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Higher-Order Complexity through R-Group Effects in Self-Assembled Tripeptide Monolayers† David M. Rampulla,‡ Nuri Oncel,§ Stuart A. Malcolm,‡ and Steven L. Bernasek*,‡ ‡
§
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, and Department of Physics, University of North Dakota, Grand Forks, North Dakota 58202 Received April 8, 2010. Revised Manuscript Received June 9, 2010
Self-assembled monolayers of tri-L-leucine and tri-L-valine formed on highly ordered pyrolytic graphite (HOPG) substrates have been examined using scanning tunneling microscopy. These monolayers exhibit markedly different structures, even though the tripeptides differ by only a minor change in the amino acid R-group. This minor change in R-group apparently affects the balance between hydrogen bonding and van der Waals interactions that control the monolayer structures. Implications of this effect for evolution of molecular complexity in prebiotic synthesis on environmental surfaces are discussed.
Introduction The adsorption of small biomolecules on graphite is an important tenet in one hypothesis that details the prebiotic synthesis of homochiral life, which suggests that interstellar dust (i.e., graphite) and UV radiation may have been responsible for the formation of enantiopure amino acids.1 Evidence from meteorites2 and laboratory experiments3 have provided some support for this hypothesis. As a material present during a potential prebiotic synthesis, it may be reasonable to suggest that graphite surfaces are important in the evolution of higher-order molecular complexity. Peptides and proteins acquire their inherent complex functionality by linking any of the 20 amino acids in chains of varying order and length, and with the large number of combinations the wide range of functional diversity among biomolecules is not surprising. Biological amino acid sequences are dictated by the arrangement of the bases of nucleotide chains in RNA molecules.4 Because the spontaneous appearance of the prototype RNA is statistically unlikely,5 the advent of molecular complexity in the absence of RNA remains puzzling. One potential route to coding amino acid sequences is through the use of a template comprising organic molecules self-assembled on a surface. To demonstrate a possible mechanism for the pre-RNA coding of amino acids, Sowerby et al. showed that monolayers of different nucleic acid bases were able to template graphite surfaces and chromatographically discriminate amino acids.6 Microscopy images also indicated that the bases assembled into distinct periodic overlayer structures, which likely contributed to amino acid differentiation. In this research, the self-assembly of two amino acid peptides, tri-L-valine and tri-L-leucine, on highly ordered pyrolytic graphite (HOPG) were studied using scanning tunneling microscopy (STM). Both peptides are trimers with nonpolar R-groups and comprise L-amino acids; in fact, the only difference between the peptides is the simplest possible change in the molecular structure of the
R-group. As seen in Figure 1, the R-group of trivaline is an isopropyl group and the R-group of trileucine is an isobutyl
† Part of the Molecular Surface Chemistry and Its Applications special issue.
(1) Engel, M. H.; Macko, S. A. Nature 1997, 389, 265–268. (2) Engel, M. H.; Nagy, B. Nature 1982, 296, 837–840. (3) Bernstein, M. P.; Dworkin, J. P.; Sandford, S. A.; Cooper, G. W.; Allamandola, L. J. Nature 2002, 416, 401–403. (4) Crick, F. H. C. Symp. Soc. Exp. Biol. 1958, 12, 138–163. (5) Shapiro, R. IUBMB Life 2000, 49, 173–176. (6) Sowerby, S. J.; Petersen, G. B.; Holm, N. G. Origins Life Evol. Biospheres 2002, 32, 35–46.
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Figure 1. Valine monomer (a) is HO2CCH(NH2)CH(CH3)2, and leucine monomer (b) is HO2CCH(NH2)CH2CH(CH3)2. Ball and stick models of (c) tri-L-valine and (d) tri-L-leucine. The only difference between the molecules is the addition of methylene groups (as indicated by arrows). Red=oxygen, blue=nitrogen, gray = carbon, white = hydrogen.
Published on Web 07/06/2010
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Figure 2. (a) STM image of tri-L-valine monolayer adsorbed on HOPG. The scan dimensions are 14.5 nm 14.5 nm. The bias voltage is 0.75 V, and the tunneling current is 0.5 nA. The region within the parallelogram indicates a structure that resembles CH2 groups of close packed hydrocarbon tails of octanoic acid. Line-1 and Line-2 indicate the regions where the line scans presented in (b) are measured.
Figure 3. (a) STM image of tri-L-valine monolayer adsorbed on HOPG. The scan dimensions are 6.9 nm 6.9 nm. The bias voltage is 0.75 V, and the tunneling current is 0.5 nA. A model of tri-L-valine peptide dimers and octanoic acid molecules adsorbed on HOPG is overlaid on the STM image. Hydrogen bonding between peptides stabilizes the dimers. Octanoic acid molecules are stabilized on the surface via hydrogen bonding between the amine groups of the trivaline peptides and the carboxyl groups of octanoic acid, and by van der Waals interaction between hydrocarbon tails of neighboring octanoic acid molecules. (b) Detailed image of the proposed monolayer structure.
group. This research describes a significant change in the morphology of the self-assembled monolayer structures of the two chemically similar yet subtly different peptide trimers.
Experimental Section The trivaline and trileucine peptides are from Bachem (>98% purity). Each peptide was dissolved in octanoic acid (g99.5% purity) from Aldrich, until the solution was completely saturated. The HOPG substrate is from Nanoscience Instruments. STM tips are fabricated through mechanical cutting of 0.25 mm diameter Pt/Ir wire (Pt/Ir = 80/20) from Nanoscience Instruments. A drop of each solution was deposited on freshly cleaved HOPG, and the STM images were measured at the solid/liquid interface under ambient conditions. STM images were obtained using a Nanosurf AG EasyScan STM system (NanoScience Instruments, Liestal, Switzerland). Images were obtained in constant current mode and under various tunneling conditions (Vb = 0.7 - 1.2 V, It = 0.31.0 nA).
Results Figures 2a and 4a are the STM images of trivaline and trileucine self-assembled monolayers (SAMs) formed on HOPG. The common feature of both STM images is the presence of bands or lamella of adsorbed molecules. Although the detailed morphologies of the lamella are different in the two images, they are considered to be formed by the respective tripeptides. Because of the higher tunnel16288 DOI: 10.1021/la101413e
ing rates through the amine and carboxyl groups on tripeptide molecules, these features appear as topographical protrusions. The lamella repeat distance measured on the trivaline SAM is about 3 nm, and the width of the bands (as seen in the line profile indicated by Line-1) is about 2 nm. This is approximately 2 times the theoretically predicted length of an individual trivaline molecule. This suggests that the bands are formed by dimerized trivalines. The troughs between peptide bands are about 1 nm in width. These troughs are too wide to result from band-to-band hydrogen bonding, and they are not consistent with moleculemolecule van der Waals interactions. In addition, the structure indicated by the parallelogram in Figure 2a resembles close packed CH2 groups7 in hydrocarbon monolayers and suggests that the solvent, octanoic acid, may be coadsorbed with trivaline. A hydrogen bonded octanoic acid lamella will occupy a length of ∼1 nm. The hydrogen bond between the carboxyl groups of trivaline and octanoic acid, and the van der Waals interaction between hydrocarbon tails of adjacent octanoic acid molecules makes this configuration rather stable. Figure 3b shows a suggested structural model based on the distances derived from the STM image shown in Figure 2a, overlaid on a smaller area STM scan of the monolayer (Figure 3a). (7) Claypool, C. L.; Faglioni, F.; Goddard, W. A.; Gray, H. B.; Lewis, N. S.; Marcus, R. A. J. Phys. Chem. B 1997, 101, 5978–5995.
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Figure 4. (a) STM image (5.8 nm 5.8 nm) of a monolayer of tri-L-leucine molecules adsorbed on HOPG. The bias voltage is 0.75 V, and the tunneling current is 0.4 nA. Line-1 and Line-2 indicate the regions where the line scans presented in (b) are measured. The average peak to peak distance in Line-2 is 0.34 ( 0.03 nm. To determine the center of each peak, the line scan is fit to a line containing 10 Gaussian peaks. The center of each Gaussian peak is assigned as the center of each peak.
Figure 5. (a) STM image (2.7 nm 2.7 nm) of a monolayer of tri-L-leucine molecules adsorbed on HOPG. The bias voltage is 0.75 V, and the tunneling current is 0.4 nA. A model of the tri-L-leucine chain is overlaid on the STM image. (b) Side and top view of the tri-L-leucine chains that illustrates the proposed face-to-face packing.
On the other hand, the lamellar repeat distance measured (from the Line-1 profile) on the trileucine SAM is 1.0 nm which is about the same as the length of a single trileucine peptide, suggesting that the band is formed by single trileucine molecules in adjacent bands bound together by hydrogen bonding between terminal amine groups and carboxyl groups of the peptide molecules. Figure 4b shows line scans taken along and across the lamellar directions as indicated by the arrows in Figure 4a. The repeat distance along the band (Line-2 profile) is 0.34 ( 0.03 nm, which is significantly smaller than 0.7 nm, the theoretically predicted width of the trivaline peptide. It is difficult to extract detailed topographical information confirming that the trileucine molecules form the band from these STM images. However, based on the repeat distances measured in these images, it is possible to suggest that individual molecules pack face-to-face and are positioned slightly off the surface plane as indicated in the proposed monolayer structural model shown in Figure 5. In the lamellar self-assembled monolayers observed for linear alkanes adsorbed on HOPG, the addition of a methylene group merely increases the width of the lamellae, and the lattice packing usually remains identical.7-10 Packing of branched alkanes, however, yields entirely different results. Tetracosane (n-C24H50) (8) Liang, W.; Whangbo, M. H.; Wawkuschewski, A.; Cantow, H. J.; Magonov, S. N. Adv. Mater. 1993, 5, 817–821. (9) McGonigal, G. C.; Bernhardt, R. H.; Thomson, D. J. Appl. Phys. Lett. 1990, 57, 28–30. (10) Rabe, J. P.; Buchholz, S. Science 1991, 253, 424–427.
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adsorbed on HOPG forms well-ordered lamellae, while the branched molecule, squalane (2,6,10,15,19,23-hexamethyl-tetracosane), forms no discernible overlayer.11 Self-assembly is widely recognized to occur due to a balance of attractive and repulsive forces between the molecules within the overlayer and between the overlayer and the surface,12-14 and with the addition of sidechain methyl groups on linear alkanes the balance is sufficiently disrupted to drive the tetracosane/squalane system to disorder. With peptides, there is the added stabilizing force of hydrogen bonding between molecules in the overlayer. Thus, the additional branching seen in trileucine versus trivaline is capable of distorting the packing, yet is apparently insufficient to entirely disrupt the packing. Previous research has shown that proteins comprising many peptides denature and self-assemble on HOPG,15 so it is likely that even increasingly complex peptides retain a sufficient degree of hydrogen bonding to laterally organize.
Conclusions Examination of the monolayer structures formed by the two closely related tripeptides tri-L-valine and tri-L-leucine adsorbed (11) Fuhrmann, D.; Graham, A. P.; Criswell, L.; Mo, H.; Matthies, B.; Herwig, K. W.; Taub, H. Surf. Sci. 2001, 482-485, 77–82. (12) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437–463. (13) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103–1169. (14) Schreiber, F. Prog. Surf. Sci. 2000, 65, 151–256. (15) Svaldo-Lanero, T.; Penco, A.; Prato, M.; Canepa, M.; Rolandi, R.; Cavalleri, O. Soft Matter 2008, 4, 965–967.
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on HOPG indicates that the monolayer morphology is strongly affected by the size of the amino acid R-group in the tripeptide. Both tripeptides form lamellar structures on the surface, with trivaline showing clear evidence of octanoic acid solvent incorporation into the structure of dimerized tripeptides. The trileucine structure does not incorporate solvent, and it forms a lamellar structure composed entirely of individual tripeptide molecules. The presence of a slightly larger R-group as compared to trivaline likely impairs the ability of the trileucine peptides to hydrogen bond between amide groups to a significant extent. In this case, there are fewer interactions working to extend the molecules as is seen in the trivaline system. These structural results indicate that
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two drastically different peptide overlayer morphologies can be expressed on graphite substrates by making only the slightest structural change to the constituent molecules. A modest change in constituent molecular structure results in higher order complexity in the assembled overlayer. Acknowledgment. This research was partially supported by the National Science Foundation, CHE-0910549. Supporting Information Available: Two large scale STM images of trivaline and trileucine monolayers. This material is available free of charge via the Internet at http://pubs.acs.org.
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