Effect of Peptide Sequence on Surface Properties and Self-Assembly

Jul 30, 2009 - 1980 Kimball Avenue, Manhattan, Kansas 66506. Received April 6, 2009; Revised Manuscript Received June 9, 2009. Peptides that undergo a...
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Effect of Peptide Sequence on Surface Properties and Self-Assembly of an Amphiphilic pH-Responsive Peptide Jeanne N. Shera and Xiuzhi Susan Sun* Bio-materials & Technology Lab, Department of Grain Science & Industry, Kansas State University, 1980 Kimball Avenue, Manhattan, Kansas 66506 Received April 6, 2009; Revised Manuscript Received June 9, 2009

Peptides that undergo a morphological change when exposed to a stimulus have been investigated for their surface and self-assembly properties. Two 15-residue sequences were designed and synthesized for the purpose of determining the role of sequence on surface properties and peptide self-assembly. The KhK (KKKFLIVIGSIIKKK) and Alternating Kh (KFLKKIVKIGKKSII) sequences were synthesized via microwave peptide synthesis according to the automated base-labile Fmoc strategy. Despite having the same amino acid content, KhK solutions exhibited an increase in contact angle with increasing pH, whereas Alternating Kh solutions demonstrated a decrease in contact angle with increasing pH. Further analysis by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed marked differences in the peptide solution and peptide particle morphology. Circular dichroism (CD) spectroscopy indicated that KhK consisted of primarily β-sheet conformations at acidic and neutral pH. In Alternating Kh CD spectra, random coil conformations were predominant at acidic and neutral pH.

Introduction Traditionally, synthetic polymers have been used for a wide range of applications including biomaterials and adhesive resins because of advantageous properties such as biocompatibility and good mechanical properties.1,2 Polymers synthesized from amino acids, or peptides, have excellent biocompatibility because they are composed of the same basic units as proteins. Previous studies have demonstrated that β-sheet structures contribute to increased adhesion and water resistance.3,4 Peptides also exhibit several conformations such as R-helices, β-sheets, and random coils depending on pH and the kind of amino acid used. The ability of peptides to spontaneously form ordered structures at the nanoscale makes them promising building blocks for nanomaterials.5,6 To understand the molecular driving force for molecular self-assembly, varied approaches have been taken toward engineering peptide structures. Tubular nanostructures have been shown to self-assemble by both synthetic and naturally occurring cyclic peptides.7,8 Another approach was to design peptides in such a way that self-assembly would occur because of electrostatic interactions between positively and negatively charged groups within the peptides.9,10 The linear building blocks consisted of more than 50% charged peptides. These peptides included negatively charged glutamic acid and aspartic acid and positively charged lysine and arginine residues. In aqueous solutions, the peptides readily assembled to form fibril structures. Another important aspect of biological self-assembly is the presence of hydrophobic interactions between molecules. Several peptide structures for selfassembly have been based on amphiphilic or surfactant-like molecules that possess a hydrophobic “tail” and a hydrophilic “head” that allow them to form ordered structures in aqueous solutions. These amphiphilic molecules include bolaamphiphile peptides11-13 and peptide-conjugated amphiphiles.14-16 * To whom correspondence should be addressed. Phone: (785) 532-4077. Fax: (785) 532-7193. E-mail: [email protected].

The various amphiphilic building blocks use different geometries for the self-assembly process. In this study, two peptide sequences were investigated for their surface, morphological, and self-assembly properties. Using solid-phase amino acid sequence synthesis, we synthesized short peptide nanomaterials based on a mixture of hydrophobic and electrostatic interactions. Our peptide sequences are inspired by the sequences present in soy protein. In both glycinin and β-conglycinin, hydrophobic residue sequences are interspersed among hydrophilic residue sequences, leading to highly ordered secondary and tertiary structures.17,18 We theorize that the hydrophobic segment(s) will promote aggregation in an alkaline pH environment, whereas the hydrophilic lysine residues can form strong hydrogen-bonding interactions with themselves. A nine-residue hydrophobic core FLIVIGSII derived from the human skeletal dihydropyridine-sensitive calcium channel was flanked by three lysine residues on each side to yield the sequence KKKFLIVIGSIIKKK (KhK).3,4,19 In the second peptide, residues of the hydrophobic core were alternated with lysine residues to yield the peptide with the following sequence: KFLKKIVKIGKKSII (Alternating Kh). The goal of this research is to study the contributions from peptide sequence that govern peptide morphology and secondary structure of these two peptides. By designing peptides with similar overall hydrophobicity, isoelectric points, and amino acid content, this information may be elucidated. Such information is valuable for tailoring protein nanomaterials for use in adhesive, biomedical, and pharmaceutical applications.

Experimental Section Materials. N,N-Dimethylformamide, N,N-diisopropylethylamine, piperidine, triisopropyl silane, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (Milwaukee, WI). Dichloromethane, anhydrous ether, and N-methylpyrrolidone were purchased from Fisher Scientific (Pittsburgh, PA). All protected amino acids, rink amide MBHA resin, and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate were purchased from EMD Biosciences/Merck Biosciences (Darmstadt, Germany). All reagents were used as received.

10.1021/bm900388b CCC: $40.75  2009 American Chemical Society Published on Web 07/30/2009

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Table 1. Net Charges and Calculated Mean Residue Hydrophobicity of Peptide Sequences sample

net charge pH (1.8)

∆Gavea

net charge pH (7.3)

∆Gavea

net charge pH (12.3)

∆Gavea

h KhK Alternating Kh

0 (4)h(3) ) 7 (2)h(2)h(1)h(2)h ) 7

-0.40 0.49 0.49

0 (4)h(3) ) 7 (2)h(2)h(1)h(2)h ) 7

-0.40 0.49 0.49

0 (0)h(0) ) 0 (0)h(0)h(0)h(0)h ) 0

-0.40 -0.51 -0.51

a

Lower ∆Gave values indicate higher peptide hydrophobicity.

Peptide Synthesis. Peptides were synthesized on a CEM Liberty microwave peptide synthesizer (CEM Corporation, Matthews, NC) according to the automated base-labile 9-fluorenylmethoxycarbonyl (Fmoc) strategy with Fmoc-protected amino acids. After synthesis, polypeptides were washed twice with anhydrous ether, dissolved in deionized water, and then freeze-dried. Subsequent to synthesis, peptides were characterized by mass spectrometry (MS) and high-performance liquid chromatography (HPLC). Desired peptide molecular weight was confirmed by matrix-assisted, laser desorption-ionization time-of-flight (MALDI-TOF) mass spectroscopy on an Ultraflex II instrument (Bruker Daltronics, Billerica, MA). Peptide purity was confirmed by a Beckman System Gold HPLC (Beckman Coulter, Inc., Fullerton, CA) on a Phenomenex Synergi 4µ Hydro-RP column (Phenomenex, Inc., Torrance, CA) with the following gradient: 10-90% B in 20 min (A: 99.9% H2O, 0.1% TFA; B: 90% acetonitrile, 9.9% H2O, 0.1% TFA). Contact Angle Analysis. Peptide solutions, 1% (w/v), were prepared in deionized water and adjusted to pH 1.8, 7.3, and 12.3 with 1 N HCl or NaOH. Peptide solutions were analyzed with the sessile drop method on a KSV Instruments CAM 100 optical contact angle meter (KSV Instruments, Ltd., Helsinki, Finland) at ambient conditions on highdensity polyethylene (HDPE) substrate. Drop profiles were fit with the Young-Laplace equation to obtain contact angles (θ°).20 Transmission Electron Microscopy (TEM) Analysis. To elucidate the morphology of KhK and Alternating Kh in solution, 1% (w/v) peptide solutions were prepared in deionized water and adjusted to pH 1.8, 7.3, and 12.3 with 1 N HCl or NaOH. Solutions were absorbed for approximately 30 s at ambient conditions onto Formvar/carboncoated 200-mesh copper grids (Electron Microscopy Sciences, Fort Washington, PA) and stained with 2% (w/v) uranyl acetate (Ladd Research Industries, Inc., Burlington, VT) for 60 s at ambient conditions before being visualized at 5800, 13 500, 34 000, and 130 000 times direct magnification. Circular Dichroism (CD) Spectroscopy Analysis. Peptide solutions, 1% (w/v), were prepared in deionized water and adjusted to pH 1.8, 7.3, and 12.3 with 1 N HCl or NaOH. CD spectra were recorded at ambient conditions on a Jasco J-815 spectrometer (Jasco Corporation, Tokyo, Japan) by using a cell with path length of 0.1 mm. Scanning Electron Microscopy (SEM) Analysis. Peptide solutions, 1% (w/v), were prepared in deionized water and adjusted to pH 1.8, 7.3, and 12.3 with 1 N HCl or NaOH. The pH-adjusted solutions were frozen and subsequently freeze-dried to trap peptide morphology at the varied pH conditions and yield dried peptide powders for SEM analysis. Dried peptide powders were sputter-coated with 60% Au/ 40% Pd with a DESK II sputter/etch unit (Denton Vacuum, LLC., Moorestown, NJ). Images were captured on a Hitachi S-3500N SEM (Hitachi Science Systems, Ltd., Hitachinaka, Japan) at an accelerated voltage of 5 kV. Hydrophobicity Calculation. Hydrophobicity is represented by ∆Gave in kcal/mol. ∆Gave ) Σ∆Gresidue/residue number. ∆Gresidue values are taken from the octanol interface scale published on Dr. Stephen White’s Web site (http://blanco.biomol.uci.edu/hydrophobicity_scales. html; University of California, Irvine).21 No published values for the hydrophobicity of lysine at pH ) 12.3 were available, so the value for leucine was selected on the basis of the number of carbons present in the acyl side chain. Lysine’s linear acyl side chain is expected to be more hydrophobic than that of leucine, but the presence of the uncharged primary amine with a lone pair of electrons would generate a dipole that would decrease the overall hydrophobicity. Thus, using the hydrophobicity value for leucine at pH ) 12.3 was deemed appropriate (Table 1).

Table 2. Contact Angle of Peptides on HDPE at Varied pH sample

contact angle pH ) 1.8

contact angle pH ) 7.3

contact angle pH ) 12.3

KhK Alternating Kh

57.18 ( 2.28 78.59 ( 1.27

76.56 ( 1.08 76.27 ( 1.66

83.52 ( 1.34 60.94 ( 1.20

Results and Discussion Two 15-residue peptides were synthesized. In the first peptide, the hydrophobic core FLVIGSII (h) was flanked by three lysine residues on each end to yield the peptide with the following sequence: KKKFLIVIGSIIKKK (KhK). In the second peptide, residues of the hydrophobic core were alternated with lysine residues to yield the peptide with the following sequence: KFLKKIVKIGKKSII (Alternating Kh). Both KhK and Alternating Kh possess isoelectric points (pI ) 10.7). Additionally, the calculated overall hydrophobicities of the peptides are identical.3 Contact angles of peptide solutions on HDPE substrate obtained at pH 1.8, 7.3, and 12.3 indicated a reverse trend for KhK and Alternating Kh solutions (Table 2). In this study, higher contact angle values indicated higher surface tension between the low-energy HDPE substrate and the peptide solution. As pH increased for KhK solutions, contact angle also increased. This indicated an increase in the surface tension between the peptide solution and the hydrophobic HDPE substrate. As pH increased for Alternating Kh solutions, the contact angle demonstrated the opposite behavior, indicating a decrease in the surface tension between the peptide solution and the HDPE substrate. The opposing contact angle trends arose from morphology changes that were caused by differences in peptide sequence. Although overall hydrophobicity of the peptides was identical, the variations in peptide sequence altered the way each peptide responded to the pH environment. At acidic pH, both KhK and Alternating Kh formed micelle-like structures (Figure 1A,B). The micelle-like structures formed by KhK had a narrower size distribution than those formed by Alternating Kh: 60-90 versus 30-120 nm. KhK possesses the ability to form relatively uniform micelle-like structures at pH ≈ 2 because the hydrophobic core is uninterrupted and has the ability to fold at the glycine residue.22 The charged lysine residues are pushed into the water phase, and the hydrophobic core is then sequestered on the inside of the micelle-like structures. Alternating Kh attempts a similar process but is hindered by the interrupted hydrophobic core, resulting in a wider size distribution and a higher contact angle on HDPE. Though they both had a similar contact angle, as pH increased to ∼7, KhK and Alternating Kh demonstrated different morphology. KhK exhibited small micelle-like structures (10-50 nm) interspersed among short fibrils (25-100 nm) (Figure 1C). Similar fibril formation was also observed by Dong and coworkers in multidomain peptides at pH 7.13 The increase in contact on HDPE and the change in morphology indicate that the hydrophobic core of KhK had less dominant interactions with HDPE and other peptide chains than at pH ≈ 2. Alternating Kh consisted primarily of small micelle-like structures (10-30

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Figure 1. (A) TEM micrograph of KhK solution at pH 2.1 (s 500 nm), (B) TEM micrograph of Alternating Kh solution at pH 2.1 (s 500 nm), (C) TEM micrograph of KhK solution at pH 7.2 (s 500 nm) and inset (s 100 nm), (D) TEM micrograph of Alternating Kh solution at pH 7.3 (s 500 nm), (E) TEM micrograph of KhK solution at pH 12.0 (s 500 nm) and inset (s 100 nm), and (F) TEM micrograph of Alternating Kh solution at pH 12.0 (s 500 nm). All TEM micrographs are at 34 000 times magnification.

nm) (Figure 1D). The small micelle-like structures present in TEM coupled with the slight decrease in contact angle relative to pH ≈ 2 demonstrate a similar inclination of lysine groups to interact with one another, rather than be forced as far away from the hydrophobic residues as possible. Fibril formation is not possible because of the short runs of lysine residues interrupted by hydrophobic residues. Above the isoelectric point (pI ) 10.7), KhK and Alternating Kh display significant differences in morphology and surface properties. At pH ≈ 12, KhK exhibited its maximum contact angle on HDPE, indicating that hydrophilic lysine residues were in contact with the hydrophobic HDPE substrate. Also, the short fibrils observed at pH ≈ 7 became longer and more robust and were up to several hundred nanometers in length. The fibrils surrounded large peptide particles (25-100 nm) (Figure 1E). Similar structures have been observed in soy protein isolate treated with NaHSO3 at 3% solid content.23 Fibril formation occurs when the hydrophobic residues of neighboring peptides collapse together to exclude water and subsequently undergo further assembly into fibrils through hydrogen bonding through the lysine end groups.24 A similar mechanism is responsible for the morphology observed for the Alternating Kh peptide at pH ≈ 12. Rather than fibrils and large peptide particles, a continuous peptide film was formed (Figure 1F). During the

Shera and Sun

Figure 2. (A) SEM micrograph of KhK at pH 2.1 (s 500 µm), (B) SEM micrograph of Alternating Kh at pH 2.1(s 500 µm), (C) SEM micrograph of KhK at pH 7.2 (s 500 µm), (D) SEM micrograph of Alternating Kh at pH 7.3 (s 500 µm) (E) SEM micrograph of KhK at pH 12.0 (s 500 µm), and (F) SEM micrograph of Alternating Kh at pH 12.0 (s 500 µm). All SEM micrographs are at 70 times magnification.

process of peptide chains collapsing together and excluding water, the short runs of both hydrophobic residues and lysine residues create a net-like structure rather than a linear one, such as that present in a fibril. Additionally, the participation of lysine residues in hydrogen-bonded structures reduced the surface tension with HDPE. Scanning electron microscopy analysis of pH-adjusted dried peptide powders underscores the morphological differences created by peptide sequence. At acidic pH, KhK and Alternating Kh showed significantly different structures (Figure 2A,B). In solution, KhK possesses the ability to form relatively uniform micelle-like structures at pH ≈ 2.22 As the KhK peptide particles became dry, the micelle-like structures coalesced uniformly to form flat, continuous particles.25,26 For the Alternating Kh, the micelle-like structures failed to coalesce effectively because of the difference in peptide structure.25 This resulted in particles with ordered bundles and voids. As pH increased to pH ≈ 7, the presence of small micelle-like structures interspersed among short fibrils visible in TEM did not seem to affect the ability of KhK to form flat, continuous particles (Figure 2C). For Alternating Kh, however, the small micelle-like structures present in the Alternating Kh TEM enhanced the ability of this peptide to form continuous particles. Coalescence was incomplete, as indicated by the presence of peptide particles that possessed voids (Figure 2D); these randomly distributed voids were much smaller than those present at pH ≈ 2.

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Scheme 1. Schematic Representation of Solution and Surface Behavior of (A) KhK (KKKFLIVIGSIIKKK) and (B) Alternating Kh (KFLKKIVKIGKKSII)

Figure 3. (A) Circular dichroism spectra of KhK at pH 1.86 (s), 7.33 (- - -), and 12.25 (- - -). (B) Circular dichroism spectra of Alternating Kh at pH 1.88 (s), 7.31 (- - -), and 12.29 (- - -).

At pH ≈ 12, KhK and Alternating Kh peptide particles had similar morphology. However, KhK formed larger flat, continuous sheets than Alternating Kh (Figure 2E). These particles were larger than those at pH ≈ 2 and 7. The continuous film present in the Alternating Kh solution TEM translated into flat, continuous peptide sheets in SEM (Figure 2F). These flat, continuous peptide sheets formed at pH ≈ 12 shared the same mechanism that was described previously for TEM solution images at pH ≈ 12: Hydrophobic residues of neighboring peptides collapse together to exclude water and subsequently undergo further assembly into continuous particles by hydrogen bonding by lysine end groups.24 Further analysis with CD spectroscopy indicated the peptide secondary structure present at each pH level. For both KhK and Alternating Kh, low contact angles corresponded to the relative increase in the presence of β-sheets. KhK consisted of primarily β-sheet conformations at acidic and neutral pH, as indicated by negative peaks at 216 nm (Figure 3A). The β-sheet character present was indicative of β-barrels observed in previous studies.27,28 The presence of a significant negative peak at 197 nm and the shifting of the β-sheet peak from 216 to 225 nm indicate a significant increase in random coil conformations at basic pH. In Alternating Kh CD spectra, random coil conformations were predominant at acidic and neutral pH, as indicated by negative peaks at 200 nm (Figure 3B). At basic pH, Alternating Kh exhibited a conformation showing a significant amount of β-sheet character relative to acidic and neutral, as indicated by a negative peak at 226 nm. Random coil conformations were present, as indicated by a negative peak at 199 nm. According to these results, we can draw a schematic illustration to explain how the KhK and Alternating Kh arrange their local conformation to adapt to the inherent surface character and how they orientate on HDPE surfaces and in aqueous solution at acidic, neutral, and basic pH (Scheme 1). At pH ≈ 2, KhK folds at the glycine residue and sequesters the

hydrophobic peptide cores at the center of the micelle-like structures. This places the charged lysine groups in contact with the water phase in solution. When in contact with HDPE, the hydrophobic cores associate with the hydrophobic surface; the charged lysine groups, then, are oriented to be in contact with the water phase, resulting in a smaller contact angle. When the solution pH increases to ∼7, smaller, micelle-like structures formed by interactions between hydrophobic cores persist in the company of short fibrils formed by hydrogen bonding between lysine end groups and thus increase in contact angle on HDPE. At pH ≈ 12, hydrogen bonding between lysine end groups causes fibrils to grow and thicken. Globular peptide clusters present at basic pH are formed by aggregation of longer KhK peptide fibrils and individual KhK peptides folded at the glycine residue.29 The highest contact on HDPE results from a greater number of hydrophilic lysine end groups in contact with the hydrophobic surface. At pH ≈ 2, Alternating Kh folds at the glycine residue in an attempt to place the greatest number of hydrophobic residues at the center of the micelle-like structures. Interruption of the hydrophobic core by charged lysine residues causes a wide size distribution of micelle-like structures in solution and a high contact angle with HDPE. When the solution pH increases to ∼7, large micelle-like structures give way to smaller micellelike structures formed by interactions between hydrophobic cores. On HDPE, the result is a slight decrease in contact angle. At solution pH ≈ 12, peptide chains collapse together and exclude water. The short runs of both hydrophobic residues and lysine residues create a net-like structure rather than a linear one, such as that present in the fibrils observed in basic KhK solutions. β-Sheet structure development indicates that lysine residues are more likely to be buried in the interior of an extended secondary structure and not in contact with the HDPE surface.

Conclusions Peptides with the same amino acid composition but different sequences created significant differences in surface, morphological, and self-assembly properties. Although the two peptides studied possessed similar overall hydrophobicity, isoelectric points, and amino acid content, local differences in the ability to form hydrophobic and electrostatic interactions within and among peptide chains caused inverse behavior of peptide

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solutions on HDPE substrates. This indicates that hydrophobicity, isoelectric points, and amino acid content are not sufficient to characterize protein properties. Micelle-like structures were observed at pH ≈ 2 in KhK and Alternating Kh TEM images. Fibril formation that was observed at pH ≈ 7 in KhK solutions was absent in Alternating Kh solutions. Fibrils continued to develop and thicken at pH ≈ 12 in KhK solutions, whereas Alternating Kh solutions formed a continuous net-like structure. KhK peptide particles were flat and continuous at all pH levels, but Alternating Kh peptide particles were porous at pH ≈ 2 and developed into flat, continuous particles at pH ≈ 12. KhK solutions demonstrated significant amounts of β-sheet structure at all pH levels, whereas Alternating Kh solutions consisted of primarily random coils at pH ≈ 2 and 7. Alternating Kh solutions demonstrated significant amounts of β-sheet structure only above the isoelectric point at pH ≈ 12. The surface and morphological differences demonstrated by these two peptides in similar pH environments provide demonstrate the significant impact of peptide sequence and location of hydrophilic lysine reisudes versus hydrophobic residues on physical properties. As a result of this study, we believe that future investigation into tailoring the sequence of responsive peptides is warranted to better understand these effects. Acknowledgment. This study was supported by the Kansas State University Targeted Excellence Award and the Center for Biobased Polymers by Design. The authors gratefully acknowledge the following individuals for their contributions: Dr. Amy Mo (Wm. Wrigley Jr. Company, Chicago, IL), Dr. John Tomich and Dr. Michal Zolkiewski (Department of Biochemistry, Kansas State University) for the use of the HPLC and CD, Dr. Yasuaki Hiromasa (Department of Biochemistry, Kansas State University) for performing MS analysis, Mr. Kent Hampton (Department of Entomology, Kansas State University) for performing SEM analysis, and Dr. Dan Boyle (Department of Biology, Kansas State University) for performing TEM analysis. Contribution No 09-284-J from the U.S. Department of Agriculture, Kansas Agricultural Experimental Station.

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