Article pubs.acs.org/Biomac
Supramolecular Assembly and Coalescence of Ferritin Cages Driven by Designed Protein−Protein Interactions Giuliano Bellapadrona,*,† Shwetali Sinkar,‡ Helena Sabanay,§ Ville Liljeström,∥ Mauri Kostiainen,∥ and Michael Elbaum*,† †
Department of Materials and Interfaces, Weizmann Institute of Science, 76100 Rehovot, Israel Indian Institute of Technology, Bombay, Mumbai Area 400076, India § Department of Chemical Research Support, Weizmann Institute of Science, 76100 Rehovot, Israel ∥ Biohybrid Materials Group, Department of Biotechnology and Chemical Technology, Aalto University, 00076 Aalto, Finland ‡
S Supporting Information *
ABSTRACT: A genetically encoded system for expression of supramolecular protein assemblies (SMPAs) based on a fusion construct between ferritin and citrine (YFP) was transferred from a mammalian to a bacterial host. The assembly process is revealed to be independent of the expression host, while dimensions and level of order of the assembled structures were influenced by the host organism. An additional level of interactions, namely, coalescence between the preformed SMPAs, was observed during the purification process. SAXS investigation revealed that upon coalescence, the local order of the individual SMPAs was preserved. Finally, the chaotropic agent urea effectively disrupted both the macroscopic coalescence and the interactions at the nanoscale until the level of the single ferritin cage.
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INTRODUCTION Improvements in understanding the rules that govern interactions and organization of biological macromolecules in complex structures motivate efforts to synthesize artificial supramolecular assemblies. DNA has been the first biological molecule exploited for synthetic design of supramolecular structures, rapidly gaining popularity as “DNA origami”1,2 due to its high physicochemical stability, well-defined hybridization properties, and the possibility of predictive in silico modeling.3 Recently, polypeptides have been used both in the “origami” fashion,4,5 building on protein folding, as well as in combination with inorganic material.6 Particularly “protein-cages”,7 namely, roughly spherical and hollow assemblies composed of oligomeric proteins, as for example viral capsids or the wellknown ferritin proteins, have been studied for their intrinsic, highly ordered self-assembly properties,8,9 and have been exploited to form ordered lattices structure in combination with metal nanoparticles. 10−14 However, the assembly processes are for the most part achieved in vitro and are so far restricted to the nanoscale both in terms of dimensions and quantity.15 In a recent work, we demonstrated the formation of supramolecular protein assemblies16 (SMPAs) inside living human tissue culture cells, purely by means of genetic encoding.17 The system takes advantage of the native selfassembly properties of human heavy-chain ferritin18 (HuFtH) and citrine,19 a variant of the yellow fluorescent protein (YFP), via a chimeric fusion of the respective genes. Ferritin subunits spontaneously assemble to form the roughly spherical ferritin © XXXX American Chemical Society
cage structure (Figure 1) with 432-pointgroup symmetry. The citrine proteins, linked to the flexible N-terminus of each
Figure 1. Schematic representation of the self-assembly and supermolecular assembly processes of the citrine(WT)-LK-HuFtH fusion protein constructs.
ferritin subunit, extend outward from the protein surface in three dimensions. Finally, citrine−citrine dimerization in an antiparallel orientation provides a stabilizing potential for close packing of the ferritin cages in an extended structure (Figure 1). That investigation highlighted the importance of symmetry, polyvalent presentation, and chemical environment at the citrine−citrine interface.17 In the present work, we transferred the citrine−HuFtH SMPAs system from the mammalian to a bacterial host in order: (i) to investigate the influence of a different genetic expression system and biological environment, namely, the Received: April 2, 2015 Revised: May 10, 2015
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DOI: 10.1021/acs.biomac.5b00435 Biomacromolecules XXXX, XXX, XXX−XXX
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calcium chloride) for 2.5 h at room temperature followed by 4 °C overnight. The bacteria were then washed 4 times in 0.1 M cacodylatebuffer and embedded in 3.4% agarose. Osmification was done in 1% OsO4, 0.5% K2Cr2O7, 0.5% K4[Fe(CN)6]3H2O, in 0.1 M cacodylatebuffer for 1 h at room temperature followed by washing twice in 0.1 M cacodylate-buffer and three times in DDW. For contrast, bacteria were impregnated with uranyl-acetate (2% in DDW for 1 h) and then washed twice in DDW. A series of dehydrating steps with increasing EtOH concentrations (30%, 50%, 70%, 96% each for 5 min twice, followed by 100% three times for 10 min) was performed. The pellets were dried in propylene oxide twice for 5 min. Epon (“hard”) was introduced gradually into the bacterial pellets again in rising concentrations (30%, 50%, for 2 h followed by 70% overnight, then 100% overnight, followed by 100% three times for 2 h the next day). The blocks were then transferred to baking-molds in Epon and baked for 2 days at 60 °C. The Epon-blocks were finally cut into 70−80 nm ultrathin slices, mounted onto grids, and stained with lead citrate and uranyl acetate. Grids were analyzed in a Tecnai T-12 Spirit transmission electron-microscope (FEI); images were recorded using a CCD camera (FEI Eagle 2kx2k, Eindhoven, Netherlands). Iron-Enriched LB Media. An iron-citrate complex solution was prepared by dissolving crystalline ferrous ammonium sulfate, Fe(II)SO4·7H2O (Sigma-Aldrich) and citric acid monohydrate (SigmaAldrich) in Ultra-Pure Milli-Q water at 10 mM and 50 mM final concentrations, respectively. The solution was then titrated with sodium hydroxide, NaOH (Sigma-Aldrich) to pH 7.0 and sterilized using 0.22 μM syringe filters. Iron-enriched LB media was obtained by adding iron-citrate complex solution to a final concentration of 0.5 mM. In vitro Iron Loading of Citrine-HuFtH SMPAs in 8 M Urea. Citrine-HuFtH SMPAs isolated from E. coli and solubilized in 50 mM HEPES-NaOH, 0.2 M NaCl, 8 M urea pH 7.2 were reconstituted in vitro with iron. A 10 mM ferrous ammonium sulfate, Fe(II)SO4·7H2O (Sigma-Aldrich) solution was prepared in Argon-purged Ultra-Pure Milli-Q water and supplied by 10 individual additions every 30 min to a 5 μM citrine-HuFtH solution in the above buffer under continuous stirring. Each iron addition corresponded to 100 iron atoms/ferritin cage. Iron oxidation upon ferritin uptake was detected by a change in the color of the solution from yellow to red. Small-Angle X-ray Scattering. The liquid samples were sealed between two Kapton foils during the SAXS measurements and the sample environment was evacuated to reduce scattering from air. The SAXS was measured using a rotating anode Bruker Microstar microfocus X-ray source (Cu Kα radiation, λ = 1.54 Å). The beam was monochromated and focused by a Montel multilayer focusing monochromator (Incoatec). The X-ray beam was further collimated by a set of four slits (JJ X-ray) resulting in the final spot size of less than 1 mm at the sample position. The scattered intensity was collected using a Hi-Star 2D area detector (Bruker). Sample-to-detector distance was 1.59 m and silver behenate standard sample was used for calibration of the length of the scattering vector q. One-dimensional SAXS data were obtained by azimuthally averaging the 2D scattering data. The magnitude of the scattering vector q is given by q = 4π sin θ/λ, where 2θ is the scattering angle.
bacterial cytoplasm, in the assembly process, and (ii) to express, purify, and extensively characterize on the large scale the citrine-HuFtH SMPAs. The observations confirmed that the information encoded in the primary sequence of the construct is sufficient to trigger the supramolecular assembly process. However, both the size of the SMPAs and the level of order were affected by expression in the bacterial host as compared to the mammalian expression host. Extraction and purification of the citrine-HuFtH SMPAs from bacteria revealed new interactions occurring at the macromolecular scale: individual, preformed, citrine-HuFtH SMPAs of defined dimensions and shapes that were released upon the lysis of the bacterial membrane coalesced in solution to form macroscopic structures. The latter maintain the local order of the constituent macromolecules, but do not achieve higher levels of order, thus highlighting the dominant role of the predefined molecular geometries despite the same driving forces for the interactions. Control over the forces underlying the two distinct supramolecular assembly and macromolecular coalescence processes occurring at the nano- and at the macroscale, respectively, was demonstrated by the use of a chaotropic agent, urea, which is able to disrupt molecular interactions at the dimeric citrine interface without unfolding either of the associating proteins.
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MATERIALS AND METHODS
Cloning and Expression of Citrine-Ferritin Chimeric Constructs in Escherichia coli. Previously described citrine(WT)-LKHuFtH and citrine(A206 K)-LK-HuFtH genes17 were cloned from the pcDNA3 (Invitrogen) vector into the pET12a (Novagen) plasmid between NdeI and BamHI by restriction-free PCR cloning. Transformed bacteria encoding either citrine(WT)-LK-HuFtH or citrine(A206 K)-LK-HuFtH were grown from a single colony for 6−7 h in a starter of 3 mL LB/Amp (100 mg/L). Next, 1 mL of the starter was transferred to 1 L of LB/Amp (150 mg/L) media and incubated for 18 h at 37 °C with shaking. Bacteria were then harvested by centrifugation at 8000 r.p.m. for 8 min; the pellet was frozen at −20 °C. In order to break the bacterial membrane and release citrine(WT)LK-HuFtH SMPAs or citrine(A206 K)-LK-HuFtH individual cages in solution, the pellet was thawed, resuspended in 25 mL of 10 mM TrisHCl pH 7.5, 5 mM MgCl2, 0.5 mM CaCl2, 0.05 mg/mL DNase I (Sigma-Aldrich), and lysed in a probe sonicator (SONICS, Microcell) at 60% amplitude (3 s ON, 7 s OFF, total time: 9 min) in ice. The lysate was then incubated at 37 °C for 1 h with shaking and additional 0.05 mg/mL DNase I (Sigma-Aldrich) was added for DNA digestion. Ethylenediaminetetraacetic acid (EDTA) and sodium chloride were added to final concentrations 5 mM and 0.5 M, respectively. The unique method for purification of citrine(WT)-LK-HuFtH SMPAs is described in the main text; purification of citrine(A206 K)-LK-HuFtH individual cages exploited thermal denaturation of the contaminant proteins by 10 min incubation at 65 °C followed by centrifugation, filtration of the supernatant with a 0.22 μM filter, and loading on a XK 16/70 column (GE Healthcare) packed with Sepharose 6 gel (GE Healthcare), which was equilibrated with 50 mM HEPES-NaOH, 0.2 M NaCl, pH 7.2. Citrine(A206 K)-LK-HuFtH was isolated as a single peak eluted around 62 mL. Imaging of bacteria and extracted SMPA was performed in a confocal scanning microscope (Olympus Fluoview 300) equipped with a PlanApo 60x/N.A. 1.20 water immersion objective. Fluorescence of Citrine(WT)- and Citrine(A206 K)- fusion constructs with HuFtH was excited by a 488 nm, 50 mW, blue solid state laser, (Coherent, OBIS). Transmission Electron Microscopy. E. coli BL21(DE3) bacteria expressing citrine(WT)-LK-HuFtH SMPAs were centrifuged at 2000 rpm for 5 min and chemically fixed using Karnovsky-fixative (2% glutaraldehyde, 3% PFA, in 0.1 M cacodylate-buffer pH 7.4 and 5 mM
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RESULTS AND DISCUSSION
Transfer of the Expression System from Mammalian Cells to Bacteria. The citrine-HuFtH supramolecular protein assemblies (SMPAs) system was transferred from the mammalian host to a standard bacterial expression strain, E. coli BL21(DE3), by cloning the citrine(WT)-LK-HuFtH and citrine(A206 K)-LK-HuFtH genes17 from the pcDNA3 (Invitrogen) vector into the pET12a (Novagen) plasmid (LK represents a flexible spacer of 17 amino acids). The A206 K mutation introduces a positively charged residue into the hydrophobic patch at the citrine dimerization interface and eliminates the dimerization potential. After transformation of bacteria, constitutive protein expression was achieved by 18 h B
DOI: 10.1021/acs.biomac.5b00435 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules growth in Luria−Bertani (LB) media containing Ampicillin (150 mg/L) at 37 °C with shaking. Expression was assayed by confocal fluorescence and differential interference microscopy and compared to expression in mammalian cells transfected with the corresponding pcDNA3 constructs. Formation of citrine-HuFtH SMPAs was observed in the transformed E. coli BL21(DE3) strain as a results of expression of the citrine(WT)-LK-HuFtH construct. SMPAs appeared to be localized at one of the two poles of the bacteria (Figure 2A−
Figure 3. Transmission electron microscopy (TEM) image of E. coli BL21(DE3) strain expressing the citrine(WT)-LK-HuFtH construct. Scale bar is 0.5 μM (500 nm).
in the short-range (closely packed protein cages), with no evidence of the global long-range and crystalline order previously observed in HeLa cells.17 The difference in the level of order, despite the same overall assembly process, can be justified both by kinetic and environmental considerations. The dynamic concentration of constituent molecules available for the assembly process, has a direct impact on crystallization; it is strongly affected by differences in the rate of protein expression and cellular volume between the bacterial and mammalian host. Furthermore, differences in molecular crowding between the two organisms determine different environmental conditions for the crystal growth. Rapid and constrained growth in the bacteria would tend to impose a quenched disorder. Purification of Citrine-HuFtH SMPAs from Bacteria. Citrine-HuFtH SMPAs were produced on a large scale in order to purify significant quantities for biochemical/biophysical characterization (see Materials and Methods). Once released from the bacteria into solution, the SMPAs appeared as a yellowish, fluffy material that slowly and spontaneously formed sediments at the bottom of a test tube. Purification of the citrine-HuFtH SMPAs from the whole extract of E. coli proteins, DNA, and debris exploited this property. The bacterial lysate was first centrifuged at low gravitational force (220 g, 1000 r.p.m.) in a swinging bucket rotor for 20 min at 15 °C. A soft yellowish pellet was formed. The supernatant was tested for the presence of citrine by UV−visible spectroscopy; however, no peak corresponding to the citrine absorption maximum at 515 nm was detected; thus the supernatant was discarded, and the pellet was resuspended in 25 mL of 50 mM HEPES-NaOH, 0.2 M NaCl, pH 7.2 buffer and then centrifuged again for 10 min at the same gravitational force. The centrifugation/resuspension cycles were repeated 6 times until in the supernatant minimal traces of contaminant DNA and proteins were detected by UV−visible spectroscopy. The final product of the purification appeared as an intense yellow suspension (Figure 4, A). Over several hours (up to 24 h) the yellow suspension tended to sediment spontaneously by gravity forming a soft yellow pellet similar to those obtained by centrifugation (Figure 4B). Addition of urea to 8 M final concentration readily solubilized the suspension (Figure 4C). Finally, the purity of the citrine-HuFtH SMPAs was confirmed
Figure 2. Confocal scanning fluorescence (A, D, G, L), differential interference contrast (B, E, H, I) and overlay (C, F, I, N) images showing the expression of the citrine(WT)-LK-HuFtH (A-C; D-F) and citrine(A206 K)-LK-HuFtH (G−I; L−N) fusion constructs in E. coli BL21(DE3) bacteria (A−C; G−I) and HeLa mammalian cervical cancer cells (D−F; L−N). Scale bar is 5 μM.
C). The peripheral localization could represent a physiological response of the host organism to the encumbrance of the SMPAs in the limited volume of the bacterial cytoplasm. Typical diameters were smaller than the corresponding structures produced in HeLa cells (Figure 2D−F), most probably due to constraints imposed by the size of the bacteria. As a fraction of cell volume, however, they appear to be much larger in the bacteria. Strikingly, expression of the citrine mutant A206 K resulted in uniform fluorescence due to free diffusion of individual citrine(A206 K)-HuFtH cages throughout the cytoplasm (Figure 2G−I); this effect is identical to that previously observed in the nuclei of transfected HeLa cells (Figure 2L−N). The comparison thus confirms that the driving force for the self-assembly process is the dimerization between citrine proteins and that the information encoded within the chimerical polypeptide chain is sufficient to trigger the supramolecular assembly process independently by the expression host. Structural information on the citrine-HuFtH SMPAs inside the bacteria, prior to purification, was obtained by thin section transmission electron microscopy (TEM). The images confirmed the previous observations by confocal fluorescence microscopy, showing localized formation of the citrine-HuFtH SMPAs at a single pole of the bacteria (Figure 3). However, the structure of the bacterial SMPAs appeared to be ordered only C
DOI: 10.1021/acs.biomac.5b00435 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 4. Citrine-HuFtH SMPAs purified from in E. coli BL21(DE3) bacteria. Suspension in 50 mM HEPES-NaOH, 200 mM NaCl, pH 7.2 buffer (A); spontaneous sedimentation over 24 h (B); solubilization in 8 M urea (C). SDS-page, 15% polyacrylamide gel, of citrine-HuFtH SMPAs and molecular weight marker (D).
Figure 5. SAXS profile of citrine-HuFtH SMPAs purified from bacteria grown in iron enriched media (citrine-HuFtH-Fe, black line) shows clear first-, second-, and third-order correlation maxima, which are interpreted as high short-range order and periodic structure. CitrineHuFtH SMPAs isolated from bacteria grown in regular media (citrineHuFtH, blue line) shows similar features, but less distinctive maxima due to the minimal amount of iron within the ferritin cores. Ureatreated samples show only very weak correlation shoulders at smaller q-values indicating a less ordered state and a larger period. The ureatreated samples in the apo-form (citrine-HuFtH − 8 M Urea, red line) or in the iron reconstituted form (citrine-HuFtH − 8 M urea, green line) do not show any features indicating a periodic ordering within the samples (A). Kratky plot of citrine-HuFtH-Fe sample (panel A, black line): SAXS profile emphasizes the first order correlation peak (q*) as well as the second and the third order correlation peaks (B). First, second and third order correlation maxima plotted as a function of q. Linear fitting to the positions of the maxima yields the average correlation distance dcorr = 18.4 nm (C).
by SDS PAGE analysis (Figure 4D). A predominant band was detected between 40 and 55 kDa, as expected for the molecular weight of a single citrine-HuFtH fusion subunit (49 kDa); the level of purity as estimated from the gel was >90%. Small Angle X-ray Scattering (SAXS) Characterization. The nanoscale periodicity of the purified citrine-HuFtH SMPAs was characterized using SAXS. Samples of citrine-HuFtH SMPAs were produced in bacteria grown either in standard or in iron-enriched LB medium and purified as above-described to suspension in 50 mM HEPES-NaOH, 0.2 M NaCl, pH 7.2 buffer. Additionally, SMPAs grown in standard medium were dispersed in 8 M urea and then loaded with iron in vitro. The liquid samples were sealed in sample holders and the measurement chamber was evacuated to minimize the scattering from air. Small angle X-ray scattering was collected on a Bruker Hi-star 2D detector. The final scattering profile presents the azimuthal average of the scattering intensity in Figure 5A. The suspension of citrine-HuFtH SMPAs purified by bacteria grown in iron enriched media (Figure 5, black line) showed the strongest correlation maxima in the scattering profile, as iron enriched ferritin cores are strong scattering centers. The correlation peaks seen in the scattering profiles indicated a nanoscale periodicity with short-range order. In the scattering profile of citrine-HuFtH-Fe (Figure 5B) the maximum at 0.038 Å−1 together with second and third order maxima roughly at 0.069 Å−1 and 0.101 Å−1 indicates a periodicity of 18.4 nm (Figure 5C), which equals to the center-to-center distance of ferritin cages (outer diameter 12.5 nm) with 5 nm spacing. The latter distance fits well with the average diameter of a dimer of citrine molecules in between the ferritin cages. In the sample purified from bacteria grown in standard LB media (Figure 5, blue line) the observed scattering profile for the citrine-HuFtH SMPAs is less pronounced, as would be expected due to smaller contrast provided by the empty ferritin cage as compared to an iron-loaded one. However, the periodicity of the structure remains unaffected at approximately 18 nm as the absence of an iron core inside the ferritin cage does not change the overall particle arrangement. Samples solubilized in 8 M urea, both in apo form (Figure 5, red line) and reconstituted in vitro with iron (Figure 5, green line), lost their distinctive scattering profile due to the absence of ordered structures, consistent with a role of urea in destabilizing the interactions responsible for the stability of the supramolecular assembly.
Microscopic Investigation of the Citrine-HuFtH SMPAs and Effect of Urea. Confocal fluorescence and differential interference contrast (DIC) microscopy were used to investigate the spontaneous sedimentation of the citrineHuFtH SMPAs and subsequent solubilization in 8 M urea. Microscopy images of the purified materials in 50 mM HEPESNaOH, 0.2 M NaCl, pH 7.2 buffer revealed that the citrineHuFtH SMPAs, formed individually in single bacteria, tend to coalesce dendritically into macroscopic aggregates immediately once released in solution after disruption of the bacterial membrane. Individual constituent SMPAs still preserve their individual size and shape (Figure 6A,B; see also Figure S1 for full-size images). The driving force for the aggregation of SMPA is likely to be found again in the interactions at the citrine−citrine dimerization interface that were implicated in the assembly of single SMPAs,17 in particular, the cluster of hydrophobic amino acids (Ala206, Leu221, Phe223). To prove this hypothesis, urea was again used to disrupt hydrophobic interactions, this time at a series of increasing concentrations. Lower concentrations of urea, 2 and 4 M, showed a limited ability to disrupt the coalesced structures (Figure 6C,D and E,F, respectively). However, higher concentrations of urea, 6 and 8 M, dispersed the individual SMPAs originally formed in the bacteria and also partially solubilized them into the constituent citrine-HuFtH cages. This led to a significantly increased background fluorescence arising from freely diffusing molecular species (Figure 6G,H and I,L, respectively). The ability of 8 M urea to dissolve the macroscopic aggregates of citrine-HuFtH SMPAs down to the level of individual 24-mer ferritin cages carrying the covalently linked 24 citrine proteins (Mw = 1182 kDa) was further assayed by size exclusion chromatography. The tertiary and quaternary D
DOI: 10.1021/acs.biomac.5b00435 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 7. Size exclusion chromatography elution profile (A) at 280 nm (blue line) and 515 nm (green line) and UV−visible absorption spectrum (B) of citrine-HuFtH SMPAs solubilized in 50 mM HEPESNaOH, 200 mM NaCl, 8 M urea, pH 7.2 buffer. (C) Elution profiles of citrine-HuFtH SMPAs (blue line), citrine(A206 K)-HuFtH (black line), HuFtH (red line) and LiDps (gray line) in 50 mM HEPESNaOH, 200 mM NaCl, 8 M urea, pH 7.2 buffer.
The earlier one at 43.8 mL corresponded to the excluded volume of the column, suggesting still the presence of large aggregated species. The later peak eluting at 61.6 mL most likely contained the single constituent citrine-HuFtH cages. For comparison, isolated, non-SMPA-forming constructs, citrine(A206 K)-HuFtH (Mw = 1182 kDa) with similar molecular weight to that of citrine(WT)-HuFtH single cages, as well as the 24-mer HuFtH18 lacking citrine (Mw = 509 kDa), and the 12-mer Listeria innocua mini-ferritin22 (Mw = 216 kDa) were incubated in 50 mM HEPES-NaOH, 0.2 M NaCl, 8 M urea pH 7.2 and injected into the size exclusion column under the same conditions used for the citrine-HuFtH SMPAs. The three proteins eluted as single peaks at 62.8, 74.4, and 80.3 mL, respectively (Figure 7C). The elution volume of citrine(A206 K)-HuFtH single cages (62.8 mL) was comparable to that of the later peak (61.6 mL) from the elution of citrine(WT)-LKHuFtH SMPAs. This confirmed that the end point of cluster dispersion by urea reaches as well the level of single citrineferritin cages. Interestingly, while in the earlier elution the 280 nm peak absorption by protein aromatic residues was significantly stronger than the absorption at 515 nm of the citrine fluorophore, in the later peak the ratio was inverted with absorption at 515 nm much higher than the 280 nm value.
Figure 6. Confocal scanning fluorescence (A, C, E, G, I), differential interference contrast (B, D, F, H, L) images showing the dispersion of citrine-HuFtH SMPAs in suspension at progressively higher concentration of urea. Citrine-HuFtH SMPAs in 50 mM HEPESNaOH, 200 mM NaCl, pH 7.2 buffer (A,B), containing 2 M urea (C,D), 4 M urea (E,F), 6 M urea (G,H) and 8 M urea (I,L). Scale bar is 20 μM.
structure of both ferritin and green fluorescent protein (GFP) is known to be preserved in 8 M urea.20,21 A XK 16/70 column (GE Healthcare) packed with Sepharose 6 gel (GE Healthcare) was equilibrated with 50 mM HEPES-NaOH, 0.2 M NaCl, 8 M urea pH 7.2 buffer at a flow rate of 0.5 mL/min; 5 mL of citrine-HuFtH SMPAs were prepared in the same buffer and injected into the column. Elution was monitored by optical absorption at two different wavelengths, 280 and 515 nm, corresponding to the absorption maxima of the protein aromatic residues and of the citrine fluorophore, respectively (Figure 7A). Two well separated peaks eluted from the column. E
DOI: 10.1021/acs.biomac.5b00435 Biomacromolecules XXXX, XXX, XXX−XXX
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When SMPAs were dispersed by 8 M urea the 515 nm absorption was stronger than the 280 nm (Figure 7, B). A comparison to SMPA with inherently dispersed citrine(A206 K)-HuFtH showed a similar trend of inverted absorption peaks. This provides further support for the assertion that the later elution peak contained single constituent citrine(WT)-HuFtH cages where the citrine molecules extend freely from the surface of the ferritin cage. The reduced 515 nm absorption in larger assemblies can be understood as a self-quenching effect due to the very high density of fluorophores, or possibly as an effect of mechanical stress on the proteins in compacted form. The absorption ratio could potentially be exploited as a reporter for the degree of superassembly.
CONCLUSIONS In summary, the transfer of the citrine-HuFtH SMPA expression system from mammalian cells to bacteria emphasized the essential importance of structural information encoded in the primary sequence, but also the differences due to different biophysical properties of the two host organisms. The comparison between self-assembly at the nanoscale and coalescence at the macroscale highlighted the role of predefined geometry despite identical protein−protein interaction driving forces. Finally, the use of the chaotropic agent urea provided a means of successfully separating the macro-scale coalescent SMPAs into the nanoscale single constituent units. ASSOCIATED CONTENT
S Supporting Information *
Enlarged images from each panel in Figure 6 are available in the Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00435.
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[email protected] *E-mail:
[email protected]. Author Contributions
G.B., M.K., and M.E. designed the experiments, which were conducted primarily by G.B. with important contributions from S.S. and V.L. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Y. Barak for advice and assistance in molecular biology. This work was supported in part by a grant from the Israel Science Foundation (grant 1369/10), and by the Gerhardt M. J. Schmidt Center for Supramolecular Architecture. Financial support from the Academy of Finland (Grants 263504, 267497, 273645), Biocentrum Helsinki, and the Emil Aaltonen Foundation is gratefully acknowledged. This work was carried out under the Academy of Finland’s Centres of Excellence Programme (2014-2019). M.E. acknowledges the historical generosity of the Harold Perlman family.
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ABBREVIATIONS SAXS, small-angle X-ray scattering; HuFtH, human ferritin heavy-chain F
DOI: 10.1021/acs.biomac.5b00435 Biomacromolecules XXXX, XXX, XXX−XXX
