Covalent defects restrict supramolecular self-assembly of homopolypeptides: case study of β2-fibrils of poly-L-glutamic acid.

Aleksandra Fulara,Agnieszka Hernik,Wojciech Dzwolak,Hanna Nieznańska,Ilia V Baskakov

doi:10.1371/journal.pone.0105660

Abstract

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.

Highlights

Poly-L-glutamic acid (PLGA) has been a major homopolypeptide model in the field of conformational transitions in proteins since the 1950s [1,2,3,4,5,6,7]
Ahelical conformation is accessible only to PLGA chains of lengths above certain critical value – acidification of short disordered oligomers of a-L-glutamic acid converts them directly into aggregated b-sheets [8,9], as is the case of long-chain ahelical PLGA subjected to high temperature (e.g.[10])
We have investigated NBA-derivatized and polylysine-doped PLGA aggregates with Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

Summary

Introduction

PLGA has been a major homopolypeptide model in the field of conformational transitions in proteins since the 1950s [1,2,3,4,5,6,7]. In neutral or basic environment, repulsive culombic interactions between charged Glu side chains favor random coil conformation of the PLGA main chain. With reprotonation of side chains at low pH, a rapid coil-to-helix transition takes place [6]. The ability to adopt, depending on pH and temperature, different conformations has made PLGA an insightful model for biophysical studies on protein folding. The reactivity and uniformity of Glu side chains triggered interest in using PLGA and its derivatives as biodegradable drug delivery systems

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