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Nature’s Complex Copolymers: Engineering Design of Oligopeptide Materials Michael R. Caplan† and Douglas A. Lauffenburger*,†,‡ Department of Chemical Engineering and Division of Bioengineering & Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Proteins evolved in nature can form precise structures exhibiting nanometer-length-scale features that have great potential for industrial application. However, many of these applications remain unrealized because of limitations in combinatorial and directed evolutionary approaches. An alternative approach that might be able to overcome these limitations is rational design of protein (or oligopeptide in the case of 7). KFE12 does indeed gel as the pH is increased past ∼5 and ceases to form a gel as the pH is increased past ∼10. Because DLVO theory mathematically superimposes hydrophobic attraction and electrostatic repulsion by summing a term describing each, it seems likely (at least in the case of KFE12) that they key to structural specificity is electrostatic repulsion destabilizing nonpreferred structures. 5. Rational Design
Figure 5. Specificity in coiled-coils produced by electrostatic interactions: (a) diagramatic representation looking end on at a tetrameric arrangement with two coils antiparallel is formed when the e and g positions are relatively hydrophobic and the b and c positions interact favorably;46 (b) exclusively parallel heterodimers form when one monomer has all e- and g-position lysines and the other all e- and g-position glutamic acids, in addition to each having a specifically placed a-position asparagine;47 (c) trimers with one antiparallel helix forms, despite g-g and e-e position repulsion, to avoid having N-terminal tryptophans sterically overlap48
solution conditions. We hypothesized that, if DLVO theory could adequately predict the behavior of KFE12, electrostatics acted by destabilization, so we tested several predictions to test whether this theory alone could predict when KFE12 would and would not assemble. As described above, DLVO theory quantitatively describes the activation barrier to assembly due to electrostatic repulsion using a function of the potential energy vs distance between two approaching colloids.57 Only if the colloids gain enough energy to overcome the
The purpose of discussing all of these principles of protein folding and specificity of supermolecular structure is with the eventual aim of rationally designing oligopeptide materials. We have discussed how primary sequence can be varied to incorporate side chains of varied hydrophobicities; how R-helices and β-sheets can be designed to produce self-assembling peptide amphiphiles; and finally, how specificity of conditions for assembly was linked to electrostatic interactions. As we stated in the Introduction, rational design requires indepth knowledge about how the amino acid sequence relates to protein structure and ultimately function. In this section, we detail a few examples of how the current understanding of these phenomena have been used to rationally design specific sequences for specific applications in the chemical and biochemical industries. As a first example showing how hydrophobicity alone can be used in rational design, Meyer et al. recombinantly produced protein hybrids with the elastin-like pentapeptide repeat attached.59 The goal was to develop a hybrid that would phase separate from aqueous solution with increasing temperature so that purifica-
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tion could be achieved merely by centrifugation. Starting with the sequence studied by Urry, Meyer et al.59 varied several of the valine side chains to achieve a spectrum of Tt values for different hybrids. Because the hybrids were produced in cells incubated at 37 °C, the molecule’s Tt value needed to be greater than 37 °C so that it would remain soluble upon production. To optimize the amount of energy required for purification, though, Meyer et al. aimed for a Tt slightly above 37 °C. Thus, the repeat unit valine-proline-glycine-X-glycine, with a 5:2:3 ratio of valine:alanine:glycine and replacement of the X-position amino acids, was chosen for further testing because it has a predicted Tt ≈ 40 °C. Other applications include localizing chemotherapy agents to tumors, which have a tendency to be slightly warmer than body temperature. Body temperature is 37 °C, so injecting a hybrid pentapeptide chemotherapy drug with Tt ≈ 38 or 39 °C would cause the hybrid to circulate until it passed through the tumor, at which time it would aggregate and precipitate. Revisiting the Petka et al. triblock protein, we notice that the intention was to create a pH-sensitive gel. This was achieved by predominantly filling the e and g positions in the heptad repeat with negatively charged glutamic acid side chains. Remember that, in a dimer coiled-coil, the e and g positions flank the hydrophobic patch created by the a and d side chains, so the e- and g-position side chains on one R-helix will be in close proximity to the e- and g-position side chains on the other R-helix, as shown in Figure 5b.16 Thus, in the case of Petka et al., when glutamic acids are paired in e and g positions, electrostatic repulsion will make assembly into coiled-coils less stable at high pH. However, as the pH is lowered below ∼6, the glutamic acid side chains begin to become protonated, and the R-helices form coiled-coils. The sequence used had several repeats where e was glutamic acid and g was lysine that either stabilize or do not destabilize the structure at pH ≈ 7. This allowed the coiled-coils to form near pH 8 as the solution was cooled to room temperature, thus achieving a thermoreversible as well as a pH-responsive hydrogel. The previous example is a good model for a material that will undergo an in situ transformation from a soluble liquid to a gel (or solid material). This is a desirable feature for use in minimally invasive medicine because agents or material to be delivered to a specific location can be injected through a miniscule needle rather than requiring a large incision to be made.60 The materials developed by Meyer et al. and Petka et al. are useful if the tissues near the implantation site (along with anything to be encapsulated) are not temperatureor pH-sensitive, but this is not always the case. In the case of encapsulating cells, for example, it is desirable to mix the cells with the material in its soluble form at pH ∼7 and 37 °C. This can also be desirable with some protein therapeutics such as growth factors that are pHor temperature-sensitive. The EAK family, because salt allows the sol-to-gel transition, has the potential for such behavior; however, we noted earlier that KFE12 gels at pH ∼7 even in the absence of exogenous salt. Now, though, we understand that this behavior is due to the molecule becoming net neutrally charged because both the glutamic acid and lysine side chains are charged at pH ∼7. Thus, we rationally changed the glutamic side chains to glutamine side chains that do not become charged. As predicted, the new molecule, FKFQFKFQFKFQ (KFQ12), is not
a gel at pH ∼7 without added salt because the molecule still has a net positive charge because of the presence of lysine side chains but absence of negatively charged glutamic acid side chains.61 It should and does gel above pH ∼10 because the lysine side chains lose their charge and KFQ12 becomes neutral. For the application, though, this is not a problem because now we can mix cells or proteins with KFQ12 at pH ∼7 without the oligopeptide gelling so the solution can undergo an in situ transformation upon injection and diffusion of physiologic saline (150 mM NaCl) into the volume occupied by the KFQ12 solution. After equilibration, a gel of KFQ12 fills the volume with any cells or drugs that have been encapsulated inside the gel. 6. Conclusions Simple repeating structures have been studied to gain an understanding of how 2°, 3°, and 4° structures form in protein chains. Pentapeptide repeat units based on elastin yielded a useful hydrophobicity scale for amino acid side chains. Heptad repeats based on leucine zippers revealed how to construct R-helices that combine together to form coiled-coils by pairing a- and d-position hydrophobic side chains. Hydrophobic-charged/polarhydrophobic-charged/polar repeats from several sources demonstrated how to design β-sheets that assemble by burying their hydrophobic faces inside filaments. From these discussions, we have established that the primary driving force for assembly is the energetic favorability for removing hydrophobic amino acid side chains from water (analogous to formation of surfactants and subsequent assembly into micelles), but that is only the first step toward structural specificity. Another key is electrostatic repulsion, which seems to destabilize structures that would place like charges in close proximity or that would bury them in hydrophobic regions without another polar or oppositely charged partner. All of these principles have been mirrored by a discussion of how they are analogous to surfactants and multiblock copolymer structures to show that chemical engineering and polymer science theories do apply; they just need to be extended to predict the behavior of more complex copolymer systems such as proteins. To further motivate this extension of copolymer research into proteins, we have shown a few examples of how currently existing knowledge has been used to achieve rational design of some simple protein materials. Pentapeptide repeats have been recombinantly attached to therapeutic proteins in order to purify them by heating and centrifugation and localize them at tumor sites after injection into the body. Heptad repeats have been joined by a random coil sequence to form an A-B-A structure in which the A blocks form coiled-coils, causing gelation, at neutral pH when cooled or at ∼37 °C when the pH is decreased from alkaline. Finally, we show how a hydrophobic-charged/polar-hydrophobic-charged/polar repeat was rationally designed to undergo a transition from soluble at pH ∼7 to gel at pH ∼7 with only the addition of physiologically normal salt solution. Acknowledgment We are extremely pleased to offer our paper for this special issue in honor of Professor John A. Quinn, who has truly been a pioneer in the application of chemical engineering principles to both fundamental understanding and useful technology in biology and medicine. His
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teaching and research career has admirably exemplified the value we perceive in employing a chemical engineering perspective to help solve biomolecular problems, and, for one of us (D.A.L.), John served as a singular mentor in the development of our nascent academic career. Acknowledgment We thank Elissa Schwartzfarb, Peter Moore, Shuguang Zhang, Roger Kamm, Davide Marini, Alan Grodzinsky, and John Kisiday for helpful discussions. The Whitaker Foundation provided a graduate fellowship for M.R.C., and funding support for this work came from NIH Grant GM55781 to D.A.L. Literature Cited (1) Xenopoulos, A.; Wunderlich, B.; Subirana, J. A. Thermal Properties of Nylons Related to Polyglycine. Eur. Polym. J. 1993, 29, 927-935. (2) Shanklin, J. Exploring the Possibilities Presented by Protein Engineering. Curr. Opin. Plant Biol. 2000, 3, 243-248. (3) Petrounia, I. P.; Arnold, F. H. Designed Evolution of Enzymatic Properties. Curr. Opin. Biotechnol. 2000, 11, 325-330. (4) Branden, C.; Tooze, J. Introduction to Protein Folding; Garland Publishing: New York, 1991: pp 15-17, 99-104, 251252. (5) Gordon, D. B.; Marshall, S. A.; Mayo, S. L. Energy Functions for Protein Design. Curr. Opin. Struct. Biol. 1999, 9, 509-513. (6) Osguthorpe, D. J. Ab Initio Protein Folding. Curr. Opin. Struct. Biol. 2000, 10, 146-152. (7) Bull, H. B.; Breese, K. Surface Tension of Amino Acid Solutions: A Hydrophobicity Scale of Amino Acid Residues. Arch. Biochem. Biophys. 1974, 161, 665-670. (8) Vorob’ev, M. M. Hydrophobic Scale of Amino Acids as Determined by Absorption Millimeter Spectroscopy with Heat Capacities of Aqueous Solutions. Z. Naturforsch. 1997, 52c, 227234. (9) Nozaki, Y.; Tanford, C. The Solubility of Amino Acids and Two Glycine Peptides in Aqueous Ethanol and Dioxane Solutions. J. Biol. Chem. 1971, 246, 2211-2217. (10) Rose, G. D.; Geselowitz, A. R.; Lesser, G. J.; Lee, R. H.; Zehfus, M. H. Hydrophobicity of Amino Acid Residues in Globular Proteins. Science 1985, 229, 834-838. (11) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; John Wiley & Sons: New York, 1973. (12) Silver, F. H.; Christiansen, D. L. Biomaterials Science and Biocompatibility; Springer: New York, 1999: p 10. (13) Creighton, T. E. Proteins: Structure and Molecular Properties, 2nd ed.; W. H. Freeman and Company: New York, 1993: pp 142-148, 223. (14) Urry, D. W. Physical Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers. J. Phys. Chem. B 1997, 101, 11007-11028. (15) Jelesarov, I.; Durr, E.; Thomas, R. M.; Bosshard, H. R. Salt Effects on Hydrophobic Interactions and Charge Screening in the Folding of a Negatively Charged Peptide to a Coiled Coil (Leucine Zipper). Biochemistry 1998, 37, 7539-7550. (16) Cohen, C.; Parry, D. A. D. R-Helical Coiled Coils and Bundles: How to Design an R-Helical Protein. Proteins: Struct., Funct., Genet. 1990, 7, 1-15. (17) Lazo, N. D.; Downing, D. T. Stabilization of Amphipathic R-Helical and β-Helical Conformations in Synthetic Peptides in the Presence and Absence of Ionic Interactions. J. Pept. Res. 1998, 51, 85-89. (18) Harbury, P. B.; Plecs, J. J.; Tidor, B.; Alber, T.; Kim, P. S. High-Resolution Protein Design with Backbone Freedom. Science 1998, 282, 1462-1467. (19) Kortemme, T.; Ramirez-Alvarado, M.; Serrano, L. Design of a 20-Amino Acid, Three-Stranded β-sheet Protein. Science 1998, 281, 253-256. (20) Choo, D. W.; Schneider, J. P.; Graciani, N. R.; Kelly, J. W. Nucleated Antiparallel β-Sheet that Folds and Undergoes Self-
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Received for review February 14, 2001 Revised manuscript received May 30, 2001 Accepted June 4, 2001 IE010149Z
