Infrared spectra of PLGA aggregates doped with PLL (black lines), or PDL (red lines) at the indicated Glu:Lys side chain molar ratios.

<p>Aggregates were formed by incubation (72h/60°C) of acidified mixtures of PLGA and PLL (PDL). Blue spectrum corresponds to β₂-fibrils formed in the absence of polylysine.</p

Covalent Defects Restrict Supramolecular Self-Assembly of Homopolypeptides: Case Study of β₂-Fibrils of Poly-L-Glutamic Acid

<div><p>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.</p></div

Spectral characteristics of NBA/EDC-modified PLGA samples.

<p>Far-UV CD spectra of PLGA samples modified with NBA (at fixed1∶3 Glu side chain: NBA molar ratio) in the presence of varying concentrations of EDC (expressed as molar ratio of EDC:Glu side chains: 0, 0.005, 0.015, 0.05, 0.15, 0.5, and 1.5) after alkalization to pH 8.3 (A) and subsequent acidification to pH 4.9 (B). Changes in light scattering intensity (at 350 nm) of NBA/EDC-modified PLGA formed at 1.5 EDC:Glu molar ratio caused by pH-adjustment are shown in the inset in panel (B).</p

TEM (top row) and SEM (bottom row) images of amyloid fibrils formed by unmodified PLGA (β₂) and selected NBA/EDC-modified PLGA samples.

<p>TEM (top row) and SEM (bottom row) images of amyloid fibrils formed by unmodified PLGA (β₂) and selected NBA/EDC-modified PLGA samples.</p

Cross-section view at a model of different inter-sheet distances and packing modes of Glu side chains in the β₁/β₂-type structural variants of PLGA aggregates with the antiparallel arrangement of strands (A).

<p>Red circles mark sites of three-center hydrogen bonds with bifurcated carbonyl acceptors. Random covalent modification of Glu side chains (within frames) cause local structural defects and result in less-densely-packed β₁ fibrils (B).</p

An FT-IR Study on Packing Defects in Mixed β-Aggregates of Poly(l-glutamic acid) and Poly(d-glutamic acid): A High-Pressure Rescue from a Kinetic Trap

Under favorable conditions of pH and temperature, poly­(l-glutamic acid) (PLGA) adopts different types of secondary and quaternary structures, which include spiral assemblies of amyloid-like fibrils. Heating of acidified solutions of PLGA (or PDGA) triggers formation of β₂-type aggregates with morphological and tinctorial properties typical for amyloid fibrils. In contrast to regular antiparallel β-sheet (β₁), the amide I′ vibrational band of β₂-fibrils is unusually red-shifted below 1600 cm^–1, which has been attributed to bifurcated hydrogen bonds coupling CO and N–D groups of the main chains to glutamic acid side chains. However, unlike for pure PLGA, the amide I′ band of aggregates precipitating from racemic mixtures of PLGA and PDGA (β₁) is dominated by components at 1613 and 1685 cm^–1typically associated with intermolecular antiparallel β-sheets. The coaggregation of PLGA and PDGA chains is slower and biphasic and leads to less-structured assemblies of fibrils, which is reflected in scanning electron microscopy images, sedimentation properties, and fluorescence intensity after staining with thioflavin T. The β₁-type aggregates are metastable, and they slowly convert to fibrils with the infrared characteristics of β₂-type fibrils. The process is dramatically accelerated under high pressure. This implies the presence of void volumes within structural defects in racemic aggregates, preventing the precise alignment of main and side chains necessary to zip up ladders of bifurcated hydrogen bonds. As thermodynamic costs associated with maintaining void volumes within the racemic aggregate increase under high pressure, a hyperbaric treatment of misaligned chains leads to rectifying the packing defects and formation of the more compact form of fibrils