Article pubs.acs.org/Biomac
Effect of ELP Sequence and Fusion Protein Design on Concentrated Solution Self-Assembly Guokui Qin, Paola M. Perez, Carolyn E. Mills, and Bradley D. Olsen* Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States S Supporting Information *
ABSTRACT: Fusion proteins provide a facile route for the purification and self-assembly of biofunctional protein block copolymers into complex nanostructures; however, the use of biochemical synthesis techniques introduces unexplored variables into the design of the structures. Using model fusion constructs of the red fluorescent protein mCherry and the coillike protein elastin-like polypeptide (ELP), it is shown that the molar mass and hydrophobicity of the ELP sequence have a large effect on the propensity of a fusion to form well-ordered nanostructures, even when the ELP is in the low temperature, highly solvated state. In contrast, the presence of a 6xHis purification tag has little effect on self-assembly, and the order of blocks in the construct (N-terminal vs C-terminal) only has a significant effect on the nanostructure when the conjugates are heated above the transition temperature of the ELP block. These results indicate that for a sufficiently hydrophobic and high molar mass ELP block, there is a great deal of design latitude in the construction of fusion protein block copolymers for self-assembling nanomaterials.
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INTRODUCTION Controlling the nanostructure of proteins incorporated within solid materials is important for a wide variety of applications in catalysis, health, defense, and energy.1−4 Control over the structure of the material may improve the density of active sites and the orientation of protein within a material, providing for rapid transport to and from the protein active site while maintaining protein function. This can enable significant performance advantages over the current state of the art.5−8 Among many approaches to nanostructure control,9−13 block copolymer self-assembly techniques provide attractive bottomup approaches to control nanostructure in materials containing functional proteins.14−16 It has been well established that block copolymers containing a polypeptide block with simple secondary structure can self-assemble into a wide variety of nanostructures with characteristic length scales from 5 to 100 nm.17−19 However, globular proteins have more complex folded shapes, which are critical to their function, and specific interactions between proteins can change the thermodynamics of self-assembly. The phase behavior of these copolymers is significantly different than block copolymers from two Gaussian coil copolymers.20 Changing the identity of the polymer block has a large impact on the type of nanostructure formed,21 while changes to the protein that do not produce a significant change in protein shape, hydrophobicity, or net charge are shown to have a small impact on the phase behavior.5 Because this self-assembly method can achieve a high protein density while maintaining rapid transport through a material, it has the potential for the fabrication of highly active biofunc© XXXX American Chemical Society
tional nanostructures. Recently, heterogeneous biocatalysts containing the globular protein myoglobin and polymer PNIPAM have been fabricated using bioconjugate block copolymer self-assembly. The myoglobin−PNIPAM conjugates can form weakly ordered micellar and lamellar nanostructures in concentrated solution and yield very high protein loadings and activity per unit area within the flow-coated films compared to established enzyme encapsulation methods.22 However, the synthesis and purification of bioconjugates in high yield remains a significant challenge.14,15,23,24 In contrast, fusion protein block copolymers combining two structural protein elements,25 an elastin-like polypeptide (ELP) and a globular protein, are easily biosynthesized and purified by thermal precipitation.26,27 ELPs are artificial biopolymers containing repeats of the pentapeptide sequence Val-Pro-GlyXaa-Gly (VPGXG), where Xaa can be any naturally occurring amino acid except Pro.28−31 The polymers are highly solvated and soluble in aqueous solutions at low temperatures, but undergo an inverse thermal transition and become insoluble at high temperature, leading to aggregation of the polypeptides above the transition temperature (Tt).29,32 Previous studies have shown that this property of the ELPs is retained after genetic fusion to other proteins, and the Tt can be modulated by the presence of the fused proteins.27,28,33,34 Our group has recently demonstrated that these fusion proteins of ELP and a Received: November 30, 2015 Revised: January 29, 2016
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DOI: 10.1021/acs.biomac.5b01604 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 1. Schematic of mCherry-ELP fusion block copolymers designed using two different ELP sequences, ELP0 and ELP1.
Table 1. Molecular Composition of mCherry-ELP Fusions fusion
mol wt (kDa)
pI
His tag
ELP0-mCherry ELP0a-mCherry ELP0b-mCherry mCherry-ELP0c mCherry-ELP1 mCherry-ELP1a mCherry-ELP1b ELP1c-mCherry
69.4 71.8 47.1 71.8 46.3 43.9 73.7 46.3
5.5 6.4 6.4 6.4 6.4 5.5 6.4 6.4
no yes yes yes yes no yes yes
ELP ELP0
ELP1
ELP mol wt (kDa)
ELP position
41.2 41.2 16.5 41.2 15.7 15.7 43.1 15.7
N-terminal N-terminal N-terminal C-terminal C-terminal C-terminal C-terminal N-terminal
model globular protein mCherry can self-assemble into high density, solid-state biofunctional nanostructures as fully biosynthetic analogues of protein−polymer conjugate block copolymers.35 Genetic engineering of the block copolymer constructs allowed for the comparison of two different molecular architectures, E10-mCherry-E10 double tail fusions and E20-mCherry single tail fusions. The topology of the fusion had a strong effect on the phase diagram, including the locations of phase transitions and types of nanostructures formed. Therefore, fusion protein design provides a rich landscape for controlling supramolecular assembly.35
ELP repeating unit (VPGVG)2IPGVG(VPGVG)2
VPGVGVPGGGVPGAG(VPGVG)3 VPGGGVPGAGVPGGGVPGVG
The engineering of a fusion protein block copolymer requires several specific choices in molecular construction as a part of the design of genes encoding for the proteins. Herein, ELPmCherry model fusion systems are explored to understand the design characteristics that lead to the greatest propensity for self-assembly of thermodynamically stable nanostructures within fusion constructs. As with other ELP fusion proteins, the block copolymer mCherry-ELP fusions can be isolated at low cost using fusion purification tags and self-assembled into solid, bioactive materials, where the degree of order depends strongly upon several design criteria. The effect of the ELP hydrophobicity, the ELP molar mass, the order of the blocks in B
DOI: 10.1021/acs.biomac.5b01604 Biomacromolecules XXXX, XXX, XXX−XXX
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UV−Vis Spectrophotometry. The absorbance spectra of fusion proteins were collected on a Cary 50 UV−vis spectrophotometer with a Peltier temperature controller. Solid fusion films were obtained by vacuum drying protein solution samples on the quartz discs. Vacuum was slowly applied at a ramp rate of 50 Torr/h. The solid materials were then rehydrated to the designed concentrations for material characterization after self-assembly. A spectrophotometric measure of protein function is calculated as A586 of the fusion sample relative to A586 of the mCherry control, where both values are normalized by A280 to control for variations in protein concentration. At least three replicates were averaged for each sample.
a fusion, and the presence or absence of a 6xHis metal affinity purification tag were all explored, providing insight into engineering self-assembled fusion protein systems for potential application in energy and biomedical studies.
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EXPERIMENTAL SECTION
Fusion Protein Design. Model globular proteins fused with coillike proteins were synthesized using the red fluorescent protein mCherry and members of the family of thermoresponsive structural polymers ELPs, as described previously.35 The high-yield expression and purification of the model protein mCherry have been wellestablished, and its fluorescent nature provides a simple and robust spectrophotometric method for fusion characterization.35 Two ELP polymers with different amino acid sequences, ELP0 and ELP1, were chosen as coil blocks and fused with mCherry to generate two series of mCherry-ELP fusions: mCherry-ELP0 and mCherry-ELP1. To generate the new fusion protein system, the model red fluorescent protein mCherry was fused with two different ELPs by genetic engineering as reported previously.35−37 The architecture of the mCherry-ELP fusion proteins in this study, including the affinity purification 6xHis tag, ELP chain size, and their fusion order, is explored by changing the amino acid sequence of blocks encoded through gene design. In total, eight proteins were designed, as summarized in Figure 1 and Table 1. All of mCherry-ELP fusion constructs were expressed in Escherichia coli (E. coli) BL21(DE3) in soluble form with a yield of about 50 mg/L. Further purification of mCherry-ELP fusions was performed using Ni-NTA affinity columns and GE AKTA Fast Protein Liquid Chromatography (FPLC). Complete biosynthesis procedures and sequences for mCherry-ELP fusions are provided in the Supporting Information (Figures S1−S8). Sample Preparation. Fusion protein solutions were dialyzed against pure water and concentrated to approximately 100 mg/mL using Millipore Ultra-15 centrifugal filters with a molecular weight cutoff of 10 kDa. The fusion proteins were then diluted to different concentrations with concentration confirmed using an Implen Nanophotometer (Implen GmbH, Germany). The mCherry-ELP fusions self-assemble through evaporation of water from solution to form nanostructured materials. Therefore, the concentrated fusion protein solutions were also cast in ∼20 μL aliquots on a Teflon sheet and gradually exposed to vacuum at a ramp rate of 50 Torr/h. Samples were held at 10 Torr overnight at room temperature to dry completely. The pellets were redissolved in water to prepare fusion protein solutions with the desired concentrations and equilibrated at 4 °C overnight. Most experiments were performed at a concentration of 50 wt % because this concentration has been previously shown to maximize the chance of observing ordered phases.20,35 Turbidometry. A Cary 50 UV−vis spectrophotometer with a Peltier temperature controller was used to investigate optical density (OD) changes of fusion solutions at 700 nm and confirm whether the mCherry-ELP fusions still retain the thermal responsive properties of ELPs. Reversibility of the thermal transition was examined by heating and cooling at 1 °C/min over the temperature range 10−50 °C. The thermal transition was defined as the temperature corresponding to a 10% reduction in the initial sample transmittance, according to previous methods.38,39 Small-Angle X-ray Scattering (SAXS). Sample preparation for SAXS studies was performed as reported previously.35 Briefly, the fusion solutions were used to fill 1 mm thick anodized aluminum washers backed with Kapton tape. The washers were covered with Kapton tape to seal. The fusion proteins were measured on Experimental Station Beamline 1−5 of the Stanford Synchrotron Radiation Lab (SSRL) at Stanford University, and some samples were also measured at Beamline 7.3.3 of the Advanced Light Source (ALS) at Lawrence Berkeley National Lab. Samples were equilibrated at 10 °C for 20 min and for 10 min at all other temperatures prior to data collection. SAXS data were collected and corrected for empty cell and dark field scattering. Acquisition times were minimized such that the effect of beam damage on sample nanostructure was undetectable. All observed transitions were reversible with temperature.
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RESULTS AND DISCUSSION The specific molecular design of mCherry-ELP fusion proteins has a large impact on their ability to self-assemble into ordered nanostructures in concentrated solutions or gels. Most prominently, SAXS studies of self-assembly show that despite their extremely high degree of sequence similarity, ELP0 and ELP1 blocks have a very large effect on the quality of ordering (Figure 2). This is surprising from a polymer science
Figure 2. SAXS patterns of mCherry-ELP fusion proteins at 50 wt % in water at 10 °C. Peaks are indexed when they correspond to a previously identified ordered structure in block copolymers. The curves are offset for clarity.
perspective, as the two ELPs have 90% similar amino acid composition and an even higher similarity when examined on a functional group basis. Well-ordered lamellar structures (peaks at q*, 2q*, and 3q*) are formed by three of the four ELP0 fusions, while only the highest molar mass ELP1 fusion (mCherry-ELP1b) forms a structure with a sharp primary scattering peak (the peak located at lowest q). Even in this case, only a primary peak and a broad shoulder are observed. The effect of changing ELP sequence is most directly evident when comparing pairs of conjugates differing only by the identity of the ELP, such as mCherry-ELP0c/mCherry-ELP1b and ELP0b-mCherry/ELP1c-mCherry. In both cases, the ELP0 material shows stronger correlations between the two blocks, as evidenced by significantly sharper scattering peaks. The full width at half-maximum (fwhm) provides a useful measure of the quality of ordering and grain size in ordered materials40 and also the strength of repulsive interactions between blocks in disordered block copolymers.41 For mCherry-ELP0c, the fwhm of the first scattering peak is 0.0114 nm−1 at 10 °C, while for mCherry-ELP1b it is 0.113 nm−1 at the same temperature, an order of magnitude larger and consistent with the fact that C
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Figure 3. Representative SAXS patterns at 50 wt % for (a) ELP0-mCherry, (b) ELP0a-mCherry, (c) ELP0b-mCherry, (d) mCherry-ELP0c, (e) mCherry-ELP1, (f) mCherry-ELP1a, (g) mCherry-ELP1b, and (h) ELP1c-mCherry. These illustrate the different phase behaviors observed at low and high temperatures in these fusion proteins. Peaks are only indexed when they correspond to a previously identified ordered structure in block copolymers. The curves are offset for clarity.
mCherry-ELP0c is well ordered while mCherry-ELP1b is only weakly ordered (Table S1 in the Supporting Information). For the second comparative pair with lower molar mass ELP, the fwhm for ELP0b-mCherry is 0.125 nm−1, while for ELP1cmCherry it is 0.220 nm−1. Even though both fusions are in the disordered state, the narrower peak for ELP0b-mCherry indicates stronger correlations and a stronger repulsive interaction between blocks.41 All of these results clearly indicate that the ELP amino acid sequence has a large impact on self-assembly. Because the ELP sequences are chemically similar, it is hypothesized that the small difference in hydrophobicity between sequences may be responsible for the observed effect. The molar mass of the ELP has a large impact on selfassembly. This is clearly illustrated by examining the pairs of conjugates ELP0a-mCherry/ELP0b-mCherry and mCherryELP1/mCherry-ELP1b. In both cases, increasing the molar mass of the ELP increases the degree of order in the nanostructures (Figure 2). The increasing ELP molar mass both increases the total molar mass of the fusion and moves the composition of the fusion closer to symmetric. Both effects are anticipated to promote ordered structures, as is well established for coil−coil block copolymers42,43 and has been similarly demonstrated for mCherry bioconjugate constructs.20 More subtle changes to molecular design, including a 6xHis tag and the termini to which the elastin is fused, were also explored. For ELP0 fusions, neither the addition of a 6xHis tag nor changing the ELP from the N to C termini of mCherry (comparison of ELP0a-mCherry to mCherry-ELP0c) has an impact on the ability to form well-ordered nanostructures. However, the addition of the 6xHis tag results in the formation of nanostructures with a smaller domain spacing, regardless of whether the ELP is at the N or C terminal position. Without a
6xHis tag, the domain spacing is 28.11 nm, but with a 6xHis tag, the domain spacing is 24.78 nm for the N-terminal fusion and 23.58 nm for the C-terminal fusion. The addition of the 6xHis tag also results in a small decrease in ordering, as suggested by the loss of the 3q* peak and the decrease in peak intensity when comparing ELP0-mCherry and ELP0a-mCherry. In the case of ELP1, removing the 6xHis tag from a conjugate with a short ELP sequence results in complete loss of all scattering peaks that are present in both the N and C terminal fusions with a 6xHis tag, indicating the absence of order and, surprisingly, the absence of the correlation hole peak as well, presumably due to very low scattering contrast between blocks. In both ELP0 and ELP1, the terminus of mCherry to which the ELP is fused has a minimal effect on self-assembly. This is expected due to the fact that the two termini are located close to one another on the same end of the mCherry β-barrel, resulting in little change in the coarse-grained shape of the fusion. The minimal effect of changing terminus is also consistent with previous results on protein−polymer conjugates, suggesting that changes in the specific arrangement of surface residues on the protein that modify its surface potential are less important than the coarse-grained shape.5 Heating of the fusion proteins results in changes in their structure as the fusion is heated above the Tt of the corresponding ELP block (i.e., the ELP not fused to the globular protein) for most of the different constructs (Figures 3 and S10). In all cases, only two different structures, a high temperature phase above Tt and a low temperature phase below Tt, are observed by SAXS. Below the thermoresponsive transition temperature of the ELP blocks, the ELP0-based fusions with long ELP chains display clearly ordered lamellar nanostructures, while the ELP1-based fusions with short chains show disordered structures. Increasing temperature up to 40 °C D
DOI: 10.1021/acs.biomac.5b01604 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules leads to changes in structure in many of the ELP0 fusions as water becomes a poor solvent for the ELP block, and in several cases results in a change in the phase observed. All ELP0-based fusion proteins form ordered structures at 40 °C, with ELP0bmCherry showing a transition from a disordered structure to a hexagonally ordered material (peaks at q*, √3q*, and √7q*). ELP0a-mCherry retains its lamellar structure upon heating, and the observation of increased birefringence (Figure S11) suggests an improved quality of ordering. In contrast, the lamellar structure is lost in mCherry-ELP0c, with strong scattering peaks being replaced by two weaker reflections that are not indexable to a typical block copolymer morphology. For most of the fusions, the structural transitions identified by SAXS do not correspond to the formation of a macrophase separated structure, as indicated by the fact that transmission remains uniformly high throughout the range of temperatures studied. However, mCherry-ELP0c shows a significant drop in transmission upon heating (Figure S10), suggesting the formation of large scale, two-phase structures, as water is excluded from the ELP nanodomains during desolvation. It is hypothesized that for this fusion construct the desolvation of ELP has led to kinetic barriers to structure formation that prevent self-assembly of a well-ordered structure as indicated by SAXS; however, the presence of multiple scattering peaks suggests that it is microphase separated. Finally, ELP0-mCherry retains all of its peaks for the lamellar nanostructure at similar q values, but shows the appearance of a new peak at slightly lower q than the original primary peak. This material is weakly birefringent throughout the entire temperature range, suggesting that even after heating the material remains self-assembled into optically anisotropic nanostructures (Figure S11). These data suggest breaking of the lamellar symmetry in the x−y direction, with the appearance of a characteristic in-plane scattering length scale while maintaining lamellar symmetry, consistent with a perforated lamellar structure. In all of the fusions, heating results in a drop in the peak intensity, even if scattering peaks remain sharp, suggesting changes in the scattering contrast, form factor, or grain size. In contrast, ELP1-based fusions show very little change in ordering between high and low temperature in SAXS. mCherryELP1, mCherry-ELP1a, and ELP1c-mCherry show no change in SAXS pattern upon heating, while mCherry-ELP1b shows only a minor increase in the intensity of the broad second order peak. All of these results further confirm that the ELP amino acid sequence and chain size play an important role in ordered nanostructure formation of the designed mCherry-ELP fusions. Insight into the impact of the ELP sequence on self-assembly can be gained by examining the thermoresponsive properties of the two ELPs in dilute solution. Previous reports have demonstrated that the phase transition behavior of the fusion can be triggered by the introduction of ELPs into other proteins.35 Here, turbidometry shows that the thermal transitions of ELP0 and ELP1 single block proteins at 1 mg/mL occurred at 29.4 and 38.5 °C, respectively (Figure 4). However, the ELP0 sequence has a molar mass of 41.2 kDa and the ELP1 has a molar mass of 15.7 kDa, so a portion of these results are due to the thermal transition temperature (Tt) increases with decreasing molar mass of the ELP proteins.35 It is also noteworthy that the transition in ELP0 is extremely sharp, occurring over just a few degrees, while the transition in ELP1 is very broad, occurring over almost 20 °C. An additional effect captured in the observed Tt difference is a difference in hydrophobicity between the two ELPs. Based on
Figure 4. Transmittance as a function of temperature in 1 mg/mL solution indicates the thermoresponsive transition of ELP-mCherry fusions (a) mCherry-ELP0c and (b) ELP1c-mCherry as well as the individual ELPs (c) ELP0 and (d) ELP1, where high transmittance (below the transition temperature, Tt) indicates individual molecules dispersed in solution, and low transmittance (above Tt) indicates formation of aggregates.
the hydrophobicity scale developed by Urry, ELP0 has a more hydrophobic sequence than ELP1. There are two points in the pentapeptide repeat unit that can be substituted to varying degrees: the first position (XPGVG) can either be valine (V) or isoleucine (I), and the fourth position (VPGYG) can be any amino acid, except proline. Work by Urry has shown that, for the first position, isoleucine is more hydrophobic than valine,44 and for the fourth position, valine is more hydrophobic than alanine (A), which is more hydrophobic than glycine (G).29 The first positions in the ELP0 repeat sequence are 20% isoleucine, 80% valine, whereas the first positions in the ELP1 repeat sequence are 100% valine, making ELP0 more hydrophobic in this regard. Furthermore, the fourth positions in the ELP0 repeat sequence are 100% valine, and the fourth positions in the ELP1 repeat sequence are 50% valine, 20% alanine, and 30% glycine, meaning that ELP0 also has more hydrophobic residues in this portion of the repeat sequence. Thus, ELP0 has a definitively more hydrophobic sequence than ELP1. These results suggest that sequence hydrophobicity as captured by Tt is a key driving force for microphase separation, even when the ELP is in the low temperature solvated state. The solution behavior of mCherry-ELP fusions shows a dependence on the location to which the ELP is fused (Figures 4 and S9). All of designed mCherry-ELP fusions exist as isolated soluble molecules in dilute solutions below the Tt of ELPs. In the series of mCherry-ELP0 fusions, no obvious thermal transition was observed by turbidometry for ELP0mCherry, ELP0a-mCherry, and ELP0b-mCherry, all of which are N-terminal fusions. However, moving ELP0 to the C terminus yields a clear thermal transition for mCherry-ELP0c, detectable by a greater than 50% drop in transmission occurring at 32.5 °C. It is notable that changes to the specific chemical structure of both protein−polymer conjugates and fusion proteins that do not affect the coarse grained structure have little effect below the thermal transition temperature in several studies;5,21 however, increasing above Tt can cause more substantial changes.35,45 This suggests that detailed chemical structure may be important as the collapse of the polymer domain forces protein packing at high density, and induces E
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Biomacromolecules extremely specific and directional local interactions. No obvious transition was observed for the series of mCherry-ELP1 fusions in the entire measured temperature range of 10−50 °C. This is consistent with the lower hydrophobicity of the ELP1 block. The function of globular protein in designed fusion proteins is largely influenced by molecular design and processing conditions. The mCherry chromophore provides a sensitive probe for the maintenance of protein fold and function, in solid materials and gels, that is independent of transport considerations.35 UV−vis spectra in Figure 5 were measured for
Table 2. Percentage of Activity Retained by Fusion Proteins percent activity retained by fusions fusion
before dehydrationa (%)
after rehydrationb (%)
ELP0-mCherry ELP0a-mCherry ELP0b-mCherry mCherry-ELP0c mCherry-ELP1 mCherry-ELP1a mCherry-ELP1b ELP1c-mCherry
69 70 104 80 67 12 56 82
56 81 78 76 87 100 94 69
a
Compares activity retained compared to native mCherry at 586 nm. Compares activity retained after rehydration as compared to the fusion protein’s activity before hydration at 586 nm.
b
both cases, the proteins show significant retention of function due to the gentle self-assembly process, although clear trends relating structure and retention of function are not apparent from the limited set of fusion proteins investigated.
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CONCLUSION Two different ELP blocks, ELP0 and ELP1, were fused with a model globular protein (mCherry) to generate different ELP0based and ELP1-based mCherry-ELP fusions. Comparisons of different fusion structures show that the hydrophobicity and molar mass of the ELP block have a large effect on selfassembly, with higher hydrophobicity and molar mass both promoting self-assembly. In contrast, the presence or absence of a 6xHis purification tag and the terminal of the mCherry to which the ELP is fused have only minor effects. In several of the fusion constructs, the thermoresponsive transition of the ELP can trigger ordering transitions or order−order transitions upon heating, and these ordering transitions can show a dependence upon the order in which the two blocks are linked (N-terminal or C-terminal fusion). Overall, this work suggests that fusion proteins can be characterized by coarse-grained principles of self-assembly, and that for a sufficiently hydrophobic ELP sequence there is a great deal of design latitude in building a fusion protein that can self-assemble into well-ordered nanostructures.
Figure 5. Absorbance spectra of different fusion proteins overlaid with the spectrum of mCherry alone, for proteins in solution: (a) Spectra for ELP0 conjugates; (b) spectra for ELP1 conjugates.
mCherry alone, ELP0-based and ELP1-based mCherry-ELP fusions, respectively, showing the different mCherry absorbance in the mCherry-ELP fusions. In ELP0-based fusion, the ELP0bmCherry fusion with shorter ELP chain preserved the highest levels of protein function, with more than 95% of the original solution-state absorbance preserved, compared to 69% for ELP0-mCherry, 70% for ELP0a-mCherry, and 80% for mCherry-ELP0c. In ELP1-based fusions, the ELP1c-mCherry fusion with the ELP chain on the N termini shows 82% preservation of protein function, compared to 67, 56, and 12% preservation for mCherry-ELP1, mCherry-ELP1b, and mCherry-ELP1a fusions, respectively (Table 2). These results indicate the ELP size and position in the fusion system impacts the globular protein function in the fusion system, even when the ELP has the same repeat sequence. Significant protein function as assayed by UV−vis absorption was also retained after the formation of solid-state nanostructures. As reported for bioconjugate materials,16,20 there is a substantial reversible loss in absorbance when the proteins are cast into solid materials. However, rehydration without refolding results in a direct quantitative comparison to assynthesized absorbance, indicating that 56−81% of the chromophore is retained for ELP0 fusions and that 69−95% of the chromophore is retained for ELP1 fusions. Therefore, in
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01604. Complete biosynthesis procedures and sequences for mCherry-ELP fusions, additional data characterizing thermoresponsive transitions, bireinfringence measurements, SAXS of all ELP-mCherry fusions in concentrated solutions, and MALDI-TOF data of all ELP-mCherry fusions (PDF).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. F
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ACKNOWLEDGMENTS
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REFERENCES
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The National Science Foundation (Award No. DMR-1253306) supported the construction of fusion proteins and characterization in solution. The Department of Energy Office of Basic Energy Sciences (Award No. DE-SC0007106) supported SAXS studies of fusion self-assembly. SAXS experiments were performed at SSRL Beamline 1−5 at Stanford University and ALS Beamline 7.3.3 at Lawrence Berkeley National Laboratory. We thank Dongsook Chang, Christopher N. Lam, and Charlotte R. Stewart-Sloan for experimental assistance with SAXS. CD was collected from Biophysical Instrumentation Facility at MIT.
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DOI: 10.1021/acs.biomac.5b01604 Biomacromolecules XXXX, XXX, XXX−XXX
