Fibrillar Structures Formed by Covalently Bound, Short, StrandedPeptides on Self-Assembled MonolayersJason W. Dugger and Lauren J. Webb*

Department of Chemistry, Center for Nano- and Molecular Science and Technology, and Institute for Cell and Molecular Biology,The University of Texas at Austin, 1 University Station, A5300, Austin, Texas 78712, United States

*S Supporting Information

ABSTRACT: The ability to maintain or reproduce biomolecularstructures on inorganic substrates has the potential to impact diverseelds such as sensing and molecular electronics, as well as the study ofbiological self-assembly and structurefunction relationships. Becausethe structure and self-assembly of biomolecules are exquisitely sensitiveto their local chemical and electrostatic environment, the goal ofreproducing or mimicking biological function in an abiologicalenvironment, including at a surface, is challenging. However, simpleand well-characterized chemical modications of prepared surfaces canbe used to tune surface chemistry, structure, electrostatics, andreactivity of inorganic materials to facilitate biofunctionalization andfunction. Here, we describe the covalent attachment of 13-residue -stranded peptides containing alkyne groups to a at gold surfacefunctionalized with an azide-terminated self-assembled monolayerthrough a Huisgen cycloaddition, or click, reaction. The chemical composition and structural morphology of these surfaceswere characterized using X-ray photoelectron spectroscopy, grazing incidence angle reectionabsorption infrared spectroscopy,surface circular dichroism, and atomic force microscopy. The surface-bound -strands self-assemble into antiparallel -sheets toform brillar structures 24.9 1.6 nm in diameter and 2.83 0.74 nm in height on the reactive surface. The results hereinprovide a platform for studying and controlling the self-assembly process of biomolecules into larger supermolecular structureswhile allowing tunable control through chemical functionalization of the surface. Interest in the mechanisms of formation ofbrillar structures has most commonly been associated with neurodegenerative diseases, such as Alzheimers and Parkinsons, butbrils may actually represent the thermodynamic low-energy conformation of a much larger class of peptides and proteins. Theprotocol developed here is an important step toward uncovering not only the factors that dictate self-assembly but also themechanisms by which this brillar class of superstructures forms.

INTRODUCTIONThe chemical functionalization of inorganic surfaces andsubstrates with folded and functional biological molecules hasthe potential for a large variety of applications, includingsensors, electrochemistry, and molecular electronics, as well asproviding a template for studying and controlling the self-assembly of biomolecules into supermolecular structures. Theimmobilization of biomolecules on surfaces enables theformation and examination of biomolecular structures thatwould not be possible in solution, such as single-moleculestudies,1 the formation of asymmetric and two-dimensionalmaterials,24 and the generation of novel materials thatincorporate both biological and abiological functions.57 Achief obstacle to the incorporation of biomolecules ontoinorganic substrates is the preservation of the structure of themolecule, which is inherently related to its function andstability. One strategy that has been employed successfully is tocover the underlying inorganic substrate with a thick polymer,such as polyethylene glycol or polylysine, reducing the surfaceto a passive support.810 Our laboratory has been interested in

nding surface chemistries that integrate the biomolecule withthe surface in such a way that the chemical and electrostaticproperties of the surface act in concert with the biomolecule togenerate a novel biological/abiological material. To this end,our group has explored the use of self-assembled monolayers(SAM) on gold surfaces to facilitate the covalent attachment ofstructured -helical peptides orientated parallel to thesubstrate.11 This method exploits the known structure andregularity of the SAM to induce a desired secondary structurein the biomolecules while preserving desirable propertiescharacteristic of the metal surface such as conductivity.12,13

Here, we report an adaptation of this methodology tocovalently bound, short (13-residue), -stranded peptides tothe gold surface, which in turn nucleate the self-assembly oflarge supermolecular brillar structures in a controlled way.

Received: December 23, 2014Revised: February 20, 2015Published: March 4, 2015

Article

pubs.acs.org/Langmuir

2015 American Chemical Society 3441 DOI: 10.1021/la5049369Langmuir 2015, 31, 34413450

Biological and synthetic brils and brillar structures haveshown promise in applications such as sensors, orderednanomaterials, mimics of extracellular matrices, and scaoldsfor cell cultures.1418 Biological brils are also of immensecurrent medical interest because they are classically associatedwith neurodegenerative diseases such as Alzheimers, Parkin-sons, and type II diabetes.1921 Because of this, there has beenextensive investigation into the circumstances under whichnaturally occurring biological peptides spontaneously self-assemble into aggregates, brils, and plaques in certain tissuesthroughout the body, and it is widely believed that one or moreaspects of this self-assembly process may be causing the diseasestate associated with that bril. However, it has recentlybecome clear that the formation of these brillar structures isnot a unique property of a particular biological peptide, butinstead seems to be a common feature of amide-basedpolypeptides in general.22 Indeed, recent computational andexperimental work on the thermodynamic stability of brils andkinetic barriers to the assembly of these structures hassuggested that brillar conformations may be the low-energystructure of polypeptides in general, regardless of sequence orlength.2326 These observations mean that the basic under-standing of biological bril formation, stability, and function is aresearch area that lies at a crossroads between materials science,biophysical chemistry, and medicine, inuencing elds asdiverse as human health and molecular electronics.Various factors that regulate bril formation and growth,

including kinetics, inhibition, promotion, sequence variation,and environmental conditions, have been investigated,2731 andit has been observed repeatedly that there are a large variety ofconformations that a single protein sequence can adopt.19 It hasbeen shown that brils formed from amyloidogenic peptides ofdierent sequences can adopt structures comprised of parallelor antiparallel -sheets, and the resulting brils can bepolymorphic.21,3234 For instance, brils have been seen toadopt a triangular structure with a large central cavity,35 a thinribbon-like structure with diameters ranging from 3.5 to tens ofnanometers,36 and even nanotube-like structures over 100 nmin diameter resulting from the helical twisting of ribbon-likebrils.30,37 Despite the extensive characterization of a variety ofthese brillar structures, there is signicant ambiguityconcerning how the complex interactions dictated by peptidesequence, hydrophobicity, hydrophilicity, and environmentregulate the nucleation and self-assembly of small biomoleculesinto more complex supermolecular structures. Understandingthese factors is a necessary prerequisite to generate controlledmaterials containing desired properties that can be exploited forinteresting or novel purposes. Taking a surface-mediatedapproach to immobilizing these biomolecules allows forunprecedented control over a number of variables that can beused to possibly tune bril growth, size, structure, surfacedensity, and chemical reactivity.In the work described here, we demonstrate the self-assembly

of superstructures using a bottom-up method to bril formationthrough the covalent attachment of short (4.4 nm, 13-residuelong) -stranded peptides to a SAM on a gold surface. This isaccomplished with a triazole linkage formed from a Huisgencycloaddition click reaction between one or two alkynegroups on the peptide and adjacent terminal azide groups onthe SAM. Grazing incidence angle reectionabsorptioninfrared spectroscopy (GRAS-IR), X-ray photoelectron spec-troscopy (XPS), and surface circular dichroic (CD) spectros-copy conrm the presence of the peptide on reacted surfaces as

well as self-assembly into antiparallel -sheets. Additionally,structural morphology and heterogeneity observed usingatomic force microscopy (AFM) show that these -sheetsorganize into larger brillar structures 24.9 1.6 nm indiameter and 2.83 0.74 nm in height. We hypothesize thatsingle peptide strands covalently bound to the SAM act asnucleation sites for the formation of -sheets, which thenproceed to organize into brils in as little as 15 min. Sampleswith longer reaction times (18 h) contain similarly sizedstructures with a higher degree of surface coverage, creating ameshlike network of brils. These studies establish thecapability to nucleate noncovalently assembled superstructuresthrough the controlled immobilization of short peptides toinorganic substrates. This strategy inherently enables tuning ofchemical reactivity, environmental conditions, and interfaceheterogeneity and generalization to other biomolecules.

MATERIALS AND METHODSSurface Preparation. Peptide-terminated SAM surfaces were

prepared on silicon (111) wafers for GRAS-IR and XPS measure-ments, on quartz slides for surface CD experiments, and on gold-on-mica substrates (150 nm of gold on mica, SPI Supplies) for AFMmeasurements. The dierent substrates were used to exploit theirunique properties in each experiment, with silicon wafers used for theirmacroscopic atness in the spectroscopic reection experiments,quartz for its UV transparency in CD studies, and mica for itsmicroscopic atness in AFM. Silicon wafers (NOVA ElectronicMaterials) were 500 m thick, polished on one side, sealed in N2(g),and stored in a dry N2(g) atmosphere glovebox until use. A 10 nmlayer of chromium, followed by 100 nm of gold (99.95% pure), wasvapor-deposited on the silicon wafers using a thermal evaporator-II(Denton) instrument at a pressure of 105 Torr. The samples werecovered in clean-room-rated silicon wafer tape (ICROS TAPE) andcut into approximately 1 cm2 pieces with a programmable Disco 321wafer-dicing saw. After removing the tape, the samples were rinsed inmethanol, sonicated in acetone for 10 min, rinsed in high purity water(HPW) with an impedance of >18 M cm (Barnstead NANOpureDiamond Life Science UV/UF) and then rinsed again in methanol,dried under a stream of N2(g), and stored in Paralm-coveredcontainers until use. This cleaning was done to remove any residuethat may have been left behind by the wafer tape used during thecutting process. Quartz substrates precut into 2.5 0.95 cm slideswere rinsed in ethanol and dried under N2(g) before the deposition of3 nm of chromium, followed by 11 nm of gold, and subsequentlystored in Paralm-covered containers until use. The quartz sampleshad the thinnest layer of gold of all the substrates in order to increasetransmission in the CD experiments. Previous studies have shown thatSAMs are still formed on gold layers as thin as 7 nm,38 and SAMs arecommonly used to functionalize gold surfaces on a variety ofsupporting substrates, including those employed in these studies.The choice of substrate and thickness of the gold layer is not expectedto aect SAM formation or subsequent reaction with the peptides.

All chemicals were purchased from Sigma-Aldrich unless otherwisenoted. Gold substrates deposited on silicon were cleaned using apiranha solution (1:3 30% hydrogen peroxide/concentrated sulfuricacid) (Caution! Explosive in the presence of organic contaminants.) for 1min; rinsed in a sequence of HPW, concentrated hydrochloric acid,HPW, and nally ethanol; and dried under a stream of N2(g). Thesesurfaces were then annealed for 5 min using a H2(g) ame. Gold-on-mica substrates were kept in manufacturers packaging (glass vials lledwith argon gas, capped, and sealed with Paralm) until use, where theywere then annealed for 5 min with a H2(g) ame. Quartz substrateswere only rinsed in ethanol and dried under N2(g) because the thinnerlayer of gold could not withstand the harsher cleaning methodsdescribed above. All subsequent steps were identical for the silicon-,quartz-, and mica-supported substrates. To prepare the SAM, thesamples were submerged in a solution of 1 mM 11-azido-1-undecanethiol diluted in anhydrous ethanol for 24 h in the dark to

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fabricate a gold surface covered in 100% azide-terminated SAM. The11 methylene groups of the thiol participate in van der Waalsinteractions with the hydrophobic portions of surrounding thiols,inducing an ordered SAM that leaves the azide groups exposed at theSAM/aqueous interface. Unbound thiols were removed by sequen-tially rinsing with HPW, dimethylformamide (DMF), HPW, andethanol and dried under a stream of N2(g).

Azide-terminated SAM surfaces were exposed to the peptideKLKXKLLLKXKLK (hereafter 6; WuXi AppTec). It has beenshown that sequences of alternating leucine and lysine residues adopt-stranded conformations in aqueous solutions and at air/waterinterfaces.39 The 6 peptide was designed to approximate thisperiodicity with the exception of the three sequential hydrophobicresidues to minimize -sheet formation in solution. Two unnaturalpropargylglycine residues (X; Wuxi AppTec) contained reactive alkynegroups for a Huisgen cycloaddition (click) reaction, where the alkynegroups in the peptide react with the azide groups at the surface to formtriazole linkages using Cu(I) as a catalyst. Sodium ascorbate was usedto reduce Cu(II) to Cu(I), and TBTA {tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine} was present to stabilize the Cu(I) in the aqueoussolution environment.40 Solutions with a total volume of 5 mL wereprepared by adding 2:1 tert-butanol/HPW solvent, followed by TBTA(0.5 mol), 6 peptide (0.5 mol), sodium ascorbate (0.6 mol), theazide-terminated surface, and nally CuSO4 (0.1 mol). Beforeaddition to the reaction solution, the peptide was vortexed and thenstirred for 20 min in the 2:1 tert-butanol/HPW solvent atconcentrations below 1.3 mg/mL to ensure complete solubility.After addition of all reagents, vials were immediately placed in an ovenat 70 C for reaction times ranging from 15 min to 18 h. Uponremoval, samples were allowed to cool in the reaction solution for 10min, and then any physisorbed reactants were removed by sequentiallyrinsing with 2:1 t-butanol/HPW, HPW, phosphate-buered saline(PBS), HPW, and ethanol and then dried under a stream of N2(g).Dried samples were stored in the dark until measured. All controlsamples were azide-terminated surfaces that where immersed in thereaction solution lacking CuSO4 and its reducing agent, sodiumascorbate, to eliminate the presence of any Cu(I) catalyst.Grazing Incidence Angle ReectionAbsorption Infrared

Spectroscopy. Surface vibrational spectroscopy was collected with aBruker Vertex 70 FTIR spectrometer equipped with a A518/Qhorizontal reection unit (Bruker) for illuminating the sample at agrazing angle of 80 with respect to the surface normal. The samplechamber was purged with N2(g) for 1 h after samples were introducedto the instrument to reduce background noise from H2O and CO2. Atotal of 400 scans of p-polarized light were collected for each samplebefore and after reaction with the 6 peptide. A mercury cadmiumtelluride (MCT) detector collected signals from the 400-4000 cm1

region, while an indium antimonide (InSb) detector collected signalsfrom 1870 to 4000 cm1 to take advantage of its higher sensitivity tothe azide, methyl, and methylene stretches in that region. A bare goldsubstrate (cleaned and annealed as described above with each set ofsamples) was used as a common reference in order to obtain absolutedierences in absorbance between samples. Following backgroundsubtraction, the spectra were attened using a rubber band correctionbaseline function in the instruments OPUS software.Circular Dichroic Spectroscopy. The conformation of the 6

peptide in solution and on the surface was characterized using aJASCO J-815 CD spectrometer.12,41,42 Samples were illuminatedbetween 190 and 250 nm at a scan rate of 50 nm/min, 1 nmresolution, and a 4 s response time. For solution studies, a 130 Msolution of peptide dissolved in HPW in a 1 mm quartz cuvette wasused to collect three scans, using a HPW solution for backgroundsubtraction. Surface CD spectra were obtained by stacking three 6-reacted quartz slides in a 1 cm cuvette lled with HPW, orientednormal to and facing the UV source. Unreacted azide-terminatedquartz slides set up in the same manner were used for backgroundsubtraction. Due to poor UV transmission through the gold andrelatively low surface concentration of peptide compared to solution, alarge number of scans were acquired to establish the spectra. Scans ofthe background slides were taken until the spectra appeared to be

qualitatively unchanged (254 scans). For the 6-reacted surfaces, atotal of 410 scans were accumulated, and beyond backgroundsubtraction, no other data manipulation was done to the spectra.X-ray Photoelectron Spectroscopy. To further characterize the

chemical composition of the surfaces, X-ray photoelectron spectrawere collected with a Kratos Azis Ultra XPS equipped with amonochromatic Al K source illuminating the sample at 1486.5 eV.Samples were grounded using copper tape and introduced into thechamber at a pressure below 2 109 Torr. High-resolution spectra ofthe carbon 1s region were collected at a resolution of 0.1 eV with a 20eV pass lter. Ejected photoelectrons were collected with ahemispherical electron energy analyzer positioned normal to thesample surface. Due to sample charging from photoelectron loss, theadventitious carbon peaks were detected at a higher than normalbinding energy. To correct for this, the spectra were shifted toreposition the adventitious carbon peak to 284.5 eV. Spectral shiftsand decompositions were done using a linear baseline followed bypeak tting to Gaussian curves using the CasaXPS software package.Atomic Force Microscopy (AFM). An Asylum Research MFP-3D

AFM was used to image the dry, reacted, gold-on-mica substrates inambient air. AFM cantilevers (Mikromasch) with typical probe radii of8 nm, 65 kHz resonance frequencies, and 0.5 N/m force constantswere used in tapping mode to minimize shear forces and tipsampleinteractions. Height and width measurements were obtained byaveraging three line proles oriented normal to the long axis of eachbril. A total of 25 measurements for each reaction time point weretaken from images 1 1 m in size. All image processing and lineproles were done using the Gwyddion SPM software package.

RESULTSChemical Composition of Peptide-Reacted Surfaces.

GRAS-IR spectra were acquired for azide-terminated surfacesbefore and after reaction with the 6 peptide and for controlsamples. Representative spectra of the CH stretching regionfor the surfaces discussed here are shown in Figure 1. Azide-terminated surfaces (Figure 1, green) were dominated byfeatures at 2851 and 2924 cm1, corresponding to symmetricand asymmetric methylene stretches, respectively.11,43,44

Peptide-terminated surfaces reacted for 30 min (Figure 1,blue) showed an asymmetric methyl stretch at 2958 cm1, aswell as an increase in intensity of the methylene stretches dueto the presence of additional CH groups contributed by thesurface-bound peptides. The peptide-terminated surface reactedfor 18 h (Figure 1, red) also showed an increase in methylenepeak intensities along with the development of symmetric andasymmetric methyl peaks at 2870 and 2958 cm1. The reactedsurfaces were signicantly dierent from control surfacesprepared by immersing the azide-terminated SAM surface inthe Huisgen cycloaddition reaction solution without any Cu(I)catalyst (Figure 1, black). These surfaces displayed methylenestretches at 2857 and 2928 cm1, indicating preservation of theSAM, but except for a small methyl peak at 2962 cm1,corresponding to a small amount of physisorbed peptide, theydid not show any modication from immersion in the controlreaction solution.While the methyl region of the IR spectrum provides

evidence of the bound peptide on the surface, the amide region(14001800 cm1) provides additional information regardingthe secondary structure of the peptides bound to thesurface.11,4548 Figure 2 shows representative infrared spectraof the amide region containing peptide backbone stretches ofthe carbonyl group (amide I, centered around 1650 cm1) andstretching of the CN bond coupled with NH bending(amide II, near 1545 cm1). A spectrum of the azide-terminatedsurface before reaction (Figure 2, green) lacked any peaks in

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this region, and the control sample (Figure 2, black) showednegligible amide I and II signals. The 18 h reacted sample(Figure 2, red) displayed a single large amide II peak at 1541cm1 and two peaks in the amide I region at 1630 and 1695cm1. This splitting of the amide I peak is characteristic of -

sheets in an antiparallel conguration and arises from transitiondipole coupling of carbonyl groups along the peptidebackbone.45,46,4850 The less-intense peaks at 1666 and 1675cm1 were attributed to random or -turn conforma-tions.45,51,52 Similar to the 18 h reaction, the 30 min reactedsample (Figure 2, blue) also showed splitting of the amide Ipeak at 1637 and 1694 cm1 along with random and -turnabsorbances at 1662 and 1679 cm1. The signal-to-noise ratioof this sample is lower than the 18 h reaction due to lowpeptide surface coverage and represents the shortest reactiontime point that allows detection by IR spectroscopy.Overall, the increased methyl and methylene peaks, as well as

the presence of amide I and II stretches in the reacted samplespectra, provide evidence of both the presence and thestructure of surface-bound peptides. The splitting of theamide I peak indicates that the peptides are self-assembledinto a -sheet conformation in an antiparallel alignment. Theless intense stretches in the amide I region attributed torandom or -turn structures could arise either from peptidesadopting conformations that stabilize the -sheets or becausethe SAM surfaces were 100% functionalized in reactive azidegroups, and it was expected that some peptides could bind tothe surface in orientations that do not facilitate the formation oforganized -sheets. Spectra of the control samples showed onlyvery small increases in methyl/methylene signals and negligibleabsorbance in the amide region, indicating that peptides onlybind to the surface in the presence of the Cu(I) catalyst in thereaction solution. Collectively, the FTIR data demonstrate thepresence of surface-bound peptide in a largely antiparallel -sheet conformation, which requires the Huisgen Cu(I) catalystto form the triazole linkage that tethers the peptide to theunderlying SAM. In addition to the methyl, methylene, andamide regions, the azide peak (2103 cm1) of the SAMfunctionalized surfaces was also monitored to conrm SAMformation, but peak degradation was not used for quantitativeanalysis due to decomposition pathways unrelated to theHuisgen reaction.In addition to vibrational spectroscopy, XPS was also used to

conrm the presence of the surface-bound peptide. Figure 3shows representative spectra of the C 1s region from an 18 h6-reacted sample and an unreacted azide-terminated SAM,including component ts. The C 1s signal from the unreactedazide surface (Figure 3, bottom) showed peaks correspondingto CC (284.5 eV) and CN (285.8 eV) bonds, representingthe alkyl chains of the SAM as well as the CN bonds of theterminal azide groups. The 6-reacted surface (Figure 3, top)contained peaks arising from photoelectron emission from CC, C(N,O), and CO bonds near 284.5, 285.8, and 287.7eV, respectively.53 The development of the carbonyl peak aswell as the increase in detected C(N,O) photoelectrons onthe 6-reacted samples relative to the unreacted surface areconsistent with the presence of amide groups on the surface.These results indicate the successful attachment of peptides tothe gold surface and are consistent with previous XPS analysisof surface-associated peptides.4,41

Structural Characterization of Surface-Bound Pep-tides. CD spectra were collected to identify any dierences insecondary structure that the peptide adopts in solution and onthe surface. Figure 4 shows the measured ellipticity of the 6peptide in HPW solvent (red), with a negative band

unstructured.39,54,55 When the peptide was bound to thesurface, its CD spectrum changed dramatically. A small positiveabsorption was present at

with bril formation occurring in as little as 15 min. Theseresults are consistent with the hypothesis that brillation occursvia nucleation at peptide strands covalently immobilized to theunderlying SAM.

Height and width measurements of 25 brils from eachreaction time point were taken using line proles orientedperpendicular to the long axis of each bril from images 1 1m in size. Collectively, the brils had an average diameter of

Figure 5. Azide-terminated SAMs reacted with the 6 peptide for 15 min (A), 30 min (B), and 18 h (C) and an 18 h control sample that lacked theclick catalyst (D). All columns contain height (top), phase (middle), and line proles (bottom). Scale bars are all 400 nm, and green arrowscorrespond to the length and direction of the line proles.

Figure 6. Distribution of bril heights (left column) and widths (right column) for 15 min (top), 30 min (middle), and 18 h (bottom) reactions.Reaction time does not aect the height and width dimensions of brils, but each time point contains a range of bril sizes with averages of 24.9 1.6 nm in diameter and 2.83 0.74 nm in height.

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24.9 1.6 nm and an average height of 2.83 0.74 nm withlengths ranging from tens of nanometers to microns.Specically, bril heights for the 15 min, 30 min, and 18 hreactions were 2.26 0.68, 3.74 0.78, and 2.48 0.76 nm,respectively. Fibril widths were 25.5 1.6, 27.2 1.7, and 21.9 1.3 nm for the 15 min, 30 min, and 18 h reactions. On thebasis of the distribution of widths and heights, reaction timedoes not seem to have any bearing on these dimensions of thebrils (Figure 6), but bril length and surface density didincrease with extended reaction times.Immobilization of the Nucleating Peptide. As part of a

preliminary investigation of the nucleation mechanismassociated with the covalently bound brillar structures, wetested the reaction conditions with another short, -strandedpeptide that contained only one propargylglycine residue(61X, Wuxi AppTec). The 61X peptide preserved thesame alternating leucine/lysine sequence (KLKXKLLLK-LKLK) as the 6 peptide but contained only one reactivealkyne group that could covalently link the peptide to the azide-terminated SAM. It was determined that the 61X peptide wasbound to the surface and adopted an antiparallel -sheetconformation, as conrmed by an increase of methyl/methylene peaks (Figure S3, Supporting Information) as wellas amide I peak splitting (Figure S4, Supporting Information)through the use of GRAS-IR. Also, solution and surface CDexperiments showed that 61X adopted a random coilconguration in solution but forms -sheets when bound tothe surface (Figure S5, Supporting Information). Additionally,AFM experiments determined that the 61X peptide alsoformed brils of similar size and surface coverage as the 6peptide (Figure S6, Supporting Information). On the basis ofthis information, it was determined that the -sheets formed onthe azide-terminated surfaces may be nucleated by surface-bound peptides that are tethered at either one or both alkynegroups present in the 6 sequence. This is qualitativelyrepresented in Figure 7.The 61X experiments also provide insight into the question

of how the surface accelerates the formation of the brillarstructures on the surface. Two likely hypotheses for this include(1) the induced conformation of surface-bound peptides, whichwould allow subsequent addition of other peptides to self-

assemble into brils, or (2) an increase in local concentration ofpeptides at the surface that overcomes the critical concentrationnormally required for brillation to occur in solution. As the61X peptide is tethered at only one point, yet still forms brilson the surface, it is unlikely that its conformation is locked inan orientation that promotes additional self-assembly ofpeptides in the same manner as the 6 peptide. Therefore,the more likely hypothesis is that the reactive surface acts as anaggregator of peptides, creating a high density of immobilizedpeptides at which the further self-assembly of peptides intobrils can take place. In fact, brillation through increased localconcentration has been observed on lipid bilayers,57 hydrophilicmica,58 and at air/water interfaces.59

DISCUSSIONThis research represents an exciting rst step in our pursuit tocreate functional abiological structures from biological materialsbased on carefully controlled chemical inputs. This work aimsto covalently attach -stranded peptides to a SAM supportedon a gold surface, where the surface-bound peptides then act asnucleation sites for the formation of noncovalently assembledbrillar superstructures (Figure 7). Covalent immobilization ofthe peptide is accomplished through a Cu(I)-catalyzed Huisgencycloaddition reaction, where a triazole linkage is formed fromreacting one or both alkyne groups on the peptide withterminal azide groups on the SAM. GRAS-IR experimentsveried the presence of the surface-bound peptide bycomparing spectra of samples before and after reaction, aswell as controls. The vibrational spectra showed that 6-peptide-reacted samples contained a greater number of methyland methylene groups than both unreacted and control[lacking Cu(I) catalyst] samples. Additionally, absorption inthe amide region of the IR spectrum (absent in unreacted andcontrol samples) showed splitting of the amide I peak intostretches at 1630 and 1695 cm1, which arise from transitiondipole coupling of carbonyl groups and point to an antiparallelarrangement of peptides within -sheets found on the surface.Surface-bound peptides were also conrmed through XPSstudies that collected a higher intensity of C(N,O) and COphotoelectrons from reacted surfaces than unreacted surfaces.Secondary structure information obtained through solution andsurface CD experiments showed that the 6 peptide wasrandomly coiled in solution but adopted a -stranded structurewhen surface-bound. This indicates that the surface is notmerely a passive support but that its chemical reactivity acts as amedium that accelerates brillation. Morphology and hetero-geneity of the surfaces were characterized using AFM andshowed that peptide-reacted substrates develop surface-nucleated brillar structures in as little as 15 min of reactiontime. Extending reaction times to 30 min produced surfaceswith a higher degree of bril coverage, and 18 h reacted sampleswere densely covered in a combination of surface-nucleatedbrils as well as brillar structures formed in solution thatsubsequently interacted with the azide-terminated SAM.Control samples that were reacted for 18 h with no Cu(I)catalyst showed no uniform surface coverage of brils, and anybrils that did occur were rare and present only as appendagesof disordered oligomeric aggregates that likely formed insolution.On the basis of the lack of any uniform distribution of

brillar structures on control surfaces, we suggest thatchemically bonding the peptide to the surface acts as anucleation site that then induces the formation of brillar

Figure 7. Schematic representation of single peptides tethered to theSAM through triazole linkages at both (top) or one (bottom) alkyne-containing residue. Additional peptides proceed to self-assemble to thebound peptide through hydrogen bonding, forming the -sheets thatcomprise the brillar structures found on the reacted surfaces.

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structures through the addition of subsequent peptide strandsthat associate through hydrogen bonding. The resulting brilsare comprised of an antiparallel arrangement of noncovalentlyassociated peptide sheets. Peptides tethered to the surfacethrough one or both triazole linkages are able to orient in amanner that facilitates -sheet formation and catalyzesbrillation in as little as 15 min. Extending reaction times to30 min leads to a higher degree of surface coverage, dominatedby brillar structures of consistent height and width. At 45 min,brils begin to form in the reaction solution, whichsubsequently bond covalently to the azide-terminated SAM.This results in surfaces terminated by a combination of surface-nucleated and solution-formed brils. Conventionally, brilgrowth in solution is known to be concentration-dependent;however, others have noted that the presence of a surfaceallows for weakly adsorbed peptides to locally concentrate andpromote brillation in environments below the criticalconcentration for the formation of brils.60,61 The results ofthe 61X experiments indicate that brillation occurs whetherpeptides are bound at one or two locations to the surface,which supports the hypothesis that increased local concen-tration of peptides, rather than surface-imposed conformations,give rise to the surface-formed brils. This assembly hypothesisalso provides a kinetic explanation for why bril formation atthe surface occurs more rapidly than in solution, as theincreased surface concentration of peptides overcomes thecritical concentration required for brillation to occur. Oncebrils have begun forming, they may elongate through simplemonomer addition of non-surface-bound peptides and theymay also fragment, creating sites for secondary nucleation.33

The exact nucleation mechanism by which the peptides self-assemble is unclear at this point, but is likely inuenced bypeptide sequence and interactions with the solution/SAMinterface, as well as the solution environment. Previous studieshave shown how the conformation of immobilized peptides isinuenced by interactions between the tethering medium,unbound portions of the peptide, and interface chemistry, aswell as solution chemistry.6264 The method by which theparticular peptides discussed here nucleate and elongate toform brils is unknown, but studies are ongoing in ourlaboratory to investigate how this complex environmentdictates this surface-mediated self-assembly mechanism.The formation of these brillar structures on the surface is an

important step in understanding the factors that dictate the self-assembly of small biomolecules. Fibrillar structures arise fromstrands of -sheets interacting to form larger brils, whereindividual strands are oriented perpendicular to the long axis ofthe bril.19,21,33 Often, these brils are reported as havingdiameters ranging from 3 to 20 nm, although it has beendemonstrated that twisting of the brils can lead to theformation of helical ribbon structures that resemble nanotubes,which can have diameters up to 160 nm.3,30,37,65 It has beenshown that sequence symmetry may favor antiparallel overparallel sheet assembly of individual -strands, and examinationof the distribution of polar/nonpolar (P/N) binary patterns ofpentapeptide sequences found in the Protein Data Bank foundthat NPNPN and PNPNP have the highest relative frequencyamong antiparallel -strands.30,66 Therefore, the symmetric,alternating aliphatic sequence of the 6 peptide is known tohave a high propensity for orienting into antiparallel -sheetsthat proceed to self-assemble into structures within the sizerange of commonly characterized brils. Further character-ization of these brillar structures is ongoing in our lab and will

employ the use of electron microscopy and two-dimensionaldiraction experiments to determine how the antiparallel -sheets are organizing themselves into brillar forms.To summarize, this work characterizes the binding of short,

-stranded peptides to an azide-terminated SAM on a goldsurface through a Huisgen cycloaddition reaction. We havefound that the peptide orients itself in an antiparallelconguration within -sheets, as determined through splittingof the amide I peak in the vibrational spectrum as well assurface CD. While the peptide has a random coil conformationin solution, the surface induces the peptide to adopt a -stranded structure through covalent immobilization. Addition-ally, surface topography shows that the peptide self-assemblesinto brillar structures 24.9 1.6 nm in diameter and 2.83 0.74 nm in height. When subjected to longer reaction times (18h), surfaces with a higher density of brillar structures areproduced. Studies are ongoing in our laboratory to furthercharacterize the structure of these brils as well as to determinethe nucleation mechanism by which they form and continue toassemble on the surface.These reacted surfaces demonstrate the production of

supermolecular structures through the use of tunable variables,including peptide sequence, concentration, solution chemistry,and surface reactivity. This work provides a general platform forstudying and controlling the natural self-assembly processes ofbiomolecules at an interface, with potential applicability towardstudying how peptides and proteins aggregate or assemble intotheir respective thermodynamic low-energy conformations.Understanding the mechanisms by which this self-assemblyoccurs is a crucial step in the exploitation of these biomolecularsuperstructures in elds such as sensors, molecular electronics,and small molecule immobilization. This research provides aunique platform to study these factors and further develop theuse of biofunctionalized inorganic surfaces as practical devices.

ASSOCIATED CONTENT*S Supporting InformationAFM of bare gold and SAM-covered gold (Figure S1); AFM ofbrils formed in solution (Figure S2); and GRAS-IR, CD, andAFM of the 61X peptide studies (Figures S3S6). Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATIONCorresponding Author*E-mail: lwebb@cm.utexas.edu.NotesThe authors declare no competing nancial interest.

ACKNOWLEDGMENTSThe authors are grateful for funding from the NSF (Grant No.CHE-1361252), the Army Research Oce (Grant No.W911NF-10-1-0280), and the Norman Hackerman AdvancedResearch Program. L.J.W. holds a Career Award at the ScienticInterface from the Burroughs Wellcome Fund and is an AlfredP. Sloan Foundation Research Fellow. We thank the TexasInstitute for Drug and Diagnostic Development (TI3D)Automation Facility for use of their circular dichroicspectrometer, the Texas Materials Institute for the support ofthe clean room facility, the Center for Nano- and MolecularScience and Technology (CNM) for the support of the AsylumResearch AFM, and the National Science Foundation for

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funding the Kratos X-ray photoelectron spectrometer (GrantNo. 0618242).

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