Chirality-selected phase behaviour in ionic polypeptide complexes

Abstract

Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.

Figure 1. Optical micrographs of polyelectrolyte complexes.

Bright-field optical micrographs showing the liquid coacervates or solid precipitates resulting from the stoichiometric electrostatic complexation of L, D, or racemic (D,L) poly(lysine) with L, D or racemic (D,L) poly(glutamic acid) at a total residue concentration of 6 and 100 mM NaCl. Complexes are formed from (a) pLK+pLE, (b) pDK+pLE, (c) p(D,L)K+pLE, (d) pLK+pDE, (e) pDK+pDE, (f) p(D,L)K+pDE, (g) pLK+p(D,L)E, (h) pDK+p(D,L)E, (i) p(D,L)K+p(D,L)E. Liquid coacervate droplets are only observed during complexation involving a racemic polymer. Scale bars, 25 μm.

Figure 2. FTIR of polyelectrolyte complexes.

FTIR spectra showing the amide I region for (a) individual polypeptides and the resulting liquid coacervates and solid precipitates, as well as (b) the polypeptides and block copolymers involved in the formation of related LCM and SCM. All samples were prepared in D₂O. Polypeptides were analysed at a concentration of 10 mM with respect to monomer, liquid coacervates and solid precipitates at a concentration of 6 mM with respect to monomer and 100 mM NaCl, while micellar complexes were prepared at a concentration of 0.186 mM polymer, with no salt. All materials show a peak at 1,644 cm⁻¹, characteristic of random coil polypeptide structure. However, the additional peaks associated with aggregated β-strands are present for the solid precipitates and SCMs. For solid precipitates formed from polypeptides with matching chirality (pLK+pLE, pDK+pDE), the main peak is located at 1,611 cm⁻¹ and is shifted to 1,613 cm⁻¹ for opposite chirality (pLK+pDE, pDK+pLE). For SCMs, this peak is located at 1,610 cm⁻¹. An additional low-intensity peak is also present near 1,680 cm⁻¹. The signal for the carbonyl stretching of the glutamic acid can also be observed at 1,564 cm⁻¹. Micelles were prepared using a polyethylene glycol-pLK block copolymer with an average N=50 and either pLE with N=50 for SCMs or N=100 for LCMs.

Figure 3. Salt and urea stability of polyelectrolyte complexes.

(a) Turbidity as a function of NaCl concentration for various liquid coacervates (open symbols) and solid precipitates (solid symbols) prepared at 1 mM total residue concentration and pH=7.0. Error bars are the s.d. from triplicate measurements. (b) Optical micrographs showing the transition from solid precipitate to liquid coacervate for pLK+pLE complexes with increasing urea concentration. Scale bars, 25 μm.

Figure 4. Secondary structure of micellar complexes.

CD spectra for (a) LCM (dark green) and SCM (light green) micelles formed from PEG-pLK+p(D,L)E and PEG-pLK+pLE, respectively, and (b) SCMs in the absence (light green) and presence of 1 M urea (dark green), respectively. LCMs show a random coil structure, while SCMs display β-sheet character in the absence of urea, but convert to a random coil structure in the presence of 1 M urea, suggestive of a LCM. LCMs were prepared at a polymer concentration of 0.01 mM total polymer concentration, while SCMs were prepared at 0.0125, mM with an average N=100 for the micelles in a, and both LCMs and SCMs were prepared at a total polymer concentration of 0.04 mM with an average N=50 for b.

Figure 5. Kinetics of micelle formation.

Total scattered intensity as a function of time for LCMs (circles) and SCMs (squares) depicting the timescale for equilibration. LCMs in water equilibrate relatively quickly, reaching 72% of their equilibrium value within 1 h, while SCMs in water show much slower kinetics, on the order of days; only reaching 25% of their equilibrium value in 1 h. However, the addition of 4 M urea to a SCM sample to disrupt hydrogen bonding between the polypeptides enables fast equilibration, similar to that of LCMs (74% of equilibrium value in 1 h). Micelles were prepared at a polymer concentration of 0.01 mM total polymer concentration using a polyethylene glycol-pLE block copolymer with an average N=50 and either pLK with N=100 for SCMs or p(D,L)K with N=100 for LCMs.

Figure 6. MD simulations of polyelectrolyte complexes.

Visualization and residue maps indicating polypeptide secondary structure from representative MD simulations for two pairs of poly(lysine) and poly(glutamic acid) peptides, N=10. Polypeptides are initially equilibrated in a random coil conformation and then allowed to complex for 1,000 ns. (a) A representative simulation of homochiral pLK complexing with racemic p(D,L)E indicates preservation of a mostly random coil structure, as would be expected for liquid coacervates, while (b) homochiral polypeptides pLK with pLE shows the evolution of β-strand structure expected for a solid precipitate. Map of secondary structure as a function of time for (c) pLK+p(D,L)E and (d) pLK+pLE.

Figure 7. Hydrogen bonding in polyelectrolyte complexes.

Quantification of the number of hydrogen bonds formed as a function of time during a 1,000 ns MD simulation of complex formation between (a) pLK+p(D,L)E and (b) pLK+pLE.