Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

Abstract

•Single-molecule force spectroscopy probes the mechanics of 1-nm-size synthetic helices•Outstanding elasticity and rewinding capabilities of aromatic amide helices•Ultrafast nondissipative winding along precise trajectories•Reducing molecular size does not compromise mechanical properties Chemistry excels in designing and controlling molecular structures. Beyond structure, programming molecular dynamics is one of the next grand challenges. The conformation dynamics of natural elastomeric proteins, for example, are responsible for their ability to behave either as pure elastic spring-like molecules that can be reversibly stretched or as shock absorbers that dissipate energy. Here, we report the outstanding mechanical performances of synthetic helical molecules much smaller than proteins. The molecules were designed for interfacing with AFM force spectroscopy to study them one at a time, a true performance for such tiny objects. We show that the elastic response of helices as small as 1 nm is among the fastest and the most robust ever described. This unprecedented elastic behavior suggests that various applications may arise from their use as building blocks in molecular machines or in new classes of artificial elastomeric materials with tailored mechanical properties. Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable. Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable. Folding is the process by which the three-dimensional shapes and properties of proteins are generated from linear peptide chains. Folding is reversible and allows for some important dynamics to take place: molecular shape changes determine mechanical and motor functions in living systems. For example, reversible conformational changes in titin and elastin have been associated with tissue elasticity.1Labeit D. Watanabe K. Witt C. Fujita H. Wu Y. Lahmers S. Funck T. Labeit S. Granzier H. 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The molecular helices behaved as springs that are energy loaded in their extended states. A detailed analysis, based, among others, on elements of stochastic calculus, allowed us to decipher precise winding/unwinding trajectories at the sub-molecular level and cooperative effects responsible for the observed elasticity. The analysis shows that at short times both winding and unwinding processes are driven by the same stochastic mechanisms. It also shows that at longer times a differentiation of mechanisms occurs. Such well-defined conformational trajectories open up new capabilities to orchestrate motion at the molecular scale. Oligoamides of 8-amino-2-quinolinecarboxylic acid Q (Figure 1C) were selected because their structures are well defined, and because their rigidity hinted at simple conformational dynamics. Qn oligomers adopt defectless helical conformations comprised of 2.5 quinoline units per turn that show high stability in a wide range of solvents.33Jiang H. Léger J.M. 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Soc. 2016; 138: 13568-13578Crossref PubMed Scopus (47) Google Scholar For this study, four oligomers comprised of 5, 9, 17, and 33 units were prepared (Figures 1C, S1, and S17–S32 and Supplemental experimental procedures). Monomers are δ- or ε-amino acids and equivalent in size to a dipeptide. Thus, even the longest sequence, Q33, is much smaller than proteins investigated so far by single-molecule force spectroscopy. All sequences bear a thiol group at their N terminus to ensure stable attachment to gold substrates, and polyethylene oxide (PEO) at the C terminus to be linked to the AFM tip for pulling. The molecules were grafted onto gold/silicon substrates at low grafting density to ensure large interspaces between them (Figure 1B, See Supplemental information for details). The AFM tip was brought into contact with the oligoamide-PEO substrate in N,N-dimethylformamide (DMF) to allow the linker to adsorb onto the tip. The caught molecule was then stretched in a controlled manner by moving the tip away from the substrate at a fixed pulling rate, and the force-extension profiles were measured. PEO behaves as a purely random coil in DMF and does not show specific features in the force curves.26Lussis P. Svaldo-Lanero T. Bertocco A. Fustin C.A. Leigh D.A. Duwez A.S. A single synthetic small molecule that generates force against a load.Nat. Nanotechnol. 2011; 6: 553-557Crossref PubMed Scopus (81) Google Scholar,27Van Quaethem A. Lussis P. Leigh D.A. Duwez A.-S. Fustin C.-A. Probing the mobility of catenane rings in single molecules.Chem. Sci. 2014; 5: 1449-1452Crossref Scopus (38) Google Scholar Any deviation from this behavior can thus be safely attributed to the oligoamide helix. Force-extension profiles revealed a consistent behavior for all four foldamers. A typical profile can be divided into different steps (Figure 2A). We attributed the initial part of the force-extension curve (up to about 100 pN) to the unraveling of the PEO random coil and the subsequent plateau to the unwinding of the helical structure. The occurrence of a plateau is characteristic of the breaking of intramolecular interactions in series and is thus usually associated to a non-cooperative unfolding. However, here, the plateau is slightly tilted toward lower forces when the distance increases (Figures 2A, 2C, and S2). It means that it gets easier and easier to open the helix in the course of the unwinding, a signature of a cooperative effect. Once an interaction is broken, the subsequent one is easier to break. At the end of the plateau, the force decreases below plateau level and fluctuates (see next section below). The length of the plateau is in a very good agreement with the theoretical difference in length between the wound and fully extended structures (Figures 1C and S3; Table S1. See Supplemental information for the details of calculation): the extended conformation is 3.8 times longer than the helical state. At the end of the plateau, the helix is completely unwound, and the force rises again as the PEO stretching continues. The force of unwinding, 101 pN for Q33 (Figure 2B), is much higher than for natural helices. For example, double-helical coiled-coil protein structures, triple-helical spectrin repeats, parallel-stacked α-helical ankyrin repeats, and myomesin α-helical linkers in muscles exhibit unwinding forces of about 20 pN at comparable loading rates.45Rief M. Pascual J. Saraste M. Gaub H.E. Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles.J. Mol. Biol. 1999; 286: 553-561Crossref PubMed Scopus (469) Google Scholar, 46Berkemeier F. Bertz M. Xiao S. Pinotsis N. Wilmanns M. Gräter F. Rief M. Fast-folding alpha-helices as reversible strain absorbers in the muscle protein myomesin.Proc. Natl. Acad. Sci. 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For Q17, we observed the same characteristic force curves with the length of the plateau being proportional to the length of the molecule (Figures 2E and S2). Q9 displayed a small plateau followed by predominant large fluctuations (Figure S5). Q5 showed only fluctuations or a single rupture peak between the two states (Figure S6), which confirms the cooperativity. The histograms of the length of unwinding signal for each helix show Gaussian-like monomodal distributions centered on a value proportional to the number of units in the helix (Figure 2E). This observation implies that all the helices adopt a unique wound structure in DMF, in agreement with earlier solution and solid-state studies.32Qi T. Deschrijver T. Huc I. Large-scale and chromatography-free synthesis of an octameric quinoline-based aromatic amide helical foldamer.Nat. Protoc. 2013; 8: 693-708Crossref PubMed Scopus (34) Google Scholar,33Jiang H. Léger J.M. Huc I. Aromatic δ-peptides.J. Am. Chem. Soc. 2003; 125: 3448-3449Crossref PubMed Scopus (252) Google Scholar Indeed, narrow monomodal distributions exclude the possibility of populated partially unfolded or differently folded conformations in solution. The unwinding force ranges from 74 to 101 pN from the smallest to the longest helix (Figure 2D). The average unwinding force increases with the number of quinoline units until it reaches a maximum value between 9 and 17 units and plateaus between 17 and 33 units. This is probably an additional signature of cooperativity in winding. Such effects had never been identified in earlier experimental ensemble studies because completely unwound conformations were never reached, only partly unwound transition states were identified. For instance, helix handedness inversion was shown to proceed through a nucleation-propagation mechanism involving the unwinding of only two units.38Delsuc N. Kawanami T. Lefeuvre J. Shundo A. Ihara H. Takafuji M. Huc I. Kinetics of helix-handedness inversion: folding and unfolding in aromatic amide oligomers.ChemPhysChem. 2008; 9: 1882-1890Crossref PubMed Scopus (68) Google Scholar,39Abramyan A.M. Liu Z. Pophristic V. Helix handedness inversion in arylamide foldamers: elucidation and free energy profile of a hopping mechanism.Chem. Commun. 2016; 52: 669-672Crossref PubMed Google Scholar Before the plateau starts, we often observed fluctuations (Figures 2A and S7). These fluctuations can be explained by the reforming of an interaction a short time after being broken, and thus occur between wound and partially unwound states. It means that during pulling, the oligoamide pulls on the AFM cantilever in the opposite direction and rewinds against the mechanical load. The average force in the hopping region was about 10 pN higher than the plateau force. This difference in force can be associated to a barrier to overcome in order to trigger the unwinding of the helix. Such a barrier has been described by molecular dynamics simulations and was shown to correspond to the spring-like deformation of the helix.54Zegarra F.C. Peralta G.N. Coronado A.M. Gao Y.Q. Free energies and forces in helix–coil transition of homopolypeptides under stretching.Phys. Chem. Chem. Phys. 2009; 11: 4019-4024Crossref PubMed Scopus (9) Google Scholar Then, the force decreases, and the plateau starts. At the end of the plateau, the force largely decreases (about 30 pN), and we again detect significant hopping states. In this phase, unwinding requires a lower force and a segment of the foldamer dynamically fluctuates between a partially unwound and the fully unwound state. This is a clear signature of the cooperativity of mechanical unwinding: when the interactions are progressively broken, the subsequent opening is facilitated. The interactions weaken progressively as neighboring interactions disappear along the extension. Weaker forces are thus sufficient to unfold the last part of the sequences, and the drop in force is significant when the length reaches less than two turns. At the upper limit of sampling rate (4,200 nm·s−1), we measured a fluctuation rate between wound and unwound states of about 7,500 s–1 at forces of 100 pN (Figure 3A). From these fluctuations, we could estimate a minimum rewinding rate of about 25,000 s−1 under a force of 100 pN (see Supplemental information for details of the analysis). This estimation is a lower limit. We were indeed limited by the response time of the cantilever and the sampling rate (Figure S14). It means that the rewinding may be even faster than our estimation. The fastest folding kinetics for single α-helices that has been reported so far by direct time-resolved experimental measurements of fluctuations was 30,000 s−1 for villin, an actin-binding protein. This rate is thus comparable to the one obtained here (25,000 s−1), but it was measured at a much lower force of 10 pN.55Zoldák G. Stigler J. Pelz B. Li H. Rief M. Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy.Proc. Natl. Acad. Sci. USA. 2013; 110: 18156-18161Crossref PubMed Scopus (58) Google Scholar As the folding rate is known to drastically decrease when the applied force increases,46Berkemeier F. Bertz M. Xiao S. Pinotsis N. Wilmanns M. Gräter F. Rief M. Fast-folding alpha-helices as reversible strain absorbers in the muscle protein myomesin.Proc. Natl. Acad. Sci. USA. 2011; 108: 14139-14144Crossref PubMed Scopus (45) Google Scholar we can conclude that the aromatic amide helices are faster to rewind than those natural single α-helices. It is worth mentioning that here we compare the aromatic amide helices, which are single isolated helices, with natural single α-helices only. Other protein structures described in the literature, such as β-sheets, trimeric α-helices in spectrin, and a series of α-helices in bacte