Hydration and Conformational Mechanics of Single, End-Tethered Elastin-like Polypeptides Alexei Valiaev,†,§ Dong Woo Lim,‡,§ Scott Schmidler,| Robert L. Clark,†,§ Ashutosh Chilkoti,‡,§ and Stefan Zauscher*,†,§ Department of Mechanical Engineering and Materials Science, Department of Biomedical Engineering, Center for Biologically Inspired Materials and Materials Systems, and Institute of Statistics and Decision Sciences, Duke UniVersity, Durham, North Carolina 27708 Received January 21, 2008; E-mail: [email protected] Abstract: We investigated the effect of temperature, ionic strength, solvent polarity, and type of guest residue on the force-extension behavior of single, end-tethered elastin-like polypeptides (ELPs), using single molecule force spectroscopy (SMFS). ELPs are stimulus-responsive polypeptides that contain repeats of the five amino acids Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. We fitted the force-extension data with a freely jointed chain (FJC) model which allowed us to resolve small differences in the effective Kuhn segment length distributions that largely arise from differences in the hydrophobic hydration behavior of ELP. Our results agree qualitatively with predictions from recent molecular dynamics simulations and demonstrate that hydrophobic hydration modulates the molecular elasticity for ELPs. Furthermore, our results show that SMFS, when combined with our approach for data analysis, can be used to study the subtleties of polypeptide-water interactions and thus provides a basis for the study of hydrophobic hydration in intrinsically unstructured biomacromolecules. Introduction peptides (ELPs) at the single molecule level. ELPs are stimuli- responsive polypeptides26 that consist of repeats of the pen- Hydration is essential for proteins to maintain a functional, tapeptide sequence Val-Pro-Gly-X-Gly (VPGXG) (X is any three-dimensional structure and thus to maintain biological activity. Protein conformation and flexibility is thus intimately linked to the hydration water structure, in which the hydrogen- (2) Baldwin, R. L. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 8069–8072. (3) Kita, Y.; Arakawa, T.; Lin, T. Y.; Timasheff, S. N. Biochemistry 1994, bonded hydration water network transmits information around 33, 15178–15189. the protein and controls its dynamics.1 The interaction of water (4) Lee, B. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 5154–5158. with hydrophobic protein surfaces (hydrophobic hydration) is (5) Privalov, P. L.; Gill, S. J.; Murphy, K. P. Science 1990, 250, 298– 299. typically manifest in a change in the clustering of water in the (6) Ooi, T.; Oobatake, M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2859– vicinity of the hydrophobic solute surface rather than in changes 2863. in water-solute interactions. Hydrophobic hydration generally (7) Spolar, R. S.; Ha, J. H.; Record, M. T. Proc. Natl. Acad. Sci. U.S.A. produces a reduction in water density and an increase in heat 1989, 86, 8382–8385. (8) Timasheff, S. N. Annu. ReV. Biophys. Biomol. Struct. 1993, 22, 67– capacity, both of which are consequences of more ordering in 97. the solvent which also causes a negative entropy change. The (9) Connelly, P. R.; Thomson, J. A. Proc. Natl. Acad. Sci. U.S.A. 1992, interaction of water with hydrophobic groups is important in 89, 4781–4785. 2–8 (10) Connelly, P. R.; Thomson, J. A.; Fitzgibbon, M. J.; Bruzzese, F. J. protein folding/unfolding and intra and intermolecular Biochemistry 1993, 32, 5583–5590 9–11 . interactions and profoundly affects the mechanochemical (11) Timasheff, S. N. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 9721–9726. properties of biomacromolecules.12–14 While computer simula- (12) Weis-Fogh, T.; Anderson, S. Nature 1970, 227, 718–721. tions15–20 provide insights into temperature,21 size,17,22 and (13) Li, B.; Alonso, D. O. V.; Daggett, V. J. Mol. Biol. 2001, 305, 581– 23–25 592. cosolvent effects on hydrophobic hydration, experimental (14) Li, B.; Alonso, D. O. V.; Bennion, B. J.; Daggett, V. J. Am. Chem. studies that address the mechanical consequences of changes Soc. 2001, 123, 11991–11998. in hydrophobic hydration on the molecular level are largely (15) Silverstein, K. A. T.; Haymet, A. D. J.; Dill, K. A. J. Am. Chem. Soc. 2000, 122, 8037–8041. missing. (16) Urbic, T.; Vlachy, V.; Dill, K. A. J. Phys. Chem. B 2006, 110, 4963– Our research is motivated by the desire to understand the 4970. mechanochemical and hydration properties of elastin-like poly- (17) Southall, N. T.; Dill, K. A. J. Phys. Chem. B 2000, 104, 1326–1331. (18) Xu, H. F.; Dill, K. A. J. Phys. Chem. B 2005, 109, 23611–23617. (19) Silverstein, K. A. T.; Dill, K. A.; Haymet, A. D. J. J. Chem. Phys. † Department of Mechanical Engineering and Materials Science. 2001, 114, 6303–6314. § Center for Biologically Inspired Materials and Materials Systems. (20) Chorny, I.; Dill, K. A.; Jacobson, M. P.; Xu, H. F. Abstr. Pap. Am. ‡ Department of Biomedical Engineering. Chem. Soc. 2004, 227, U1010–U1010. | Institute of Statistics and Decision Sciences. (21) Huang, D. M.; Chandler, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, (1) Nakasako, M. Philos. Trans. R. Soc. London, B 2004, 359, 1191– 8324–8327. 1204. (22) Southall, N. T.; Dill, K. A. J. Phys. Chem. B 2001, 105, 2082–2083. 10.1021/ja800502h CCC: $40.75 XXXX American Chemical Society J. AM. CHEM. SOC. XXXX, xxx, 000 9 A Published on Web 07/23/2008 ARTICLES Valiaev et al. amino acid except Pro),27,28 and undergo, in solution, a lower in the configurational entropy of elastin chains, and (ii) by critical solution temperature (LCST) transition. Below their changes in the interfacial free energy associated with hydro- LCST, ELPs are soluble in water, but when the temperature is phobic hydration of elastin side groups upon extension. The raised above their LCST, a sharp phase transition occurs that latter model is supported by recent molecular dynamics (MD) leads to desolvation and aggregation of the polypeptides. simulations of elastin with explicit water.14 These simulations Changes in the ambient temperature, ionic strength, or pH can showed that an ELP molecule in water behaves more as an trigger this reversible, inverse transition.28 ELPs are attractive amorphous polymer chain, with only local secondary structure.28 for different applications that require molecular level control Furthermore, these simulations showed that interaction of water of polymer structure because they are genetically encodable and with hydrophobic side chains of ELP modulates the elasticity can be synthesized easily by heterologous overexpression from of the ELP molecule significantly. a synthetic gene, with precise control over their composition 26,29 Single molecule force spectroscopy (SMFS) by AFM and and chain length. Furthermore, the LCST of ELPs can be optical tweezers have enabled the study and manipulation of - ° tuned precisely to a temperature of interest between 0 100 C, single macromolecules attached to surfaces.40–44 SMFS experi- which is difficult to achieve with other synthetic polymers that ments with globular proteins, DNA, and synthetic macromol- exhibit LCST behavior. ecules have largely focused on the analysis of molecular We previously showed that ELPs that are chemically grafted “fingerprints” that arise from force-induced changes in secondary onto self-assembled monolayers (SAMs) on gold, exhibit an and tertiary structures40,45–48 and changes in intrachain hydrogen interfacial hydrophilic-hydrophobic phase transition in response bonding.44,49 Nevertheless, systematic experiments on the single to increased temperature or ionic strength.30–32 Understanding molecule level that link differences in the elastic behavior of the mechanochemical properties of ELPs is important because polypeptides to changes in their hydration behavior are missing. ELPs can be exploited for force generation33 and as molecular switches with tunable properties34 and can provide the building Force-induced molecular stretching of polypeptides in SMFS blocks for engineered protein elastomers.26 experiments causes a change in the equilibrium conformation Although waters of hydrophobic hydration are thought to play of the macromolecule and potentially increases its solvent- an important role in the elasticity of elastin,14,35 details of their accessible surface area by exposing previously solvent-inac- conformational mechanics on the molecular level remain largely cessible regions. Especially for hydrophobic polypeptides, such unexplored.36,37 Three main models have been proposed to as ELPs, molecular-level stretching likely leads to hydrophobic explain elastin’s elasticity: (1) The model by Flory and Hoeve38 hydration of the molecule as increasingly more hydrophobic 13 considers elastin to behave like a random chain polymer with surface is exposed. We argue that the force required to stretch entropic (configurational) elasticity. While this model can a single ELP molecule in an aqueous solvent thus primarily explain the high mobility of the elastin backbone, it neglects reflects two components: (1) the restoring force that arises from the details of solvent-polypeptide interactions. These are, the stretch-induced entropic elasticity of the polypeptide back- however, important, as elastin only shows elasticity in the bone, and (2) the force that arises from changes in the presence of solvents.14 (2) An alternative model, proposed by solvent-polypeptide interactions. The polypeptide conformation Urry,28,39 suggests that elasticity arises