Tetrameric β3-Peptide Bundles

There is considerable current interest in the design of non-proteinaceous quaternary structures with defined oligomeric states, as these materials have potential as nanomaterial scaffolds, drug delivery tools and enzymatic platforms.[1, 2] We recently described a series of β3-peptides, exemplified by the sequences of Zwit-1F and Acid-1Y (Figure 1A), that assemble into octameric β-peptide bundles of known structure and high stability.[3–6] The 314 helices comprising these octamers form three distinct faces: a leucine face whose side chains form the bundle core, a salt bridge face of alternating β3-ornithine and β3-aspartate resides, and an aromatic face containing two β3-tyrosine or β3-phenylalanine residues.[7–9] Figure 1 (A) Helical net representation of Zwit-1F, Acid-1Y, Zwit-VY and Acid-VY. (B, C) Zwit-VY and Acid-VY self-association monitored by analytical ultracentrifugation and fit to monomer-n-mer equilibria. Samples were prepared in 10 mM NaH2PO4, 200 mM NaCl (pH ... It is well established that natural coiled coil bundle stoichiometries are controlled by the identity and conformation of side chains buried at the bundle interface.[10] For example, substitution of valine or isoleucine for leucine at the GCN4 dimer interface leads to trimeric and tetrameric bundles.[10] Here we show that β3-peptide bundle stoichiometry is also controlled by side chain identity within the bundle core. Specifically, substitution of valine for leucine at the octamer interfaces of Zwit-1F and Acid-1Y generates valine derivatives, Zwit-VY and Acid-VY. These second generation β-peptides assemble into discrete and stable tetrameric bundles that were characterized by analytical ultracentrifugation (AU), circular dichorism (CD), 1-anilino-8-naphthalenesulfonate (ANS) binding, and deuterium exchange NMR.[4, 6, 11] First we used analytical ultracentrifugation (AU) to determine whether Zwit-VY and Acid-VY formed bundles possessing a discrete stoichiometry in solution (Figure 1). The Zwit-VY AU data fit best to a monomer-n-mer equilibrium where n = 4.07 with an RMSD of 0.00670 (Figure 1B). A comparable fit (RMSD = 0.00672) resulted when n was set to equal 4 (Figure S2A). The ln Ka value calculated from these two fits are 37.70 ± 0.07 and 38.2 ± 0.8, respectively. Significantly poorer fits (RMSD > 0.008) resulted when n was set to equal 5 or 6.[12] The AU data collected for Acid-VY fit optimally to a tetramer where n = 3.94 with an RMSD value of 0.00861 (Figure 1C). The Acid-VY data was fit to a variety of other oligomeric assemblies, all of which afforded poorer fits to the AU experimental data and corresponding increased RMSD values (Figure S2).[12] We next used wavelength-dependent circular dichroism (CD) spectroscopy to characterize concentration and temperature-dependent changes in the secondary structures of Zwit-VY and Acid-VY (Figures 2 and S3). Zwit-VY underwent a concentration-dependent increase in 314-helical structure (as judged by the molar residue ellipticity at 209 nm, MRE209[13]) between 12 and 100 µM (Figure 2A), consistent with a concentration-dependent equilibrium between a partially structured monomer and a folded oligomer. This behavior mimics the behavior of previously characterized β-peptide bundles Zwit-1F, Zwit-1F*, and Acid-1Y.[5, 6] A plot of MRE209 as a function of Zwit-VY concentration fit well to a monomer-tetramer equilibrium with ln Ka = 38.3 ± 0.5 (Figure 2A), in agreement with the value calculated from the AU data (38.2 ± 0.8). Acid-VY also displayed a concentration-dependent increase in 314-helical structure (as judged by the MRE minimum at 209), fitting best to a monomer-tetramer equilibrium with a ln Ka of 39.3 ± 0.5 (Figure S3).[12] In addition, the temperature dependent CD spectra of both Zwit-VY and Acid-VY show concentration-dependent increases in Tm that imply self-association (Figures 2B and S4C).[14] The Tm (defined as the maximum in a plot of δMRE209•δT−1) of a 50 µM Zwit-VY solution (88% folded) is 85 °C, and for a 80 µM (88% folded) solution of Acid-VY is also 85 °C. Unexpectedly, these values are higher than the Tm values of 70 °C and 78 °C previously observed for Zwit-1F (100 µM, 65% folded) and Acid-1Y (100 µM, 92% folded), respectively.[5] In summary, analytical ultracentrifugation measurements, as well as wavelength and temperature-dependent CD experiments indicate that β-peptides Zwit-VY and Acid-VY assemble into a 314-helical tetramer. Figure 2 Zwit-VY self-association monitored by circular dichroism spectroscopy (CD). (A) Plot of MRE209 as a function of [Zwit-VY]monomer fit to a monomer-tetramer equilibrium. (B) Plot of δMRE209•δT−1 for the concentrations of ... We used 1-anilino-8-naphthalenesulfonate (ANS) to further probe features of the tetrameric Zwit-VY and Acid-VY hydrophobic cores. The fluorescence of ANS increases upon binding to hydrophobic surfaces.[15] A significant increase in ANS fluorescence (between 60 and 100 fold) upon addition of protein indicates a loosely folded, exposed hydrophobic core as shown for the α-lactalbumin molten globule.[16] Most well-folded or unfolded proteins do not provide favorable ANS binding sites and minimal fluorescence changes are observed in these cases.[16] The relative fluorescence of 10 µM ANS increased from a value of 1.1 at 12.5 µM Zwit-VY (79% tetramer) to a value of 1.6 at 350 µM Zwit-VY (98% tetramer, Figure S5). The increase in relative ANS fluorescence upon addition of Acid-VY ranged from 1.1 at 25 µM (91% tetramer) to 1.2 at 300 µM (98% tetramer). Taken with the CD data, the minimal concentration-dependent increase in ANS fluorescence upon addition of Zwit-VY and Acid-VY suggests that both tetramers possess minimally exposed hydrophobic cores. Finally, we used hydrogen/deuterium exchange NMR to characterize the kinetic stability of the Zwit-VY and Acid-VY tetramers.[17] The 1H NMR spectra of both samples show dispersion of the amide N-H resonances (0.8 ppm) that is higher than that observed for the β-peptide monomer Acid-1YL2A,L,11A (Figures 3 and S6)[5] although smaller than that observed for Zwit-1F (1.4 ppm) and Acid-1Y (1.5 ppm). As was observed for the isosteric octameric bundles Zwit-1F and Acid-1Y,[4, 6] the pattern of the amide N-H resonances of Zwit-VY and Acid-VY are nearly identical, suggesting that the quaternary structures of the two bundles are similar.[5, 6] Figure 3 Kinetic stability of the Zwit-VY tetramer as determined by hydrogen/deuterium exchange analysis. (A) 500 MHz 1H NMR spectra of 0.6 mM Zwit-VY acquired at the indicated times after a lyophilized Zwit-VY sample was reconstituted in phosphate-buffered D ... The amide (N-H) exchange rate constants (kex) derived from the deuterium exchange NMR experiment correlate with the availability of amide protons to exchange with bulk solvent. Slow exchange rate constants are found for amide protons that are protected from exchange due to participation in stabilizing hydrogen bond interactions. The rate of disappearance of peaks a – d in a 600 µM sample of Zwit-VY (Figure 3A) fit a first order kinetic model with rate constants (kex) between 1.6 × 10−3 and 5.5 × 10−4 s−1 (Figure 3B). Comparison of these values to the rate constant for exchange of an amide N-H in poly-β-homoglycine (krc) allows the calculation of a protection factor (P = krc/kex)[18] that facilitates comparisons between protein and β-peptide quaternary systems.[5, 18, 19] The protection factors calculated for a 600 µM (98% folded) sample of Zwit-VY fall between 4 × 103 and 1.8 × 104.[12] These values are comparable to the range calculated for a 1.5 mM (97% folded) solution of Zwit-1F of 3.4 × 103 and 2 × 104.[5, 6] A similar trend in protection factors was also observed for Acid-VY.[12] The minimal difference in protection factors of Zwit-VY and Zwit-1F provides additional evidence that the valine β-peptide derivatives assemble into a discrete tetrameric structure possessing kinetically stable hydrophobic cores.[5, 17, 20, 21] In summary, here we show that β-peptide bundle stoichiometry can be controlled by the identity of side chains buried at the subunit interface in a manner analogous to that observed in natural coiled coil proteins.[10] These results provide a second, critical step in the “bottom-up” assembly of β-peptide assemblies possessing defined sizes, reproducible structures, and sophisticated function.[22]