Biophysical Characterization of a β-Peptide Bundle: Comparison to Natural Proteins

We recently described the high-resolution X-ray structure of a helical bundle composed of eight copies of the β-peptide Zwit-1F (Figure 1A,B).¹ Like many proteins in Nature, the Zwit-1F octamer contains parallel and antiparallel helices, extensive inter-helical electrostatic interactions, and a solvent-excluded hydrophobic core. Here we explore the stability of the Zwit-1F octamer in solution using circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AU), differential scanning calorimetry (DSC), and NMR. These studies demonstrate that the thermodynamic and kinetic properties of Zwit-1F closely resemble those of natural α-helical bundle proteins.

Figure 1.

(A) Helical net representation of the Zwit-1F monomer. β³-Amino acids are designated by the single letter corresponding to the equivalent α-amino acid. O signifies ornithine. (B) Zwit-1F octamer structure determined by X-ray crystallography.¹ (C) Plot of MRE₂₀₅ as a function of [Zwit-1F] fit to a monomer–octamer equilibrium. Inset: CD spectra (MRE in units of 10³ deg·cm²·dmol⁻¹) at the indicated [Zwit-1F] (μM).

CD spectroscopy indicates that Zwit-1F is minimally 3₁₄-helical in dilute solution (as judged by the molar residue ellipticity at 205 nm, MRE₂₀₅)² but undergoes a large increase in helical structure between 20 and 200 μM (Figure 1C). The concentration dependence of MRE₂₀₅ fits a monomer–octamer equilibrium with an association constant of 4.0 × 10³⁰ M⁻⁷ (ln K_a = 70.5 ± 1.9).³ This value matches the result of AU analysis, which fits a monomer–octamer equilibrium with ln K_a = 71.0 ± 0.9.³ Taken together, the AU and CD data support a model in which the unfolded Zwit-1F monomer is in equilibrium with the folded octamer.⁴

Few known natural proteins assemble as octamers. Examples include the histones⁵ (hetero-octamer), TATA binding protein⁶ (octamer in 1 M KCl), and the well-characterized hemerythrin (ln K_a = 84).⁷ Although Zwit-1F is less stable than hemerythrin, it is smaller in mass (13.1 vs 110 kDa) and interaction surface area (7000 vs 15 000 Ų).^1,8 To compare the stability of Zwit-1F to that of proteins of diverse size and stoichiometry, we calculated the free energy of association per Ų of buried surface area (ΔG_area). The ΔG_area of Zwit-1F is higher than that of hemerythrin, the tetrameric aldolase, and natural helical bundle proteins GCN4 and ROP (Table 1). In fact, ΔG_area for Zwit-1F is close to the average value (7.0 ± 2.8 cal·mol⁻¹·A⁻²) observed for protein complexes burying at least 1000 Ų of surface area upon association.^9,10 This comparison implies that the lower affinity of Zwit-1F is due to its small size and not an inherent instability of β³-peptide complexes.

Table 1.

Comparison of Protein Association Parameters^a

protein (stoichiometry)	MWmonomer	ΔGarea
Zwit-1F (8)	1.6 kDa	5.9
hemerythrin (8)	13.8 kDa	3.37
aldolase (4)	39.2 kDa	3.911
GCN4 (2)	4.0 kDa	4.812
ROP (2)	7.2 kDa	≥3.013

^aΔG_area values in units of cal· mol⁻¹·Å⁻². Interaction surface areas and ΔG_area calculated as described in Supporting Information.

Temperature-dependent CD studies (Figure 2A) show Zwit-1F to exhibit a concentration-dependent T_m, an inherent property of protein quaternary structure.¹⁴ The Zwit-1F T_m, which increases from 57 °C at 50 μM to 95 °C at 300 μM, is comparable to T_m values of thermostable proteins such as ubiquitin (T_m = 90 °C) and bovine pancreatic trypsin inhibitor (T_m = 101 °C).¹⁵ The Zwit-1F T_m is significantly higher than the T_m of GCN4 (41–78 °C at 1–880 μM)¹⁶ and ROP (58–71 °C at 0.5–160 μM).¹⁷ We note, however, that the unfolding of Zwit-1F is less cooperative: the width of the temperature derivative of the CD signal at half-maximum is 40 versus 20 °C for GCN4 or 15 °C for ROP.^16,17

Figure 2.

(A) Temperature-dependent CD analysis of Zwit-1F. Plot of MRE₂₀₅ as a function of temperature at the indicated Zwit-1F concentration (μM). (B) DSC analysis of Zwit-1F unfolding fit to a subunit dissociation model. Raw data are shown as black circles.³

A high T_m is not a definitive measurement of thermodynamic stability, so DSC was used to further characterize Zwit-1F unfolding (Figure 2B). At 300 μM concentration (where Zwit-1F is 87% octameric), the temperature-dependent heat capacity (C_P) peaks near the T_m identified by CD. This peak is embedded in a sloping baseline (∂C_p/∂T = 5.1 cal·mol⁻¹·K⁻²) 3.1 mcal·g⁻¹·K⁻²) that is similar to the C_P versus temperature plot of monomeric β³-peptides, for which no cooperative unfolding peak has yet been observed.² For most natural proteins, (∂C_p/∂T) is about 1 mcal·g⁻¹·K⁻² in the folded state,¹⁵ but GCN4 (∂C_p/∂T = 3.6 mcal·g⁻¹·K⁻²)¹⁶ and some ROP mutants (∂C_p/∂T = 4–5 mcal·g⁻¹·K⁻²)¹³ have sharply sloped pretransition baselines like Zwit-1F.

The DSC data fit well to a process defined by a two-state transition with dissociation of eight subunits using the program EXAM.^3,18 The fitted enthalpy and heat capacity change per mole octamer are 107.4 ± 0.3 kcal·mol⁻¹ and 1.4 ± 0.1 kcal·mol⁻¹·K⁻¹, respectively. Substituting these values into the Gibbs–Helmholz equation³ yields an equilibrium constant of 5.3 × 10³¹ (ln K = 73.3 ± 1.4) at 25 °C, in excellent agreement with values derived from CD and AU data. The integrated calorimetric unfolding enthalpy (ΔH_Cal) for Zwit-1F is 7.2 cal·g⁻¹, within the range observed for natural globular proteins (5.2–11.8 cal·g⁻¹),^19,20 but somewhat lower than GCN4 (7.7 cal·g⁻¹)²¹ and ROP (9.5 cal·g⁻¹).¹⁷

The NMR spectra of many well-folded natural and designed proteins are characterized by differentiated amide resonances and slow hydrogen/deuterium exchange.²² The amide N–H resonances in the ¹H spectrum of Zwit-1F, under conditions where the sample is 97% octameric, span 1.4 ppm (Figure 3A). While this span is narrower than that observed in the NMR spectra of large proteins such as α-lactalbumin (3 ppm), it is comparable to that seen for coiled-coil proteins GCN4 and ROP (1.3 and 2.2 ppm, respectively).^13,23,24 In contrast to Zwit-1F, the amide resonances of the poorly folded, monomeric β-peptide Acid-1Y^A2,11 span only 0.5 ppm.³ These results indicate that the Zwit-1F fold in solution creates distinct electronic environments for the amide backbone protons.

Figure 3.

(A) 500 MHz ¹H NMR spectra of 1.5 mM Zwit-1F, acquired in phosphate-buffered “H₂O” (9:1 H₂O/D₂O) or at the indicated times after reconstitution of a lyophilized Zwit-1F sample in phosphate-buffered D₂O. (B) Peak heights of the indicated resonances (normalized to the peak at 6.70 ppm) fit to exponential decays.³ Bars indicate standard error.

Participation in a hydrogen bond can protect an amide N–H from exchange with bulk solvent; since exchange occurs from the unfolded state, a slow amide exchange rate constant (k_ex) correlates with protein stability in solution.²² Exchange is often characterized by a protection factor (P) equal to k_rc/k_ex, where k_rc is the rate constant for exchange of a random coil amide N–H under similar conditions. When a lyophilized sample of Zwit-1F is redissolved at 1.5 mM concentration in D₂O, 9 of 14 resolvable peaks require more than 4 h to become indistinguishable from baseline. The time dependence of exchange corresponds to exchange rate constants between 0.6 × 10⁻⁴ and 2.9 × 10⁻⁴ s⁻¹. Using β-alanine (βG in our nomenclature) as a random coil model,^3,25 these values of k_ex correspond to a protection factor of 2 × 10⁴ for Zwit-1F. Thus, amide protons in Zwit-1F are less protected than those in large protein cores, where P ≥ 10⁵.^22,26 However, the protection factor for Zwit-1F, like the span of amide resonances, is comparable to ROP (10⁵ at 250 μM)¹³ and GCN4 (10⁴ at 1.0 mM).^23,24 Acid-1Y^A2,11 undergoes amide N–H exchange in less than 10 min, showing that slow exchange requires a stable β-peptide fold.³

The biophysical experiments presented here describe the thermodynamic and kinetic stability of the Zwit-1F octamer in solution. The data allow us to quantify the similarity of Zwit-1F to GCN4 and ROP, two small, well-folded α-amino acid helix bundle proteins. In fact, the T_m, ΔG_area, and ΔH_Cal for Zwit-1F are even comparable to much larger natural proteins. Taken together with the recent high-resolution structure of Zwit-1F,¹ these studies show that β-amino acid heteropolymers can assemble into quaternary complexes that resemble natural proteins in both solid-state structure and solution-phase stability. We note that our characterizations do not preclude some molten globule character of the Zwit-1F core in solution.²⁷ Nonetheless, these studies establish Zwit-1F as a remarkably protein-like stepping stone in the path toward fully synthetic mimics of biological molecules.