotide primers for fGALA2, fGALA4, and fGALA6 constructs were 50 -CCG CCG CTC GAG TTA AGC TTC AGC TAA AGC TTC AGC TAA AGC CTC CGG GCC-30 , 50 -CCG CCG CTC GAG TTA TTC AGC TAA AGC TTC AGC TAA ATG TTC AGC TAA AGC TTC AGC TAA AGC CTC CGG GCC-30 , 50 -CCG CCG CTC GAG TTA TTC AGC TAA AGC TTC AGC TAA AGC TTC AGC TAA AGC TTC AGC TAA ATG TTC AGC TAA AGC TTC AGC TAA AGC CTC CGG GCC-30 respectively, where the bases in bond represent the NdeI and XhoI restriction enzyme cleavage sites. Amplified wild-type ferritin DNA was templated to remove the whole C-terminal fragment region ranging between the E-helix domain (161Gly-170Leu) and C-terminus (175Asp). And three different length of GALA genes were substituted for those eliminated C-terminal fragment, resulting in fGALAn hybrid construct. The amplified DNA was inserted into the NdeI/XhoI e digested expression vector pET-28a(þ) by T4 DNA ligase. This vector construction adds a 20-residue tag (MGSSHHHHHHSSGLVPRGSH) to the aminoterminus of the gene product in order to facilitate protein purification. Each of constructed expression vectors were sequenced and confirmed. 2.2. Protein expression and purification The proteins were over-expressed in E. coli BL21(DE3) cells. Cells were grown at 310 K to an OD600 of 0.5 in LB medium containing 50 mg/mL kanamycin and protein expression was induced by 1.0 mM isopropyl-b-D-thiogalactopyranoside (IPTG). Cell growth continued at 293 K for 18 h after IPTG induction and cells were harvested by centrifugation at 4200g (6000 rev min1; Sorval GSA rotor) for 10 min at 277 K. The cell pellet was resuspended in ice-cold lysis buffer (50 mM TriseHCl pH 8.0, 100 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride) and homogenized with an ultrasonic processor. The crude lysate was centrifuged at 70 400g (30 000 rev min1; Beckman 45Ti rotor) for 1 h at 277 K and the recombinant protein in the supernatant fraction was purified in two chromatographic steps. The first step utilized the hexahistidine tags by metal-chelate chromatography on Ni-NTA resin (Qiagen). Next, size exclusion chromatography (SEC) was performed on a Superdex 200 10/ 300 GL column (GE Healthcare) previously equilibrated with buffer containing 50 mM TriseHCl (pH 8.0), 100 mM sodium chloride, and 1 mM mercaptoethanol. The homogeneity of the purified protein was assessed by SDS-PAGE. The protein solution was concentrated using an YM10 ultrafiltration membrane (Amicon). The protein concentration was estimated using Bradford assay method with bovine serum albumin as a standard. 2.3. Analysis of oligomeric state of proteins
Fig. 1. Schematic diagram of acid-inducible disassembly of engineered protein cage by GALA peptide. GALA peptides (yellow) are introduced to pore sites on cage scaffold without loss of assembly. Coil-to-helix transition of GALA (magenta) is triggered by acid and induces the conformational change around artificial pores, leading to dissociation of caged particles. Our engineered protein scaffold retains intact at physiological pH but reversibly dissociate at pH 6.0. The dissociated subunits re-associate simply by returning to physiological pH. Each subunits of caged protein is represented by hexagons, and yellow and magenta colours on protein subunits denote the random coiled and helical structures in GALA, respectively.
After purification, each of all proteins in this study was incubated for 1 h at room temperature in the pH range 3.0e8.0, and then analyzed by size exclusion chromatography (Superdex 200 10/300 GL column). Oligomeric states were judged from the elution volume (Ve) and bands on denatured SDS-PAGE. Regardless of buffer condition, all equilibrated buffer contained 100 mM sodium chloride and 1 mM mercaptoethanol, and injection volume on SEC did not exceed about 2 mL. Before analyzing proteins in this research, standard protein curve was prepared so that 24-mer of wild-type ferritin (Mr 440 kDa), BSA (Mr 67 kDa), and Cytochrome C (Mr 14 kDa) were eluted at Ve ¼ 9e10 mL, 15e16 mL, and 20e21 mL, respectively. Protein elution profiles on SEC were monitored by measuring the absorbance at 280 nm. When needed, the protein samples were concentrated on Centricon 30 membrane (Amicon). 2.4. Dynamic light scattering (DLS) and circular dichroism (CD) DLS experiments were performed using a model DynaPro-801 instrument from Protein Solutions (Lakewood, New Jersey). The data were measured at 297 K with the protein at 1 mg/mL concentration in a variety of buffer conditions containing
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100 mM sodium chloride and 1 mM mercaptoethanol. CD scans were performed on a Jasco J-715 spectropolarimeter equipped with a Jasco peltier temperature controller. We used far-UV circular dichroism to characterize the protein folding/ unfolding and thermal stability. Samples at concentration of about 0.075 mg/mL in 50 mM potassium phosphate (pH 6.0 and 7.0, respectively) and 100 mM NaCl were scanned between 190 and 260 nm at 25 C at a scanning speed of 10 nm/min in 0.1 cm path length quartz cells. To evaluate thermal stability, we monitored the ellipticity at 222 nm from 5 C to 100 C at a heating rate of 0.5 C/min. 2.5. Electrophoresis and dialysis Denaturing electrophoresis was carried out on 15% poly-acrylamide gels, and proteins were stained with Coomassie Blue or silver stain (Bio-Rad). Samples were diluted with an 5-fold concentrated sampling buffer (0.25 M Tris/HCl buffer, pH 6.8), which contained SDS, b-mercaptoethanol, and glycerol. After destaining, if needed, densitometry was performed on a Computing Densitometer (Molecular Dynamics). We changed pH by buffer exchange with dialysis membrane tube (Mr. cutoff 3000 Da, Millipore) at room temperature for at least 4 h.
3. Results and discussion 3.1. Design of protein scaffold for acid-sensitive disassembly Structural analysis of ferritin in atomic levels revealed that two kinds of pores existed around the 3- and 4-fold axis, respectively (Fig. 2B). Around the 4-fold axis, six pores, which were composed of the adjacent four E-helices, were investigated for possible incorporation of GALA peptide (Fig. 2B). To substitute the E-helix with GALA peptide (Fig. 2B and C), we linked GALA peptide to the end of DE loop (160Ala) by genetic modification (Fig. 3A and B). SDS-PAGE analysis revealed that alteration of the native pores by deletion of the E-helices did not affect the expression and solubility of the subunits (Fig. 3C). Since the sequences of DE loop were not modified and sequentially followed by GALA, we expected that GALA could form the artificial pores on behalf of the E-helices (Fig. 3C). To examine if the sequence length of GALA peptide affected on acid-sensitive disassembly, three kinds of ferritin-GALA hybrids with variation of Glu-Ala-Leu-Ala amino acid repeat were prepared by the same redesign (fGALA2, fGALA4, and fGALA6 respectively) (Fig. 3A). Each constructs showed the high expression and solubility levels, as did the native ferritin (Fig. 3D), implying that genetic modification for incorporation of GALA peptide did not hinder the correct folding of subunits. Because lysis buffer maintained pH 8.0, a random coiled structure of GALA peptide at least did not affect the folding process of the subunits. 3.2. Correct assembly of native ferritin and ferritin-DE mutant at a wide range of pH 4.0e8.0 Generally, ferritin caged proteins have been reported to dissociate only under the strongly acidic condition and reversibly re-associate at or above neutral pH [30e32]. Horse spleen apoferritin dissociates under the extreme pH conditions below 2.8 and reversibly re-associates at pH value above 3.0 [30,31]. Rhodobacter capsulatus bacterioferritin is 100% 24-mer over the pH range 3.0e10.0 and dissociates at pH below 2.0 as well [32]. These assembly behaviors have been utilized in the medical imaging so that GdIII chelates, GdHPDO3A could be entrapped within ferritin
Fig. 2. Ribbon diagram of monomer subunit and native pore structure in ferritin caged protein, and schematic diagram of fGALA hybrid around symmetry axis. (A) Subunit is composed of four-helix bundle and fifth E-helix. Each of five helices are displayed with different colours. (B) Individual subunits of 24-mer caged particle are represented with
different colours around 3- and 4-fold symmetry axis, respectively (left and right). Pore structures around 4-fold symmetry axis were magnified by dashed circle after rotated by 90 along the horizontal axis. For clarity, one monomer in the relation of the 4-fold symmetry is abbreviated and three E-helices are coloured by green. (C) Incorporation of GALA peptide into protein cage. The view point is the same as Fig. B. Random coiled structure of GALA peptide in pH 7.4 (yellow) does not hinder assembly of protein cage, but transition to helical structure at pH 6.0 yields molecular repulsion (magenta) between GALA peptide in narrow artificial pores. This figure is prepared with PyMOL (DeLano, 2002).
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Fig. 3. Various mutants of ferritin caged proteins and secondary structure analysis. (A) Summary of various ferritin mutants and GALA peptide sequences. GALA sequences are denoted by one-letter code. (B) Secondary structure analysis of human ferritin light chain reveals five helices on sequences. The cylinders correspond to helices and deleted E-helix region is highlighted with a rectangular. (C) Denatured SDS-PAGE analysis for E-helix-deleted ferritin shows the high level of expression and solubility in E. coli system. () and (þ) represent before and after IPTG induction, respectively. (D) Solubility test of fGALA hybrids in E. coli expression system. fG2, 4, and 6 represent each of fGALA2, 4, 6 hybrids.
interior by dissociation at pH 2.0 and subsequently re-association at pH 7.0 [10]. However, we are not aware of any ferritin proteins that remain assembled at the neutral pH but disassemble at pH 6.0. At first, we examined the pH-dependent disassembly of the native human ferritin light chain using size exclusion chromatography (SEC) and denatured SDS-PAGE. Each protein samples were dialyzed at room temperature against the different pH values ranging from 3.0 to 8.0, and then concentrated to 1 mg/mL by ultrafiltration membrane. To make the same environmental condition as protein samples, the SEC column was pre-equilibrated with corresponding buffer and 2 mL of samples was loaded on every injection. Below pH 4.0, most ferritin existed as unassembled building blocks as judged from the elution volume, Ve ¼ w20 mL after size exclusion chromatography, and any cage structures were not observed (Fig. 4). However, in the pH range of 4.0e8.0, the peak appeared in the earlier elution volume (Ve ¼ w9 mL) and corresponded to the size of 24-mer ferritin (Fig. 4), indicating the native ferritin subunits self-assembled and retained its cage structure in these pH range. This result demonstrated that disassembly in human ferritin light chain was triggered only by harsh acidic condition (below pH 4.0), as were other ferritin proteins. Since we tried to build the artificial pores by alteration of the native pores, we needed to examine if the native pore structures modulate assembly of cage. Therefore, pH-dependent profile of the truncated ferritin (ferritin-DE) was investigated with variation of pH. The elution peaks resembled those of the native ferritin (Fig. 4) at a wide range of pH values, as ferritin-DE assembled intact at pH 4.0e8.0 and dissociated below pH 4.0. This suggested that we could alter the pore-forming structural motif without loss of assembly. Especially, because the E-helices were positioned in the hollow sphere of the cage, the outer diameter of ferritin-DE was expected not to differ in that of the native ferritin, and actually there was no
difference in the elution volume of 24-mer between native and ferritin-DE. The hydrodynamic diameters measured by DLS for the native and ferritin-DE were 12.4 0.4 nm and 12.9 0.8 nm at the pH 7.0, respectively, and there was no noticeable difference in size between the native and ferritin-DE. But in the pH value below 4.0, DLS analysis could not measure the exact size due to the high polydispersity (more than 90%), which seemed to be another evidence of disassembly. 3.3. Effect of GALA peptides on cage assembly in pH 7.0 and 8.0 Because our engineered protein should retain intact cage assembly at the physiological pH, we investigated the effect of GALA peptides on assembly of the cage at or above neutral pH. Each
Fig. 4. Size exclusion (SEC) chromatography profile of native ferritin and mutant ferritin-DE. Straight lines correspond to SEC profiles at various pH range (4.0, 5.0, 6.0, 7.0, and 8.0), and dashed lines do at pH 2.5 and 3.0. There was no difference in elution profiles between native and ferritin-DE.
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Fig. 5. SEC profiles of fGALA4, 6 hybrids. (A) Oligomeric states of fGALA4, 6 hybrids are analyzed on SEC column at various pH solutions. Population of each species is analyzed on denatured SDS-PAGE. Circles, squares, and triangles denote the different oligomeric states of protein particles, respectively. In the view of acid-dependant profile, only fGALA2 hybrid coincided with the native ferritin rather than other two hybrids. (B) Superimposed SEC profiles of each fGALA4, 6 hybrid at various pH conditions. Whenever protein samples were injected, their volume and concentration were adjusted to the equal value for comparison between profiles. (C) Decreased peak areas corresponding to 24-mer in Fig. B are plotted at various pH conditions to predict the transition pH range of caged particles. The calculated area at 24-mer peaks is postulated to be proportional to the amount of assembled cage.
of three different fGALAn (n ¼ 2, 4, and 6) hybrids were dialyzed against both pH 7.0 and 8.0, and subsequently each samples were concentrated to 1 mg/mL. 2 mL of samples were injected on the pre-equilibrated SEC column with both pH 7.0 and 8.0 buffer, and collected fractions from the individual peaks were analyzed by denatured SDS-PAGE. At both pH values 7.0 and 8.0, all fGALAn hybrids retained their cage structures as judged from the elution volume (Ve) (Fig. 5A). Especially, the elution peak Ve ¼ w9 mL coincided with that of the native ferritin at pH 4.0e8.0 (Fig. 4), so it corresponded to the size of 24-mer. Dynamic light scattering analysis also indicated all fGALAn hybrids to have molecular weight of w500 kDa (12.5 0.3 nm in diameter) with a polydispersity range of 20.0e24.5%. Because the calculated monomer weight including the N-terminal tag is about 22 kDa, it corresponded to 24-mer cage particle. From these results, we could prove that all fGALAn hybrids existed as 24-mer at pH 7.0 and 8.0, and the artificial pores at least did not disrupt the cage structure in these pH range. And the length of GALA in the artificial pores was not likely to affect the assembly, either. 3.4. Acid-sensitive dissociation of protein cage in pH 6.0 To demonstrate the acid-sensitive disassembly predicted by our designing, each fGALAn sample was prepared at various acidic conditions by dialysis as described above and then, analyzed on the SEC columns and denatured SDS-PAGE. In contrast to at pH 7.0 and 8.0, as pH decreased from 7.0 to 6.0, two fGALA4 and fGALA6
hybrids, not fGALA2, yielded the shifted elution peaks on SEC column, implying the conformational transition from 24-mer to small building blocks (Fig. 5A and B). In this study, two fGALA4 and fGALA6 hybrids were named fGALA4, 6 hybrids to distinguish this behavior from that of fGALA2 hybrid because we did not notice any difference between two fGALA4 and fGALA6 hybrids. Two fGALA4, 6 hybrids revealed the apparent dissociation under weak acidic conditions as following. At pH 6.8, the peak corresponding to Ve ¼ w20 mL in fGALA4, 6 hybrids, increased as much as the height of 24-mer peak decreased, and this revealed the partial dissociation was triggered. Interestingly, at more acidic condition, pH 6.5, another peaks of Ve ¼ w15e17 mL were subsequently eluted with considerably high population after the 24-mer peak (Fig. 5A). These fractions corresponded to the intermediate oligomers because they eluted faster than Ve ¼ w20 mL but slower than Ve ¼ w9 mL. Since the heights of 24-mer and smallest building block decreased at the same time, these fractions were regarded as mixture of partially dissociated particles or subassembly of the smallest building blocks (Fig. 5B). And the polydispersity value of DLS analysis at pH 6.5 increased more than 90.0% so that it implied the species of cage particles was highly heterogeneous. Remarkably, below and at pH 6.0, the caged particles of fGALA4, 6 hybrids dissociated completely so that only single peak was observed at Ve ¼ w17 mL (Fig. 5A and B). According to comparison with our standard markers, elution of this single peak was slower than BSA (Mr 67 kDa) but faster than cytochrome C (Mr 14 kDa), so
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we could demonstrate that cage particles dissociated completely at pH 6.0 and below pH 6.0. DLS analysis also revealed that the molecular weight was w70 kDa with 4.2 0.2 nm in diameter (whereas the calculated molecular weight of cage was about 530 kDa), supporting disassembly of cage particles. When compared with the native ferritin in Fig. 4, our engineered ferritin cage dissociated completely at pH 6.0, whereas the native ferritin did below pH 4.0. Since most natural ferritin proteins dissociate in harsh acidic condition such as at pH 3.0 [7e9], this is the first example of complete dissociation of ferritin cage at pH 6.0 without loss of assembly at neutral pH, and these results showed our design could impart the acid-sensitive disassembly to the engineered cage protein. Since the individual peak areas on SEC profiles were proportional to the population of the corresponding species, we tried to estimate a transition pH value by comparison between SEC profiles. The decreased ratio of cage assembly was determined by calculation of 24-mer peak areas at each of pH values (Fig. 5B and C). Although the aggregation tendencies of concentrated samples increased at acidic conditions, such aggregates were removed by the high speed ultracentrifugation before injection and only the soluble fractions were analyzed on SEC column. Whenever analyzing the proteins samples on SEC column, since we adjusted the sample concentration and the injection volume to the equal values (1 mg/mL and 2 mL, respectively) so that it was possible to compare the elution profiles. A sigmoidal curve fitted to this data, reveals that two fGALA4, 6 hybrids undergo a transition from an assemble state to a disassemble state in the pH range of 6.0e7.0, and w80% dissociation of cage particles was observed near pH 6.5 for two fGALA4, 6 hybrids (Fig. 5C). On the other hand, fGALA2 did not dissociate its cage in the pH range of 4.0e8.0, but disassembled completely to be small building blocks only below pH 4.0 (data not shown). This acidity-dependant disassembly of fGALA2 resembled that of the native ferritin (Fig. 4). Relative to two fGALA4, 6 hybrids, the artificial pores in fGALA2 was built by only two repetition of Glu-Ala-Leu-Ala amino acids, and this implied that the length of GALA was too short to have any stable a-helical structure or to yield molecular interaction between the adjacent GALA peptides.
3.6. Unfolding of subunits coupled to dissociation of caged particles Since the ferritin proteins are composed only of the a-helices, they have the characteristic profile of minima at 208 and 222 nm on circular dichroism spectra. In the stability study of R. capsulatus bacterioferritin, the CD and tryptophan fluorescence emission spectra demonstrated that any secondary or quaternary structural change were not observed in the pH range 3.0e8.0, but the a-helix content at or below pH 2.0 decreased substantially [32]. Another CD spectra of the recombinant horse ferritin light chain reported a decrease in the a-helix content at acidic conditions (at or below pH 2.0) as well [31]. These reports explain unfolding of the subunits is coupled to dissociation of the native ferritin at strongly acidic conditions although a great deal of ordered structures are still present in the subunits and unfolding mechanism still remains veiled [31,32]. Similarly, in the native ferritin of our scaffold, we have also observed the representative CD spectra of a-helical proteins at pH 6.0 and 7.0 (Fig. 6A), and a-helix content also decreased substantially below pH 4.0 (data not shown).
3.5. Re-assembly capability The design strategy proposed by Wang and coworker reported that their engineered cage protein could not re-assemble after dissociation because disassembly of protein cage lead to aggregation [14,15]. However, the re-assembly capability is important in application to drug delivery system because drug or cargo should be capsulated by the dissociated subunit during reassembly process. If dissociation and re-association process of the caged particles are irreversible, it would be impossible to capsulate the cargo inside cage particles. Hence, we tried to investigate if the disassembled subunits could re-assemble into caged particle simply by buffer change. The corresponding peak of dissociated subunits was pooled after the sample was injected on SEC column at pH 6.0. To neutralize the sample, the pooled fraction was dialyzed against the buffer containing 50 mM TriseHCl (pH 7.0) and 100 mM NaCl, and then concentrated to 1 mg/mL. When the sample was injected again on SEC column equilibrated at pH 7.0, we could observe the same 24-mer peak as Fig. 5A, indicating that acid-sensitive dissociation was reversible and the dissociated subunits could re-associate into caged particles by pH neutralization. Therefore, this result shows the first example of engineered caged scaffold to be reversibly re-assembled by neutralization.
Fig. 6. Far-UV circular dichroism spectra for native and fGALA4, 6 hybrids. (A) CD spectra of the native ferritin at pH 6.0 and 7.0. Since ferritin subunits are composed of only a-helices, caged scaffold shows two minima at 208 and 222 nm. (B) CD spectra of fGALA4, 6 hybrids at pH 6.0 and 7.0. Loss of 208 nm minimum and a shift in 222 nm minimum at pH 6.0 indicate unfolding events.
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Fig. 7. SEC profiles of fGALA4, 6 hybrids at various protein concentrations. Equal volume of protein samples with variation of its concentration (0.075, 0.15, 0.3, and 0.6 mg/mL, respectively) are injected on SEC column at pH 7.0. These profiles reveal that assembly of caged particles is independent of protein concentration.
Interestingly, as we expected, our engineered cage protein revealed the different CD profile at pH 6.0. Before triggering dissociation, two fGALA4, 6 hybrids also had the similar CD profile to that of the stable caged particles at pH 7.0 (Fig. 6B). However, when disassembly process was accomplished at pH 6.0, a remarkable decrease of a-helix content at 208 nm minimum and a shift in 222 nm minimum were observed, representing variation of secondary and quaternary structure (Fig. 6B). Especially, robust variation of minima at 208 nm accounted for unfolding process of subunits. Since this variation of CD spectra meant unfolding of subunits, and elution profile on SEC column explained dissociation of cage, we could conclude that unfolding of the subunits in two fGALA4, 6 hybrids were coupled to dissociation of caged particles. The native ferritin did not show any CD spectral variation in pH 6.0 so that the unfolding was anticipated to be triggered by coil-tohelix transition of GALA. 3.7. Effects of protein concentration and ionic strength One variant with the substitution Lys169 / Arg in the recombinant human ferritin heavy chain has been reported to exist as dimer in low protein concentration, but to be readily reassembled after concentration by membrane ultrafiltration [33]. To examine if our fGALA4, 6 hybrids form caged particles only in the high concentrated state, we have compared the oligomeric states at the different protein concentrations of 0.075, 0.15, 0.3, and 0.6 mg/ mL, respectively. Due to the relatively low solubility of the samples, it was impossible to concentrate more than 1 mg/mL. At pH 7.0, although protein concentrations were increased up to eight times higher for each of two fGALA4, 6 hybrids, only the caged particles were observed on the profiles of SEC column (Fig. 7), and assembly of two fGALA4, 6 hybrids was not affected by the protein concentration. In addition, when the acidity increased, acid-sensitive disassembly of two fGALA4, 6 hybrids did not depend on the protein concentration either (data not shown). If unfolding of the subunits is modulated by electrostatic forces, variation of ionic strength would affect the acid-sensitive disassembly of cage particles. To investigate the influence of ionic strength on acid-sensitive disassembly, we increased the NaCl concentration from 100 mM to 500 mM or decreased from 100 mM to zero, but did not observe any different behavior on SEC profiles (data not shown). Hence, we could conclude that assembly process of our protein scaffold was not affected by either protein or salt concentration. 3.8. Thermal stability of fGALA4,
6
hybrids
Because incorporation of artificial pores had possibility to lower the thermostability of our scaffold, we monitored the ellipticity at 222 nm as the protein samples were heated and calculated
Fig. 8. Temperature stability profiles of native ferritin and fGALA4, 6 hybrids. The ellipticity value at 222 nm is monitored to evaluate the degree of unfolding by heating. (A) The native ferritin does not show any transition event over a wide range of temperature both at pH 6.0 and 7.0. The slow increase of molar ellipticity during heating accounts for the high degree of stability in native ferritin. (B) In contrast to the native ferritin, fGALA4, 6 hybrid reveals unfolding with a sigmoidal profile (Tm 78.0 1.0 C) at pH 7.0. As pH is lowered more to pH 6.0, additional transition point is observed at 39.0 0.5 C. At pH 6.0, any transition event is not observed.
apparent midpoint unfolding temperatures (Tm). The native ferritin proteins remain intact over a wide range of temperature and this high thermostability has enabled ferritin to be utilized widely as nanocontainer in material synthesis [9]. In the native ferritin of our study, we could not measure any apparent melting temperatures at pH 6.0 and 7.0 (at which cage assembly retained intact) because the ellipticity values increased slowly and gradually (Fig. 8A). Relatively small variation of ellipticity in a wide range of temperature explained high thermostability of the native protein. In contrast, our acid-sensitive scaffold revealed the apparent sigmoidal curve with a Tm 78.0 1.0 C at pH 7.0 (Fig. 8B) and these transition inflections could account for unfolding of scaffold by heating. Although the different profile between the native and our scaffold was anticipated to be due to incorporation of the artificial pores into scaffold, the acid-sensitive scaffold was still thermostable at pH 7.0. Remarkably, at pH 6.5, which corresponded to the early transition state of disassembly, acid-sensitive scaffold had two melting temperature at 39.0 0.5 C and 75.0 1.0 C (Fig. 8B). According to the analysis of SEC profiles on above, the acid-sensitive scaffold existed as mixture of several oligomeric states in the transition pH range so we interpreted this unfolding profile as superimposition of several profiles, which corresponded to each oligomeric species. The first
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transition temperature, Tm 39.0 0.5 C, was likely to imply unfolding of the intermediate oligomers, which were less stable than assembled cage particles. Moreover, this profile suggested potentially another proof that the coil-to-helix transition of GALA could destabilize the assembly of caged particles. At pH 6.0, which corresponded to complete dissociation of our scaffold, any apparent transition between folding and unfolding was not observed (as we expected), because there were not any stable secondary and quaternary structures (Fig. 8B). After unfolding of the subunits and subsequent dissociation of cage particles were triggered, dissociated form had the lowered thermostability than caged scaffold. However, our acid-sensitive scaffold was sufficiently stable in a wide range of temperature before acidity-inducible disassembly was initiated. 4. Conclusions In summary, we demonstrated the engineered cage protein can dissociate completely at pH 6.0 but reversibly re-assemble at pH 7.0. In this study, ferritin scaffold was genetically modified by our strategy so that artificial GALA peptides were incorporated into caged protein. Coil-to-helix transition of GALA peptide inducible by acid was suggested to impart acid-sensitive disassembly function to cage protein. Our design strategy overcomes the limitation that depended on the specific structural motif of viral capsids. The disassembly and re-assembly process of caged protein were sensitive to weak acid like pH 6.0 so that engineered cage protein would be potentially applicable as acid-inducible molecular switch. Acknowledgements This research was supported by the Pioneer Research Center Program (2009-0081523) and Global Research Laboratory Project of MEST. Appendix Figures with essential color discrimination. Many of the figures in this article have parts that are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/j.biomaterials.2010.03.016. References [1] Kramer RM, Li C, Carter DC, Stone MO, Naik RR. Engineered protein cages for nanomaterial synthesis. J Am Chem Soc 2004;126:13282e6. [2] Uchida M, Klem MT, Allen M, Suci P, Flenniken M, Gillitzer E, et al. Biological containers: protein cages as multifunctional nanoplatforms. Adv Mater 2007;19:1025e42. [3] Flenniken ML, Willits DA, Harmsen AL, Liepold LO, Harmsen AG, Young MJ, et al. Melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture. Chem Biol 2006;13:161e70. [4] Johnson JE, Speir JA. Quasi-equivalent viruses: a paradigm for protein assemblies. J Mol Biol 1997;269(5):665e75. [5] Bancroft JB, Hills GJ, Markham R. Study of the self-assembly process in a small spherical virus. Formation of organized structures from protein subunits in vitro. Virology 1967;31(2):354e79. [6] Ausar SF, Foubert TR, Hudson MH, Vedvick TS, Middaugh CR. Conformational stability and disassembly of Norwalk virus like particles: effect of pH and temperature. J Biol Chem 2006;281(28):19478e88.
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