Effect of the Peptide Secondary Structure on the Peptide

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Effect of the Peptide Secondary Structure on the Peptide Amphiphile Supramolecular Structure and Interactions † ‡ § || ^ z z § Dimitris Missirlis,*, , , , Arkadiusz Chworos, ,# Caroline J. Fu, Htet A. Khant, Daniel V. Krogstad, ,£ and ‡ § || Matthew Tirrell , , ,£ ‡ Department of Chemical Engineering, University of California, Santa Barbara, California 93106, United States § Materials Research Laboratory, University of California, Santa Barbara, California 93106, United States Department) of Bioengineering, University of California, Berkeley, California 94720, United States ^ Department of Physics, University of California, Santa Barbara, California 93106-9530, United States #The Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences in Lodz, Sienkiewicza 112, Lodz 90363, Poland z Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, United States £Department of Materials, University of California, Santa Barbara, California 93106, United States bS Supporting Information

ABSTRACT: Bottom-up fabrication of self-assembled nano- materials requires control over forces and interactions between building blocks. We report here on the formation and architecture of supramolecular structures constructed from two diﬀerent peptide amphiphiles. Inclusion of four alanines between a 16-mer peptide and a 16 carbon long aliphatic tail resulted in a secondary structure shift of the peptide headgroups from R helices to β sheets. A concomitant shift in self-assembled morphology from nanoribbons to coreshell worm-like micelles was observed by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). In the presence of divalent magnesium ions, these a priori formed supramolecular structures interacted in distinct manners, highlighting the importance of peptide amphiphile design in self-assembly.

’ INTRODUCTION One approach to enable innate folding of synthetic peptides Self-assembly is a powerful approach to construct advanced that emulate the folding in the whole protein and simultaneously induce the formation of nanosized, peptide-based materials is to functional materials with control over dimensions on the 2,3 nanoscale.1 Peptide-based building blocks have gained the atten- modify them with a hydrophobic fatty acid tail. The resulting tion of bioengineers because their natural origin provides a chimeric molecules, termed peptide amphiphiles (PAs), self- biocompatible and functional platform for construction of assemble because of hydrophobic interactions and stabilize biomaterials.2 Moreover, advances in peptide synthesis enable secondary-structure motifs on their surface as a result of peptide 4 such technologies for scale up to production levels. The chal- tethering and crowding. Apart from the predominant role of lenge, however, remains to control the self-assembly process hydrophobic interactions of the tails, peptide headgroup inter- fi actions are important for controlling stability and the shape in the accurately at the molecular level to fabricate well-de ned materials, whether these are nanosized colloids for drug delivery, aggregated state.4 6 Solution ionic strength and pH have often macroscopic scaffolds for tissue engineering, or coatings.2 been used as the trigger for self-assembly through screening of Nature serves as inspiration for the fabrication of new materi- charges between neighboring peptides.7,8 The effect of ion type als that rely on self-assembly and exhibit well-defined properties. and valency has also been investigated, revealing the possibility of Structural proteins have evolved as molecular building blocks using it to tune interpeptide forces.9,10 Overall, it has become that come together based on interactions, often mediated by evident that the understanding of peptidepeptide interactions small folded peptide segments. These peptides are potential is a key not only in determining how peptides behave at the candidates for the design and fabrication of three-dimensional molecular level but also in controlling the macroscopic proper- structural systems. Moreover, they possess inherent biofunction- ties of the formed materials.4,10,11 ality able to guide or interfere with cell behavior depending upon the application in mind. However, short peptides isolated from Received: March 2, 2011 the protein generally lose their capacity to form active, near- Revised: March 26, 2011 native secondary structures. Published: April 13, 2011

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Figure 1. Chemical structure of PAs used in this study.

We recently demonstrated that peptide folding of the short folding stability of the peptide headgroups in micelles (panels B 16-mer peptide p53 (derived from the MDM2-binding site and D of Figure 2). For C A p53, heating the PA solution above 14 29 16 4 þ of tumor suppressor protein p53) could be manipulated by the 60 C in the presence of divalent Mg2 resulted in nonreversible selection of appropriate linkers that covalently attached it to a precipitation of the micelles, hence the increase in molar palmitic tail.4 The resulting PAs self-assembled into elongated ellipticity at 215 nm above that temperature. ff micelles that exhibited a di erent type and extent of peptide When bound to MDM2, p531429 forms an amphipathic fi R secondary structure. Among the ndings, we observed an inter- helix, exposing a hydrophobic face containing Phe19, Trp23, R β 12 esting shift from a predominantly -helical fold to a -sheet and Leu26 side chains. The peptide in solution is mostly structure, when four alanines were introduced between the unstructured, but following PA assembly, its conformational N terminus of p531429 and the palmitic tail. Considering that freedom is reduced and folding is energetically favored. Con- both peptide structure and supramolecular assembly are critical sidering the above, we hypothesized that the peptide has a natural ffi R to developing an e cient delivery system of this inhibitory tendency to form an helix, as is the case for C16p53. Interest- peptide, we examine here in detail the supramolecular structure ingly, inclusion of alanines induces β-sheet formation of the of these two PAs and study how these are influenced by a high peptide headgroups. This result is nontrivial considering that þ concentration of Mg2 ions. Our results reveal striking differ- alanines are frequently found in R-helical segments of proteins ences in self-assembly stemming from the folding of the peptide and, therefore, are considered as R-helix-promoting amino into distinct secondary-structure motifs, which have general acids.13 However, alanine-rich peptides forming β sheets have implications for designing biomaterials as well as for studying also been documented in nature, most notably in silk proteins.14 peptide misfolding diseases. We believe that the positioning of alanines close to the hydrophobic core of the PAs and the tendency to minimize the interfacial area between the core and the aqueous phase induce tight peptide ’ RESULTS AND DISCUSSION packing and favor intermolecular hydrogen bonding. The small side chain of alanine, which is beneficial for the formation of R helices, Self-Assembly of PAs. The chemical structures of the PAs can also be viewed as advantageous for tightly packing β sheets. used in our study consist of an aliphatic palmitic tail and an Indeed, a number of studies have used alanines as spacers in PAs to oligopeptide fragment (Figure 1). The two PAs differ solely by 1517 promote fiber formation through β-sheet stacking. four alanines interposed between the palmitic tail and the amino The capacity of the peptide headgroup to adopt two different acid sequence LSQETFSDLWKLLPEN from peptide p53 . 14 29 secondary-structure motifs implies that folding of supramolecu- Hydrophobic forces predominantly drive the self-assembly of lar structures is amenable to control. Several examples exist in the these PAs above the critical association concentration (cac) of R approximately 2 μM;4 the corresponding peptides do not self- literature, where a peptide switches between an -helix and a β-sheet conformation in either a reversible or a nonreversible associate at concentrations up to 0.5 mM. Despite the similarity 5,18 in architecture and amino acid content, the inclusion of four manner. Such transformations also bring in mind protein misfolding scenarios, where a certain protein or peptide segment alanines between p531429 and the palmitic tail had a striking aggregates in β-sheet fibrils. These amyloid fibers have been effect on headgroup interactions and peptide folding, as demon- 19 strated using circular dichroism (CD) spectroscopy (Figure 2). implicated with various pathologies. Here, the peptides studied CD spectra in 10 mM PBS, well above the cac of the PAs, adopt a controlled conformation in response to self-assembly. It showed that the peptide adopted a mostly R-helical conforma- is noteworthy that this occurs at particular conditions β fi tion in C16p53 assemblies, whereas it formed sheets in (concentration, pH, temperature, etc.), in which the unmodi ed C A p53 assemblies (panels A and C of Figure 2). In the peptides show limited interactions. We therefore propose that 16 4 þ presence of Mg2 ions, the type of secondary structure was not PAs hold potential not only as building blocks of biomaterials but altered and only a small stabilizing effect in folding was observed, also as tools for studying peptide folding and peptidepeptide 20 particularly for C16p53. Peptide melting curves showed high interactions.

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Figure 2. CD spectroscopy of C16p53 and C16A4p53 in the presence and absence of 1 M MgCl2. (A) N-Bromosusuccinimide (NBS)-oxidized C16p53 ﬀ R O 2þ b in phosphate-bu ered saline (PBS) folds into an helix ( ), which is slightly stabilized in the presence of Mg ( ). The inset shows original C16p53 ﬁ CD spectra, which contain contributions from Trp23 stacking interactions. (B) Melting curves for C16p53 con rm enhanced folding stability in the 2þ b O β 0 presence of Mg ( ) compared to PBS ( ). (C) C16A4p53, on the contrary, folds into sheets with comparable CD spectra in PBS ( ) and PBS with 9 0 9 1 M MgCl2 ( ). (D) Melting curves of C16A4p53 in PBS ( ) and PBS with 1 M MgCl2 ( ).

Imaging of Supramolecular Structures. The aforemen- preferentially linear, and twisting occurs presumably to accom- tioned differences in the peptide headgroup structure and, modate close packing of R helices. therefore, peptide peptide interactions between C16p53 and In contrast to C16p53, C16A4p53 aggregates were readily C16A4p53 had a pronounced effect on the supramolecular imaged even at lower concentrations (Figure 3C). In this case, structure, as observed using cryogenic transmission electron structures that resemble worm-like micelles of uniform thickness fi microscopy (cryo-TEM) (Figure 3). The C16p53 structures were and nite length were imaged. Figure 3D presents the diameter scarce after sample preparation, presumably because of aggregate measurements of four different micelles. The average diameter instability during the blotting procedure or preferential binding obtained from 40 such micelles from six different TEM micro- of the PAs on the blotting paper. A couple of structures can be graphs was found to be 8.5 ( 0.9 nm (average ( standard seen in Figure 3A and several more in Figures S1 and S2 of the deviation). This corresponds well with coreshell micelle mor- Supporting Information. Careful examination of C16p53 struc- phology, with the core composed of the hydrocarbon chains and tures revealed twisted ribbon morphology with a pitch that varied the shell made up of extended peptide headgroups (Figure 4B). between 50 and 100 nm among different aggregates. The This fibrillar structure is similar to those reported by analogous diameter of these aggregates oscillated between a minimum PAs rich in alanines immediately after the acyl chain (C16Ax value of 5 nm and a maximum value of 8 nm (Figure 3B and peptide) reported in the literature.16,21,22 Hydrogen bonding Figure S1 of the Supporting Information). These dimensions occurs between adjacent PAs and runs axially along the micelles. excluded the possibility that the imaged structures are two It is not clear at this point whether there is a twist in the β sheets intertwined coreshell micelles and suggested instead nanor- or they run parallel to the central axis.11,23,24 However, the ibbon morphology. Coreshell micelles twisted together would minimum in the CD spectrum at 214 nm suggests linear PA exhibit a width oscillating between a diameter and the double of stacking.23 Independent of the extent of disorder in β-sheet that value. Moreover, individual micelles fraying at the ends of formation along the micelle long axis, interpeptide hydrogen the supamolecular structures were not observed, as previously bonding in C16A4p53 is expected to increase stability of the 27 R seen in such aggregates. Taking into account that an helix is supramolecular structures, as compared to C16p53 where hydrogen formed (Figure 2A) and that Trp23 fluorescence indicates water bonding occurs intramolecularly. shielding and a more hydrophobic environment,4 we propose the The differences in the supramolecular structure observed structure schematically presented in Figure 4A. In this model, the between the two PAs shed light on our previously reported amphipathic R helix folds on itself, interacting with the acyl chain, findings with respect to their stiffness, obtained by dynamic light 4 while another PA symmetrically shields the acyl chain from scattering (DLS) measurements. For C16p53, we determined water. The macroscopic association of C16p53 PAs is then that the persistence length exceeded 50 nm, while C16A4p53

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Figure 3. Cryo-TEM micrographs of (A) C16p53 and (C) C16A4p53 in 10 mM PBS. C16p53 structures self-assembled into twisted ribbon structures, ﬁ whereas C16A4p53 appeared as core shell worm-like micelles. Pixel intensity pro les to determine aggregate width at positions indicated by the arrows in (A) and (C) are shown in (B) for C16p53 and (D) for C16A4p53, respectively. Scale bars = 100 nm.

Figure 4. Schematic representation of supramolecular assemblies formed by (A) C16p53 and (B) C16A4p53 in 10 mM PBS. The presence of magnesium ions increases the pitch of (C) C16p53 twisted structures, whereas it causes lateral aggregation of (D) C16A4p53 worm-like micelles. The dotted lines in A and B represent hydrogen bonds, and the dot-dash lines in A and B represent the longitudinal axis of the supramolecular structures. micelles had a persistence length of less than 20 nm, despite both the cause of this diﬀerence is the qualitatively diﬀerent morphology PAs having similar hydrodynamic diameters. Here, we show that of the supramolecular structure.

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and C16A4p53 PA micelle suspensions: C16p53 aqueous suspensions remained clear, whereas C16A4p53 suspensions rapidly turned turbid (inset in Figure 5A). We performed light scattering measurements to gain a better understanding of the process. Kinetics of the scattering intensity at 90, measured as counts of scattering events detected per second, showed that aggregation of C A p53 micelles occurred within seconds following the 16 4 þ addition of Mg2 (Figure 5A). Instead, the scattering intensity of C16p53 dropped slightly because of dilution with the MgCl2 solution and remained constant over at least 30 min. Diffusion þ coefficients before Mg2 addition were 2.08 10 8 and 3.52 8 2 10 cm /s for C16p53 and C16A4p53 structures, respectively, corresponding to hydrodynamic diameters of 236 and 139 nm. In the case of C16A4p53, the jump in the scattering intensity was concurrent with a rapid increase in the diffusion coefficient, as evidenced by the autocorrelation curves measured immediately after mixing with MgCl2. Interestingly, DLS measurements over the next 48 h did not indicate a shift in autocorrelation curves, suggesting the formation of stable aggregates (bottom of Figure 5B). Furthermore, in this time period, no precipitation was observed. On the contrary, the autocorrelation curves for 2þ C16p53 structures in the presence of Mg gradually shifted toward longer decay times, corresponding to higher diffusion coefficients (top of Figure 5B), while the solution remained transparent. We note that the observed increases in the diffusion coefficient could originate from viscosity increases parallel to structure modification, and therefore, we have refrained from reporting data on the hydrodynamic size. To understand the effect of magnesium ions on the two PAs, we probed their structure following MgCl2 addition using both AFM and cryo-TEM. AFM of the PAs in the presence of divalent 2þ þ Mg ions allowed visualization of C p53 aggregates (Figure 6), Figure 5. DLS was used to study the effect of Mg2 on PA self- 16 assembly. (A) Scattering intensity at 90 was monitored with time which were again scarce in samples prepared for cryo-TEM μ (Figure 7). C16p53 exhibited the same twisted ribbon morphol- following mixing (t = 2 min) of 200 M PA solution with MgCl2 solution O 0 ( ,C16p53; ,C16A4p53). The inset shows PA solutions immedi- ogy as in the absence of magnesium ions. In this case, however, fi 2þ fi ately following MgCl2 addition ( nal Mg concentration = 1 M). structures seemed to be longer, a nding that agrees well with (B) Normalized autocorrelation curves obtained by DLS for (top) DLS results. The aggregate height oscillated from 2.5 to 5.0 nm, C16p53 and (bottom) C16A4p53. with a periodicity ranging from 100 and 200 nm (Figure 6). The height of the aggregates was lower than the width measured using We next performed atomic force microscopy (AFM) in semi- cryo-TEM (510 nm; Figure 7). This difference could originate wet conditions and observed the twisted ribbon morphology of from compression of the structures on the mica surface by the AFM C16p53 (Figure S3 of the Supporting Information). The absence tip. Alternatively, flattening of the aggregates on the surface of mica of excess water and/or the deposition process could have altered could occur. The measured pitch was similar between the two þ the structure morphology, while inclusion of buffers was not techniques and higher than that observed in the absence of Mg2 . possible because of their crystallization upon drying. Although C16A4p53 imaged by AFM under liquid in the presence of ff AFM in liquid overcomes these issues, the net surface charge MgCl2 showed a strikingly di erent image compared to C16p53 of the micelles is negative and they are repelled by the negati- (Figure 8). Aggregates of micelles with lengths similar to those þ vely charged mica surface, making imaging challenging. The observed by cryo-TEM in the absence of Mg2 (Figure 3B) were addition of divalent ions addressed this problem and will be visualized. Association of the micelles appeared to be along the discussed below. long axis of the micelles (Figure S4 of the Supporting In- þ Effect of Mg2 Addition in PA Solutions. Interactions formation). The micelle diameter was uniform, and a height of between the building blocks in PA supramolecular assemblies approximately 10 nm was measured, which corresponds well are dependent upon the presence of ions between these blocks. with the width obtained by cryo-TEM. The bundling of indivi- Divalent ions are often employed to form ionic bridges between didual micelles was also imaged using cryo-TEM (Figure 9) and neighboring PAs in fibrillar aggregates, with the aim of stabilizing negative-stain TEM (Figure S5 of the Supporting Information). þ the structure.9,10 We studied the effect of magnesium ions at a Our results highlight major differences upon Mg2 ion addi- high concentration after the PAs had already assembled in PBS. tion between the two different PAs. These differences are not þ Moreover, an excess of Mg2 ions induced the formation of likely to be a result from direct binding or interactions of cross-links between the PA structures and a mica surface, magnesium ions with alanine side chains because these are facilitating AFM imaging in liquid. nonpolar and hydrophobic. Instead, the differences are expected þ Visual examination following the addition of Mg2 at a con- to originate from the different peptide secondary structures ff ff centration of 1 M revealed major di erences between C16p53 induced. The two di erent folded patterns not only possess

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Figure 6. (A) Atomic force micrograph of C16p53 structures associated with a mica surface in the presence of 1 M MgCl2. (B) Measurements from one such structure revealed (C) a regular pitch of approximately 180 nm and a height of 5 nm at the higher points.

induce a similar effect. Our results were obtained with magnesium as the divalent ion because this is the prevalent divalent ion in the intracellular matrix, present in numerous protein and RNA structures. Studies of PA systems have sometimes revealed significant changes in interactions depending upon the ion type.8 10,26 It would be of interest to test the effect of divalent ion size, especially on C16p53 structures, where presumably ions interact with less accessible, glutamic acid anionic side chains. In contrast to previous reports of structures with similar morphologies where the self-assembled structure was stabilized by β sheets,20,27 31 the peptide here adopts a mostly R-helical conformation. Pashuck and Stupp recently reported on the formation of twisted ribbons, which upon aging, formed helical 29 ff ribbons. The e ect of aging on C16p53 structures is under investigation; however, during preparation of samples reported here, a heating step of 1 h at 60 C was performed, which makes the formation of kinetically trapped structures unlikely. The concentration of the sample has also been implicated in determining supramolecular structure morphology28 and merits further investigation. In contrast to the twisted ribbon morphol- fi ogy, the brillar, core shell morphology observed for C16A4p53 has often been observed in PA systems.7,11 In summary, we have presented in detail the process of self- Figure 7. Cryo-TEM image of C16p53 PAs in the presence of 1 M assembly into supramolecular structures of two p531429 PAs MgCl2, forming twisted ribbons. Measurements of structure width and differing solely by a four-alanine “linker”. The presence of this periodicity are shown in the inset. The globular structures present in the “linker” had a dramatic effect on the peptide secondary structure image are surface ice contamination. Scale bar = 100 nm. in the self-assembled state, shifting the energy balance from R favoring the formation of an helix in the case of C16p53 to ff β di erent overall morphology but also dictate the positioning of favoring the formation of sheets for C16A4p53. This shift in the anionic residues within the supramolecular structure. In the turn determined the morphology of the supramolecular struc- case of C16A4p53, p531429 is extended with Glu28 on the outer tures as well as their assembly in the presence of a divalent cation þ surface of the corona. Magnesium ions could therefore act as (Mg2 ). Such differences are important for the design of cross-links between adjacent micelles (Figure 4D) and result in p531429 peptide delivery systems based on PAs and have the observations of TEM (Figure 9) and AFM (Figure 8). For implications for a number of similar systems encountered in C16p53, elongation of the twisted ribbons may result from a the literature. Previously, we have shown that C16p53 PAs readily stabilizing effect of ionic cross-linking between adjacent PAs in enter cells following disassembly into isolated PAs;32 altering the structure. Lateral aggregation of the ribbons is unlikely interactions between PAs through incorporation of linkers consti- considering their geometry and the contact points between tutes a potential control mechanism over stability and delivery. them;25 energetically costly untwisting would be necessary to Furthermore, our results revealed promise for use of PAs to study

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Figure 8. (A) Atomic force micrograph of C16A4p53 structures associated with a mica surface in the presence of 1 M MgCl2, revealing extensive association of PA micelles. (B and C) Micelle height was between 10 and 11 nm.

purity was determined using analytical high-performance liquid chro- matography (HPLC) on a reverse-phase C4 column (Vydac). PAs with purity greater than 95% were used. To prepare micelles, PAs were dissolved in a 1:1 mixture of chloroform and methanol. The organic solvents were then evaporated under N2 flow, eventually forming a film on a glass vial wall. The film was dried in vacuum and, subsequently, hydrated in water or buffer at 60 C for 1 h. All samples were stored at 4 C and used within 1 week of preparation. Spectroscopy. CD spectroscopy was performed on a Jasco J-815 spectropolarimeter. PA solutions (100 μM) were analyzed using 1 mm path-length cuvettes, while the temperature was controlled within 0.1 C using a Peltier device. DLS. DLS was performed on a system (Brookhaven Instruments) that consisted of an avalanche photodiode detector to measure scattering intensity from a 632.8 nm HeNe laser (Melles Griot) as a function of the delay time. The temperature was maintained at 25.0 ( 0.1 C using a circulating water bath. The first-order autocorrelation function was measured at 90 (q = 0.0189 nm 1). The autocorrelation function was then fit using a second-order cumulant to extract the average decay Γ Γ 2 rate, 1. The quantity 1/q was then taken as the apparent diffusion coefficient, Dapp. The scattering intensity was measured as scattering events per second (counts/s) at 90. Figure 9. Cryo-TEM image of C16A4p53 PAs in the presence of 1 M AFM. A typical AFM experiment follows the deposition of 50 μLof MgCl2. Association along the long axis of individual micelles was evident. Scale bar = 100 nm. PA solution on a freshly cleaved V1-grade mica surface (from Ted Pella) for 10 min in a closed humid chamber. After deposition, samples were washed with appropriate buffer. Samples were either imaged under peptidepeptide interactions and their aggregation processes. Inter- buffer conditions in an AFM liquid chamber or dried in the stream of molecular interactions are ampliﬁed in PAs because of condensation nitrogen and imaged under air. The AFM images were recorded in the of the peptide on the surface of the supramolecular structures. Even amplitude modulated tapping mode using a MultiMode microscope though contributions of the alkyl chains are always present, the equipped with an E scanner controlled by Nanoscope IIIA (Veeco, Santa eﬀective concentration required to observe peptidepeptide inter- Barbara, CA). For experiments performed under liquid, silicon nitride actions is greatly reduced and energy contributions can be extracted probes (MLCT-AUHM) with a resonance frequency of ∼8kHzandspring through systematic selection of spacers and conditions. constant k of ∼0.03 N/m (Veeco Probes, Santa Barbara, CA) were used. Images in air were collected using silicon probes (NSC12/without Al ’ MATERIALS AND METHODS coating) with a resonance frequency of ∼160200 kHz and spring constant k of ∼4.56.5 N/m (MicroMasch, Wilsonville, OR). Images were pro- Materials. PAs were synthesized as previously described.33 Their cessed and analyzed using NanoScope software (Veeco, Santa Barbara, identity was verified by electrospray ionization mass spectrometry, and CA) and leveled by a first-order plane fit to correct the sample tilt.

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TEM. All PA samples used for TEM imaging were prepared at a (8) Anderson, J. M.; Andukuri, A.; Lim, D. J.; Jun, H. W. ACS Nano concentration between 1.0 and 2.5 mg/mL and were imaged at 200 kV 2009, 3, 3447–3454. with a JEOL JEM2010F (Figure 3 and Figure S1 of the Supporting (9) Stendahl, J. C.; Rao, M. S.; Guler, M. O.; Stupp, S. I. Adv. Funct. Information) and a FEI Technai G2 Sphera microscope (Figures 7 and 9 Mater. 2006, 16, 499–508. fi ff and Figure S5 of the Supporting Information). Images were recorded (10) Green eld, M. A.; Ho man, J. R.; de la Cruz, M. O.; Stupp, S. I. – digitally with a Gatan Ultrascan 4000 charge coupled device (CCD) Langmuir 2010, 26, 3641 3647. (11) Paramonov, S. E.; Jun, H. W.; Hartgerink, J. D. J. Am. Chem. Soc. camera on JEM2010F and 1000 CCD camera on Technai microscopes. – Images were analyzed using the Gatan Digital Micrograph software. 2006, 128, 7291 7298. (12) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Negative-stain samples were prepared by placing a 3 μL droplet of PA Levine, A. J.; Pavletich, N. P. Science 1996, 274, 948–953. solution onto a glow-discharged Formvar-coated copper grid for 5 min. fi μ (13) Blaber, M.; Zhang, X. J.; Matthews, B. W. Science 1993, 260, The excess liquid was wicked away by lter paper. A 3 L droplet of 2% 1637–1640. (w/v) aqueous phosphotungstic acid was placed on the grid and left for (14) Numata, K.; Kaplan, D. L. Adv. Drug Delivery Rev. 2010, 62, 5 min before similarly being wicked away. The samples were left to dry 1497–1508. overnight before imaging. (15) Rajangam, K.; Behanna, H. A.; Hui, M. J.; Han, X.; Hulvat, J. F.; Cryo-TEM samples were prepared using the environmentally con- Lomasney, J. W.; Stupp, S. I. Nano Lett. 2006, 6, 2086–2090. trolled FEI Vitrobot Mark IV (24 C, 100% humidity). A 3.5 μL droplet (16) Tovar, J. D.; Claussen, R. C.; Stupp, S. I. J. Am. Chem. Soc. 2005, of the sample was pipetted onto a glow-discharged Quantifoil or lacey 127, 7337–7345. carbon-coated copper grid. The sample was blotted once with filter (17) Beniash, E.; Hartgerink, J. D.; Storrie, H.; Stendahl, J. C.; Stupp, paper for 2 s before being plunged into liquid-nitrogen-cooled liquid S. I. Acta Biomater. 2005, 1, 387–397. ethane. The samples were placed in a Gatan cryo-holder and were kept (18) Altman, M.; Lee, P.; Rich, A.; Zhang, S. Protein Sci. 2000, 9, below 170 C throughout imaging. Samples were imaged using low- 1095–1105. – dose mode. (19) Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333 366. ’ (20) Meijer, J. T.; Roeters, M.; Viola, V.; Lowik, D. W. P. M.; Vriend, ASSOCIATED CONTENT G.; van Hest, J. C. M. Langmuir 2007, 23, 2058–2063. – S (21) Hung, A. M.; Stupp, S. I. Langmuir 2009, 25, 7084 7089. b Supporting Information. Additional cryo-TEM (Figures (22) Cui, H.; Pashuck, E. T.; Velichko, Y. S.; Weigand, S. J.; S1 and S2), AFM (Figure S3) images of C16p53 PAs and AFM Cheetham, A. G.; Newcomb, C. J.; Stupp, S. I. Science 2010, 327, (Figure S4), and negative-stain TEM images of C16A4p53 555–559. (Figure S5). This material is available free of charge via the (23) Pashuck, E. T.; Cui, H.; Stupp, S. I. J. Am. Chem. Soc. 2010, 132, Internet at http://pubs.acs.org. 6041–6046. (24) Jiang, H. Z.; Guler, M. O.; Stupp, S. I. Soft Matter 2007, 3, ’ AUTHOR INFORMATION 454–462. (25) Nyrkova, I. A.; Semenov, A. N.; Aggeli, A.; Boden, N. Eur. Phys. Corresponding Author J. B 2000, 17, 481–497. *Telephone: þ49-6221-54-4969. E-mail: [email protected]. (26) Dai, Q.; Dong, M.; Liu, Z.; Prorok, M.; Castellino, F. J. J. Inorg. Biochem. 2011, 105,52–57. Present Addresses (27) Muraoka, T.; Cui, H.; Stupp, S. I. J. Am. Chem. Soc. 2008, 130, † Department of Biophysical Chemistry, Institute for Physical 2946–2947. Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, (28) Cui, H.; Muraoka, T.; Cheetham, A. G.; Stupp, S. I. Nano Lett. Heidelberg D-69210, Germany. 2009, 9, 945–951. (29) Pashuck, E. T.; Stupp, S. I. J. Am. Chem. Soc. 2010, 132, 8819–8821. ’ ACKNOWLEDGMENT (30) Castelletto, V.; Hamley, I. W.; Hule, R. A.; Pochan, D. Angew. Chem., Int. Ed. 2009, 48, 2317–2320. We thank B. Lin for critical comments on the manuscript and (31) Aggeli, A.; Nyrkova, I. A.; Bell, M.; Harding, R.; Carrick, L.; T. Neumann for help with AFM in air. This work was supported McLeish, T. C.; Semenov, A. N.; Boden, N. Proc. Natl. Acad. Sci. U.S.A. by the National Heart, Lung, and Blood Institute Grant 5 U54 2001, 98, 11857–11862. CA119335-04, the MRSEC Program of the National Science (32) Missirlis, D.; Khant, H.; Tirrell, M. Biochemistry 2009, 48, Foundation, under award DMR05-20415, and National Insti- 3304–3314. tutes of Health (NIH) Grant P41RR02250. (33) Berndt, P.; Fields, G. B.; Tirrell, M. J. Am. Chem. Soc. 1995, 117, 9515–9522. ’ REFERENCES (1) Zhang, S. Nat. Biotechnol. 2003, 21, 1171–1178. 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6170 dx.doi.org/10.1021/la200800e |Langmuir 2011, 27, 6163–6170