Chirality-selected phase behaviour in ionic polypeptide complexes

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.

| Microscopy of sequence controlled complexes. Optical micrographs showing the liquid nature of complex coacervates formed from sequence-controlled polymers of alternating D and L monomers (i.e., (Kk)14W and EW(Ee)14e where K and E indicates an L monomer and k and e indicate a D monomer, and a tryptophan W was added for concentration determination) at a total monomer concentration of 6.8 mM and 100 mM NaCl. Scale bar is 25 µm.

Supplementary Fig. 4 | FTIR of polyelectrolytes, polyelectrolyte complexes and micellar polyelectrolyte complexes.
Transmission FTIR spectra showing the amide I region for the individual (a) poly(lysine) and (b) poly(glutamic acid) polymers, (c) liquid coacervates, (d) solid precipitates, (e) micellar complexes, and (f) a comparison of the various solid and liquid, bulk and micellar complexes. All samples were prepared in D2O, and unless specified, polypeptides with a degree of polymerization N = 100 were used. Polypeptides were analyzed 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. Micelles were prepared using PEG-pLK with an average N = 50 and either pLE with N = 50 for SCMs or N = 100 for LCMs.
(a) Poly(lysine) shows a single peak at 1644 cm -1 , characteristic of a random coil structure. (b) Glutamic acid shows an identical backbone signal, as well as a second peak at 1564 cm -1 , associated with the carbonyl stretching of the side chain. No variations in the FTIR signal were observed as a function of varying chirality. (c) Liquid coacervates show a similar random coil structure. (d) However, the spectra for solid precipitates contain signals for both random coil polypeptides and aggregated β-strands. For complexes formed from polypeptides with matching chirality (pLK+pLE, pDK+pDE) the main peak is located at 1611 cm -1 , and is shifted to 1613 cm -1 for opposite chirality (pLK+pDE, pDK+pLE), suggesting a decrease in the size of the βsheet network. 1 An additional low intensity peak is also present near 1680 cm -1 . (e) The spectra for solid-and liquid-core micelles (SCM, LCM) display the same features as the analogous bulk materials, but the β-strand peak location for the SCM is shifted to 1610 cm -1 , suggesting a more extended β-sheet network than for the bulk materials.    Figs. 4a and 4b). In addition to the random coil peak at 1644 cm -1 associated with the peptide backbone carbonyl stretch, an additional signal is observed at 1564 cm -1 for the side chain stretching of the glutamic acid carbonyl (Supplementary Fig. 4b). The spectra for liquid coacervates matched the random coil structure of the individual polypeptides very closely, again with no variations as a function of chirality. This random coil structure is expected for the individual, charged polypeptides, and is consistent with previous characterization of the polymer structure in liquid coacervates closely approximating that of an ideal Gaussian chain. 4 However, for the solid precipitates, we observe signals characteristic of desolvated β-strands and amyloids. 3,[5][6][7] The main amide I peak is present at 1611 cm -1 for solid precipitates formed from polypeptides with matching chirality (pLK+pLE, pDK+pDE), and is shifted to 1613 cm -1 for complexes formed from polypeptides with opposite chirality (pLK+pDE, pDK+pLE). This type of blue-shift in the amide I band has been attributed to decrease in the size of the β-sheet network. 1 An additional, low intensity peak near 1680 cm -1 is also attributable to the presence of βsheet structure. Very similar trends are observed for polypeptides present as block copolymers, and the resulting solid-core and liquid-core micellar complexes (SCM, LCM). Interestingly, the main β-sheet peak for the SCMs is Turbidity was used to investigate the stability of liquid and solid complexes as a function of both salt (NaCl) and denaturant (urea) concentration. While low concentrations of salts have been shown to enhance complex formation ( Supplementary Fig. 5), higher concentrations of salt ultimately destabilize the phase transition. The concentration above which phase separation no longer occurs is known as the critical salt concentration.
Supplementary Fig. 5 presents the turbidity data for both liquid coacervates and solid precipitates. As can be seen, the trends in turbidity are more clearly defined for the case of liquid coacervates. This is expected because the surface area of liquid droplets is a more easily defined and reproducible result than the surface area associated with scattering from a solid precipitate. However, in both cases we observe significant variations in the critical salt concentration as a function of chirality. A summary of these results is presented in Supplementary Table 2, along with data that has been scaled to correct for variations in the molecular weight of the polymers.
Taking into account these molecular weight effects, the stability of liquid coacervates formed from two racemic polymers (i.e., p(D,L)K+p(D,L)E) was significantly lower than for complexes formed from only a single racemic polymer. For solid precipitates we observe significantly higher critical salt concentrations for complexes formed from polymers of opposite chirality (i.e., pLK+pDE and pDK+pLE), than from those with matching chirality (i.e., pLK+pLE and pDK+pDE).
While the effects of salt on complexation correlate with electrostatic and entropic forces, urea acts directly on peptide hydrogen bonds. Urea appeared to have little effect on liquid coacervates. This result was expected because of the random coil structure of the polypeptides, and thus the minimal contributions of hydrogen bonds to the structure. However, the addition of sufficient quantities of urea to solid precipitates resulted in the melting of solid complexes to a liquid state that very closely resembles liquid coacervates ( Supplementary Fig. 6).
Interestingly, we observe the same trends in stability against dissolution in urea as were observed with respect to salt.

Supplementary Note 3: Chirality effects in micellar polyelectrolyte complexes.
By fitting the CD data it was shown that SCMs have 100% β-sheet structure as can be seen in Supplementary   Figs. 7a and 7c. Conversely, micelles formed using a racemic charged polypeptide maintained the random coil structure of the uncomplexed polypeptides illustrated in Supplementary Fig. 7b.
Examination of the effects of 1 M urea on LCM and SCM samples enabled characterization of peptide secondary structure via CD. As suggested by the phase transition in the bulk from a solid precipitate with β-sheet character to a liquid, the micellar systems transitioned from a SCM, which displayed 100% β-sheet character, evidenced by the characteristic minimum at 215 nm ( Supplementary Fig. 7c), to a structure with 99.8% random coil micelle, reminiscent of a LCM, and characterized by a maximum near 220 nm and a minimum below 205 nm ( Supplementary Fig. 7d). These results suggest that while electrostatic interactions provide a base level of stability, the way in which polypeptide chirality affects the number and strength of hydrogen bonding interactions can be used to tune material properties. Thus, an analogous solid to liquid transition occurs in microphaseseparated systems, as was observed in bulk homopolymer systems.