Poly-L-glutamic acid (PLGA) forms amyloid-like β2-fibrils with the main spectral component of vibrational amide I' band unusually shifted below 1600 cm(-1). This distinct infrared feature has been attributed to the presence of bifurcated hydrogen bonds coupling C═O and N-D (N-H) groups of the main chains to glutamate side chains. Here, we investigate how decreasing the chain length of PLGA affects its capacity to form β2-fibrils. A series of acidified aqueous solutions of synthetic (l-Glu)n peptides (n ≈ 200, 10, 6, 5, 4, and 3) were incubated at high temperature. We observed that n = 4 is the critical chain length for which formation of aggregates with the β2-like infrared features is still observed under such conditions. Interestingly, according to atomic force microscopy (AFM), the self-assembly of (L-Glu)n chains varying vastly in length produces fibrils with rather uniform diameters of approximately 4-6 nm. Kinetic experiments on (L-Glu)5 and (L-Glu)200 peptides indicate that the fibrillation is significantly accelerated not only in the presence of homologous seeds but also upon cross-seeding, suggesting thereby a common self-assembly theme for (L-Glu)n chains of various lengths. Our results are discussed in the context of mechanisms of amyloidogenic fibrillation of homopolypeptides.

Poly-L-glutamic acid (PLGA) often serves as a model in studies on amyloid fibrils and conformational transitions in proteins, and as a precursor for synthetic biomaterials. Aggregation of PLGA chains and formation of amyloid-like fibrils was shown to continue on higher levels of superstructural self-assembly coinciding with the appearance of so-called β2-sheet conformation manifesting in dramatic redshift of infrared amide I′ band below 1600 cm−1. This spectral hallmark has been attributed to network of bifurcated hydrogen bonds coupling C = O and N-D (N-H) groups of the main chains to glutamate side chains. However, other authors reported that, under essentially identical conditions, PLGA forms the conventional in terms of infrared characteristics β1-sheet structure (exciton-split amide I′ band with peaks at ca. 1616 and 1683 cm−1). Here we attempt to shed light on this discrepancy by studying the effect of increasing concentration of intentionally induced defects in PLGA on the tendency to form β1/β2-type aggregates using infrared spectroscopy. We have employed carbodiimide-mediated covalent modification of Glu side chains with n-butylamine (NBA), as well as electrostatics-driven inclusion of polylysine chains, as two different ways to trigger structural defects in PLGA. Our study depicts a clear correlation between concentration of defects in PLGA and increasing tendency to depart from the β2-structure toward the one less demanding in terms of chemical uniformity of side chains: β1-structure. The varying predisposition to form β1- or β2-type aggregates assessed by infrared absorption was compared with the degree of morphological order observed in electron microscopy images. Our results are discussed in the context of latent covalent defects in homopolypeptides (especially with side chains capable of hydrogen-bonding) that could obscure their actual propensities to adopt different conformations, and limit applications in the field of synthetic biomaterials.