Noncationic Rigid and Anisotropic Coiled-Coil Proteins Exhibit Cell

Jul 21, 2015 - Numerous cationic peptides that penetrate cells have been studied intensively as drug delivery system carriers for cellular delivery. H...
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Noncationic Rigid and Anisotropic Coiled-Coil Proteins Exhibit CellPenetration Activity Norihisa Nakayama,†,‡ Kyoji Hagiwara,‡,§ Yoshihiro Ito,‡ Kuniharu Ijiro,‡,∥ Yoshihito Osada,‡ and Ken-Ichi Sano*,†,‡,⊥ †

Graduate School of Environmental Symbiotic System Major and ⊥Department of Innovative Systems Engineering, Nippon Institute of Technology, Miyashiro, Saitama 345-8501, Japan ‡ Nano Medical Engineering Laboratory, RIKEN, Wako, Saitama 351-0198, Japan § Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo 156-8506, Japan ∥ Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido 001-0021, Japan S Supporting Information *

ABSTRACT: Numerous cationic peptides that penetrate cells have been studied intensively as drug delivery system carriers for cellular delivery. However, cationic molecules tend to be cytotoxic and cause inflammation, and their stability in the blood is usually low. We have previously demonstrated that a rigid and fibrous cationic coiled-coil protein exhibited cellpenetrating ability superior to that of previously reported cellpenetrating peptides. Making use of structural properties, here we describe the cell-penetrating activity of a rigid and fibrous coiled-coil protein with a noncationic surface. A fibrous coiled-coil protein of pI 6.5 penetrated 100% of the cells tested in vitro at a concentration of 500 nM, which is comparable to that of previously reported cell-penetrating peptides. We also investigated the effect of cell-strain dependency and short-term cytotoxicity.

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It is conceivable that the fiber toxicity is closely related to the fact that these materials are not biodegradable. Recently, we investigated whether rigid and fibrous structured, biodegradable materials have potential as drug delivery system (DDS) carriers for cellular delivery.15 Proteins are of course biodegradable, and those with a high aspect ratio and structural rigidity are categorized as fibrous proteins. We focused on tropomyosins, which are anisotropic with high structural rigidity. Tropomyosins have many isoforms.16 Although their typical isoforms are 40 nm in length and 2 nm in diameter,17 their persistence length is calculated to be 50 to 200 nm,18 which is longer than their actual length. Tropomyosins consist of a twostranded α-helical coiled-coil structure along their entire length.19 The isoelectric point (pI) of human skeletal muscle α-tropomyosin (Hu-sk α-Tm) was experimentally determined to be 4.5, and a C-terminal-deleted protein did not show cellpenetrating activity. We designed and produced an artificial cationic coiled-coil protein named coiled-coil protein carrier (CCPC) 140.15 CCPC 140 consists of 140 amino acid residues and forms two-stranded parallel α-helical coiled-coil structures through its entire length. CCPC 140 has the structural frame of Hu-sk α-Tm, but most of the acidic amino acids (18 residues) located at the surface are substituted by basic amino acids.15

INTRODUCTION A number of natural and synthetic cationic peptides have been widely explored for the cellular delivery of therapeutic molecules.1 The first peptide reported to have cell-penetrating activity was TAT, which was isolated from the HIV TAT protein.2,3 Over the last 25 years, numerous peptide sequences have been isolated and designed to exhibit cell-penetrating activity.4 Cell-penetrating peptides (CPPs) usually consist of 5−30 amino acid residues1 and can be classified into proteinderived (such as TAT, penetratin, and transportan) and synthetic peptides (such as polylysine and polyarginine and their derivatives).4,5 Most synthetic peptides have cationic surface properties because electrostatic interactions between positively charged CPPs and the negatively charged headgroups of phospholipids or proteoglycans are thought to be required for the cellular uptake of CPPs.6 Although the advantages of cationic CPPs in cellular delivery have attracted much attention, the inflammatory nature and cytotoxicity of cationic CPPs have been hurdles in their clinical application.7,8 Furthermore, the stability of cationic CPPs in the blood is low, and they tend to accumulate in the lung.9 To overcome undesired toxicity and inflammation, stearyl- and chloroquine-modified CPPs have been develped, and these CPPs do not exhibit short-term cytotoxicity and inflammation both in vitro and in vivo.10 It is well known that asbestos and carbon nanotubesrigid and anisotropic structured materialshave excellent cellpenetrative properties, but they also show fiber toxicity.11−14 © 2015 American Chemical Society

Received: April 2, 2015 Revised: July 15, 2015 Published: July 21, 2015 8218

DOI: 10.1021/acs.langmuir.5b01219 Langmuir 2015, 31, 8218−8223

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Figure 1. Amino acid sequence comparison of α-helical coiled-coil proteins used in this study. The seven positions of the coiled-coil motif are described as a to g. Substituted amino acids are displayed. CHT type II). The CCPC variants were fractionated from the hydroxyapatite column using 0.1, 0.2, 0.3, and 0.5 M phosphate buffer with 150 mM NaCl. The pH of the phosphate buffer was 6.0 and 8.0 for CCPC 140 pI 8.6 and CCPC 140 pI 6.5, respectively. The purified protein concentration was determined by the microbiuret method.20 Circular Dichroism. Circular dichroism (CD) spectra were obtained on a Jasco J-820 spectropolarimeter using a cell with a 1 mm path length. Measurements were conducted in PBS, and the concentration of the CCPC 140 pI variants was 0.2 mg/mL. The temperature dependence of CD was monitored at 222 nm at 1 °C intervals with an equilibration time of 1 min. The helical content of the CCPC 140 pI variants was evaluated with the following equation:

The pI of CCPC 140 is 10.6, meaning that CCPC 140 is highly cationic.15 When fluorescently labeled CCPC 140 was added to HeLa cells, we were able to detect fluorescence in 100% of the cells, even at a final CCPC 140 concentration of 3.1 nM, using image-based cytometry.15 This indicated that the cellpenetrating activity of CCPC 140 was more efficient than that of previously reported CPPs.15 Moreover, CCPC 140 has shown cell-penetrating activity in all of the cell types we have tested.15 Using deletion analysis, we have shown that the length of the deletion was directly associated with the structure and rigidity of the protein, and the relative α-helical content at the experimental temperature (37 °C) positively correlated with the cell-penetrating activity.15 CCPC 140 did not show short-term cytotoxicity in A549 human lung adenocarcinoma epithelial cells, but its surface properties are highly cationic.15 To avoid potential inflammation and cytotoxicity, we designed and expressed new CCPC 140 variants with pI 6.5 and 8.6. As in our previous report, cationic CCPC 140 penetrated 100% of the cells at a concentration of 3.1 nM,15 whereas both CCPC 140 pI 6.5 and pI 8.6 required 500 nM protein to penetrate 100% of the cells. However, the variants’ cell-penetrating activity was comparable to that of previously reported CPPs.

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helical content =

[θ ]temp − [θ ]85 ° C [θ ]5 ° C − [θ ]85 ° C

In Vitro Cell Penetration Assay. CCPC 140 variants were labeled with AlexaFluor 488 as previously reported.15 Fluorescently labeled proteins were diluted in PBS and added to cultured cells. In this study, we used three cell lines, A549 human lung adenocarcinoma epithelial cells, HeLa human cervical carcinoma cell line, and K562 human erythromyeloblastoid leukemia cells. A549 and HeLa cells were maintained in DMEM medium supplemented with 10% fetal calf serum (FCS), and K562 cells were maintained in RPMI 1640 medium supplemented with 10% FCS. Cells were observed under a fluorescence microscope and a TALI image-based cytometer (Life Technologies). In the cytometric experiment, the fluorescence intensity distribution was evaluated from more than 1000 cells in nine randomly selected fields. Cell proliferation was assessed by the WST-1 (4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetra-zolio]-1,3-benzene disulfonate) (Takara, Kyoto, Japan) assay. CCPC 140 and its pI variants were added at a final concentration of 5 μM. The cells were then incubated for 72 h and assayed using a WST-1 kit.

EXPERIMENTAL SECTION

DNA Constructs and Expression of CCPC 140 Derivatives. The protein-coding DNA sequence of CCPC 140 derivatives was optimized for codon usage in Escherichia coli and synthesized by Operon Biotechnology Inc. (Tokyo, Japan). The DNA sequences were designed to include NcoI and BamHI sites at the ends of the coding sequence for subcloning. We used E. coli BL21(DE3)pLysS for protein expression. Transformed cells were cultured in Luria-Bertani medium (1 wt % bacto-tryptone, 0.5 wt % yeast extract, 0.5 wt % NaCl) supplemented with 100 μg/mL carbenicillin at 37 °C. For induction, isopropyl β-D-1-thiogalactopyranoside was added at a final concentration of 0.2 mM when the optical density at 600 nm reached 0.6− 0.8. Cells were collected by centrifugation 3−5 h after induction, washed with 50 mM Tris-HCl, pH 8.0, and 1 mM EDTA, and stored at −80 °C. Purification of CCPC 140 Derivatives. Protein-expressing cells were resuspended in 50 mM Tris-HCl, pH 8.0, 5 mM dithiothreitol, and protease inhibitor cocktail (EDTA-free, Roche) and lysed by ultrasonication. The cell debris was removed by centrifugation. The supernatant was held at 95 °C for 15 min and then cooled to room temperature. The supernatant was collected again after centrifugation and dialyzed against phosphate-buffered saline (PBS). The dialyzed protein solution was purified on a hydroxyapatite column (BioRad,

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RESULTS AND DISCUSSION Coiled-coil proteins have a heptad repeat amino acid sequence with each position assigned as a to g.21,22 CCPC 140 has 18 acidic amino acids at the b, c, and f positions of Hu-sk α-Tm replaced with basic amino acids and a calculated pI of 10.6. To investigate the effect of surface electrostatic properties on the cell-penetrating activity, we changed the amino acid sequence of CCPC 140 to give pI 6.5 and 8.6 as follows. The amino acid sequence of the first two sections of the heptad repeat was unchanged. Then, the basic amino acids at positions b, c, and f were replaced with acidic amino acids (Figure 1). The pI of the designed sequences was calculated with ProtPram tools. The probability of the coiled-coil formation was confirmed by the Lupas’ method (COILS),23 and the probability of the dimeric 8219

DOI: 10.1021/acs.langmuir.5b01219 Langmuir 2015, 31, 8218−8223

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Figure 2. Secondary structure of CCPC 140 pI 6.5 and 8.6. (Left) CD spectra of CCPC 140 pI 6.5 (red) and pI 8.6 (blue). (Right) Thermal unfolding profiles of CCPC 140 pI 6.5 (red) and pI 8.6 (blue).

Figure 3. Cell-penetrating activity of CCPC 140 pI variants. (A−C, E) Fluorescent signal distribution of 100 nM (A) and 500 nM (C) AlexaFluor 488-labeled CCPC 140 pI 6.5, 100 nM AlexaFluor 488-labeled CCPC 140 pI 10.6 (B), and 500 nM AlexaFluor 488-labeled CCPC 140 pI 8.6 (E) in A549 cells. (D, F) Merged phase contrast cell image and fluorescent image of 500 nM AlexaFluor 488-labeled CCPC 140 pI 6.5 (D) and pI 8.6 (F). Scale bars represent 20 μm.

typical α-helical coiled-coil structure under most physiological conditions.25 Our previous results have indicated that the relative α-helical content at the experimental temperature in vitro (37 °C) closely correlated with the cell-penetrating activity.15 Judging by the thermal melting profiles, CCPC 140 pI 6.5 and CCPC 140 pI 8.6 have relative α-helical contents of 84 and 86%, respectively. Correspondingly, CCPC 140 has a relative α-helical content of 91%.15 These α-helical contents are sufficiently high for structural rigidity and superior cellpenetrating activity compared to that of cationic CCPC 140 deletion variants.15 We evaluated the cell-penetrating activity using fluorescently labeled (AlexaFluor 488) CCPC 140 pI

coiled-coil formation was calculated by the Multicoil program.24 We then constructed expression vectors and expressed the designed coiled-coil proteins in E. coli to purify them. Each purification protocol for the designed CCPC pI variants was modified according to the surface charge property because when we used phosphate-buffered solution to elute the protein from the hydroxyapatite column near the pI, the column got clogged. We examined the effect of the amino acid changes on the structural stability of the CCPC variants by far-ultraviolet CD spectra and thermal melting profiles (Figure 2A,B). The CD spectra showed negative peaks at 208 and 222 nm, indicating a 8220

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Langmuir variants (Figure 3). The fluorescently labeled CCPCs used in this study were labeled by one or two fluorophores per molecule on average from a calculation made by measuring the protein concentration using the microbiuret method20 and the absorbance of the AlexaFluor 488 dye at 495 nm (ε = 71 000). One possible effect on the CCPC molecule is a decrease in primary amines at the surface of the molecule (mainly the lysine side chain). This can cause a decrease in the pI of the molecules; however, we aimed to develop lower pI CCPC derivatives in this study. The effect of lowering the pI on the cell-penetrating activity can be a partial decrease in the cellpenetrating activity of CCPCs. Also, a common problem of labeling proteins is that some structure disturbance may occur. However, in the case of tropomyosins, preceding studies on fluorescently labeled tropomyosins have indicated that both their structure and function were maintained.26,27 After incubating A549 human lung adenocarcinoma epithelial cells for 72 h with fluorescently labeled CCPC 140 variants at a final concentration of 500 nM, we were able to detect fluorescence in 100% of the cells by cytometry (Figure 3C−F). All of the cell penetration assays were carried out in the presence of 10% fetal bovine serum. As in our previous report, a significant fluorescent signal was detected in 100% of the cells by imagebased cytometry when cells were incubated with CCPC 140 even at a concentration of 3.1 nM.15 However, both CCPC 140 pI 6.5 and pI 8.6 required 500 nM fluorescently labeled protein to detect a significant signal in 100% of the cells. These results indicate that the effective concentration for the cell-penetrating activity of the CCPC 140 pI variants is higher than 100 times that of cationic CCPC 140. However, it is slightly lower than that of the cationic but mostly randomly coiled CCPC 55,15 which is a CCPC 140 deletion derivative, and of cationic CPPs such as TAT and polyarginine.28,29 In contrast, acidic Hu-sk αTm 140, pI 4.8, did not show any cell-penetrating activity.15 Taken together, the highly cationic surface properties of CCPC 140 are very efficient for cell penetration, but the anionic surface properties of Hu-sk α-Tm 140 are not efficient for cell penetration, even though it forms a rigid and anisotropic structure. The slightly cationic CCPC 140 pI 8.6 and almost neutral CCPC 140 pI 6.5, both rigid and anisotropic proteins, exhibited cell-penetrating activity equal to or greater than that of unfolded cationic peptides. This strongly indicates that the molecular structure, in terms of rigidity and anisotropy, is extremely important for the cell-penetrating activity in the absence of surface cationic properties. We next examined the dependency of the cell-penetrating activity on cell strain. We applied 500 nM CCPC 140 pI 6.5 or pI 8.6 to the HeLa human cervical carcinoma cell line and to K562 human erythromyeloblastoid leukemia cells (Figure 4). At this concentration, the CCPC 140 pI variants showed cellpenetrating activity in both cell lines tested. These results indicate that the CCPC 140 pI variants have cell-penetrating activity in a variety of cell types, consistent with the previously reported cationic CCPC 140.15 We also evaluated the short-term cytotoxicity (72 h) of CCPCs (Figure 5). As in our previous study, cationic CCPC 140 did not show short-term cytotoxicity in A549 cells at 1 μM15 and also did not show short-term cytotoxicity even at 5 μM in this study (Figure 5). Both CCPC 140 pI 6.5 and pI 8.6 did not show short-term cytotoxicity either. On the other hand, CCPC 140 showed cytotoxicity both in HeLa and K562 cells at 5 μM (Figure 5). The short-term cytotoxicity in both cells seemed to depend on the pI because the lower pI variant

Figure 4. Cell-strain dependency. Cell-penetrating activity of CCPC 140 pI variants in HeLa cells after 24 h of administration (A) and in K562 cells (B) after 72 h of administration. (a) and (c) display a merged differential interference contrast cell image and a fluorescent image of AlexaFluor 488-labeled CCPC 140 pI 6.5 and pI 8.6, respectively. (b) and (d) indicate fluorescent images of AlexaFluor 488-labeled CCPC 140 pI 6.5 and pI 8.6, respectively. Scale bars represent 20 mm.

Figure 5. Short-term cytotoxicity of CCPCs. Viability of cells incubated in the presence of 5 μM CCPCs for 72 h. Bars represent the standard deviation (n = 3).

resulted in higher cell viability. LD50 of CCPC 140 in K562 cells was calculated to be 1.28 μM (Figure S1). In fact, CCPC 140 pI 6.5 did not show cytotoxicity in HeLa or K562 cells even at 5 μM. Our findings suggest that the factors affecting cell penetration can be divided into two categories: (1) molecular structure, rigidity, and anisotropy and (2) the surface cationic properties. Without structural rigidity and anisotropy, cationic CPPs and CCPC 55 form undefined structures and require a micromolar order of molecules to penetrate 100% of the cells. In contrast, the CCPC 140 pI variants with structural rigidity and anisotropy (without cationic surface properties, even at pI 6.5) possess cell-penetrating activity and require a submicromolar order of molecules to penetrate 100% of the cells. The 8221

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usefulness of cationic polymers is usually hampered by inflammation, cytotoxicity, lower stability, a shorter retention time in the blood, and lung accumulation. 7,8 These disadvantages are thought to originate from cationic polymers. CCPC 140 and its pI variants did not show short-term cytotoxicity in A549 cells.15 But the short-term cytotoxicity in HeLa and K562 cells seemed to depend on the pI of CCPCs. Also, consistent with the cationic CCPC 140, the CCPC 140 pI variants were able to penetrate cells in the presence of serum. Recently, coiled-coil proteins have been demonstrated to selfassemble into multimers with a high degree of specificity. This feature of coiled-coil proteins is extensively explored with the aim of developing new molecular targeting tools in DDS.30−35 By taking advantage of the cell-penetrating activity of CCPCs, we should be able to expand intracellular molecular targeting.

OUTLOOK We previously created an artificial protein, named CCPC 140, which had superior cell-penetrating activity because it is a fibrous protein with a high aspect ratio, structural rigidity, and a cationic surface. In this study, we designed and produced CCPC 140 variants of pI 6.5 and 8.6 to avoid problems related to inflammation, cytotoxicity, and instability in the blood. Both variants showed less cell-penetrating activity compared to the cationic CCPC 140; however, they penetrated 100% of the cells in vitro even at a concentration as low as 500 nM (comparable to previously reported CPPs). The CCPC 140 pI variants were able to penetrate more than one type of cell, and CCPC 140 pI 6.5 did not show short-term cytotoxicity in all of the cells tested. These variants have great potential as carriers for the cellular delivery of drugs both in vitro and in vivo. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b01219. Determination of LD50 of CCPC 140 in K562 cells (PDF)

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(1) Copolovici, D. M.; Langel, K.; Eriste, E.; Langel, U. Cellpenetrating peptides: design, synthesis, and applications. ACS Nano 2014, 8 (3), 1972−94. (2) Frankel, A. D.; Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 1988, 55 (6), 1189−93. (3) Green, M.; Loewenstein, P. M. Autonomous functional domains of chemically synthesized human immunodeficiency. Cell 1988, 55 (6), 1179−88. (4) Heitz, F.; Morris, M. C.; Divita, G. Twenty years of cellpenetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. 2009, 157 (2), 195−206. (5) Nakase, I.; Takeuchi, T.; Tanaka, G.; Futaki, S. Methodological and cellular aspects that govern the internalization mechanisms of arginine-rich cell-penetrating peptides. Adv. Drug Delivery Rev. 2008, 60 (4−5), 598−607. (6) Nakase, I.; Tadokoro, A.; Kawabata, N.; Takeuchi, T.; Katoh, H.; Hiramoto, K.; Negishi, M.; Nomizu, M.; Sugiura, Y.; Futaki, S. Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis. Biochemistry 2007, 46 (2), 492−501. (7) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Controlled Release 2006, 114 (1), 100−9. (8) Zhang, J. S.; Liu, F.; Huang, L. Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity. Adv. Drug Delivery Rev. 2005, 57 (5), 689−98. (9) Sakurai, F.; Nishioka, T.; Yamashita, F.; Takakura, Y.; Hashida, M. Effects of erythrocytes and serum proteins on lung accumulation of lipoplexes containing cholesterol or DOPE as a helper lipid in the single-pass rat lung perfusion system. Eur. J. Pharm. Biopharm. 2001, 52 (2), 165−172. (10) Suhorutsenko, J.; Oskolkov, N.; Arukuusk, P.; Kurrikoff, K.; Eriste, E.; Copolovici, D. M.; Langel, U. Cell-penetrating peptides, PepFects, show no evidence of toxicity and immunogenicity in vitro and in vivo. Bioconjugate Chem. 2011, 22 (11), 2255−62. (11) Donaldson, K.; Murphy, F. A.; Duffin, R.; Poland, C. A. Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma. Part. Fibre Toxicol. 2010, 7, 5. (12) Kam, N. W.; Dai, H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J. Am. Chem. Soc. 2005, 127 (16), 6021−6. (13) Sharma, C. S.; Periyakaruppan, A.; Barr, J.; Wise, K.; Thomas, R.; Wilson, B. L.; Ramesh, G. T. Single-walled carbon nanotubes induces oxidative stress in rat lung epithelial. J. Nanosci. Nanotechnol. 2007, 7 (7), 2466−72. (14) Zhu, L.; Chang, D.; Dai, L.; Hong, Y. DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells. Nano Lett. 2007, 7 (12), 3592−7. (15) Nakayama, N.; Hagiwara, K.; Ito, Y.; Ijiro, K.; Osada, Y.; Sano, K. Superior cell penetration by a rigid and anisotropic synthetic protein. Langmuir 2015, 31 (9), 2826−32. (16) Pittenger, M. F.; Kazzaz, J. A.; Helfman, D. M. Functional properties of non-muscle tropomyosin isoforms. Curr. Opin. Cell Biol. 1994, 6 (1), 96−104. (17) Whitby, F. G.; Phillips, G. N., Jr. Crystal structure of tropomyosin at 7 Angstroms resolution. Proteins: Struct., Funct., Genet. 2000, 38 (1), 49−59. (18) Phillips, G. N., Jr.; Chacko, S. Mechanical properties of tropomyosin and implications for muscle regulation. Biopolymers 1996, 38 (1), 89−95. (19) McLachlan, A. D.; Stewart, M. Tropomyosin coiled-coil interactions: evidence for an unstaggered structure. J. Mol. Biol. 1975, 98 (2), 293−304. (20) Itzhaki, R. F.; Gill, D. M. A micro-biuret method for estimating proteins. Anal. Biochem. 1964, 9, 401−410. (21) Lupas, A. Coiled coils: new structures and new functions. Trends Biochem. Sci. 1996, 21 (10), 375−82.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

N.N. and K.H. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by Takeda Pharmaceutical Company Limited; we are grateful for their consistent encouragement and support. We also thank Akiko Yumoto from Nano Medical Engineering Laboratory of RIKEN for her technical assistance. This work was also partially supported by the Network Joint Research Center for Materials and Devices and a special research grant from the Nippon Institute of Technology to K.-I. S.

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ABBREVIATIONS DDS, drug delivery system; CPPs, cell-penetrating peptides; CCPC, coiled-coil protein carrier; CD, circular dichroism 8222

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Langmuir (22) Smillie, L. B. Structure and functions of tropomyosins from muscle and non-muscle sources. Trends Biochem. Sci. 1979, 4, 151−5. (23) Lupas, A.; Van Dyke, M.; Stock, J. Predicting coiled coils from protein sequences. Science 1991, 252 (5009), 1162−4. (24) Kim, P. S.; Berger, B.; Wolf, E. MultiCoil:A program for predicting two- and three-stranded coiled coils. Protein Sci. 1997, 6 (6), 1179−89. (25) Greenfield, N. J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2007, 1 (6), 2876−90. (26) Miki, M.; Miura, T.; Sano, K.; Kimura, H.; Kondo, H.; Ishida, H.; Maeda, Y. Fluorescence resonance energy transfer between points on tropomyosin and actin in skeletal muscle thin filaments: does tropomyosin move? J. Biochem. 1998, 123 (6), 1104−11. (27) Miki, M.; Hai, H.; Saeki, K.; Shitaka, Y.; Sano, K.; Maeda, Y.; Wakabayashi, T. Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments. J. Biochem. 2004, 136 (1), 39−47. (28) Saito, H.; Honma, T.; Minamisawa, T.; Yamazaki, K.; Noda, T.; Yamori, T.; Shiba, K. Synthesis of functional proteins by mixing peptide motifs. Chem. Biol. 2004, 11 (6), 765−73. (29) Takayama, K.; Tadokoro, A.; Pujals, S.; Nakase, I.; Giralt, E.; Futaki, S. Novel system to achieve one-pot modification of cargo molecules with oligoarginine vectors for intracellular delivery. Bioconjugate Chem. 2009, 20 (2), 249−57. (30) Deacon, S. P.; Apostolovic, B.; Carbajo, R. J.; Schott, A. K.; Beck, K.; Vicent, M. J.; Pineda-Lucena, A.; Klok, H. A.; Duncan, R. Polymer coiled-coil conjugates: potential for development as a new class of therapeutic ″molecular switch″. Biomacromolecules 2011, 12 (1), 19−27. (31) Griffiths, P. C.; Paul, A.; Apostolovic, B.; Klok, H. A.; de Luca, E.; King, S. M.; Heenan, R. K. Conformational consequences of cooperative binding of a coiled-coil peptide motif to poly(N-(2hydroxypropyl) methacrylamide) HPMA copolymers. J. Controlled Release 2011, 153 (2), 173−9. (32) Kverka, M.; Hartley, J. M.; Chu, T. W.; Yang, J.; Heidchen, R.; Kopecek, J. Immunogenicity of coiled-coil based drug-free macromolecular therapeutics. Biomaterials 2014, 35 (22), 5885−96. (33) Wu, K.; Liu, J.; Johnson, R. N.; Yang, J.; Kopecek, J. Drug-free macromolecular therapeutics: induction of apoptosis by coiled-coilmediated cross-linking of antigens on the cell surface. Angew. Chem., Int. Ed. 2010, 49 (8), 1451−5. (34) Pola, R.; Laga, R.; Ulbrich, K.; Sieglova, I.; Kral, V.; Fabry, M.; Kabesova, M.; Kovar, M.; Pechar, M. Polymer therapeutics with a coiled coil motif targeted against murine bcl1 leukemia. Biomacromolecules 2013, 14 (3), 881−9. (35) Pechar, M.; Pola, R.; Laga, R.; Braunova, A.; Filippov, S. K.; Bogomolova, A.; Bednarova, L.; Vanek, O.; Ulbrich, K. Coiled coil peptides and polymer-peptide conjugates: synthesis, self-assembly, characterization and potential in drug delivery systems. Biomacromolecules 2014, 15 (7), 2590−9.

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