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

Date Published: August 21, 2014

Publisher: Public Library of Science

Author(s): Aleksandra Fulara, Agnieszka Hernik, Hanna Nieznańska, Wojciech Dzwolak, Ilia V. Baskakov.

http://doi.org/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.

Partial Text

PLGA has been a major homopolypeptide model in the field of conformational transitions in proteins since the 1950s [1]–[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]. However, α-helical conformation is accessible only to PLGA chains of lengths above certain critical value – acidification of short disordered oligomers of α-L-glutamic acid converts them directly into aggregated β-sheets [8]–[9], as is also the case of long-chain α-helical PLGA subjected to high temperature (e.g.[10]). The ability to adopt, depending on pH and temperature, different conformations has made PLGA an insightful model for biophysical studies on protein folding. On the other hand, the reactivity and uniformity of Glu side chains triggered interest in using PLGA and its derivatives as biodegradable drug delivery systems (e.g. [11]–[12]), components of tumor targeting gene carriers (e.g. [13]), pH-switchable multi-topology vesicles [14], and multilayer films (in complexes with polylysine) for enhancement of cell adhesion (e.g. [15]). Apart from these biomedical applications of PLGA and its derivatives, the polypeptide, along with a number of other polymerized α-amino acids, has found an important explanatory function in the context of amyloid-related disorders such as Alzheimer’s or Parkinson’s diseases. These maladies distinguish themselves by in vivo deposition of amyloid fibrils – abnormal linear β-sheet-rich aggregates of misfolded proteins or peptides [16]–[17]. In 2002, Fändrich and Dobson demonstrated that several homopolypetides including PLGA form amyloid-like fibrils [18]. This finding led them to propose that the capacity to form such fibrils is not restricted to a handful of disease-associated proteins but rather is a generic property of polypeptides, and is primarily driven by main-chain interactions. The authors reported infrared spectra of PLGA fibrils formed through incubation at low pD (4.08) and 65°C. In the conformation-sensitive amide I′ band vibrational region, the spectra revealed a pair of peaks at 1616 (strong) and 1683 cm−1 (weak) typically assigned to antiparallel intermolecular β-sheet. While these fingerprint features are commonly found in spectra of aggregated polypeptides they contrast with the infrared characteristics of similarly prepared β-aggregates described by Itoh and colleagues [19] who showed that PLGA may adopt two different antiparallel β-sheet structures: one with the spectral features similar to those described by Fändrich and Dobson (termed β1) and another (β2) with the amide I′ band position characteristically redshifted below 1600 cm−1. X-ray diffraction analysis pointed to different packing modes in β1 and β2 aggregates [19]. While Itoh et al. did not study the β2 architecture in the context of amyloid fibrils this has become subject of recent investigations carried out by our group, as well as by Keiderling′s, Kubelka’s and Bouř’s groups [20]–[24]. Detection of β2-structures through infrared absorption coincides with formation of twisted supermolecular assemblies of PLGA fibrils [21]. In all our previous investigations employing high quality linear PLGA chains [20]–[21] only β2-aggregates were observed, although the aggregation conditions were essentially the same or very similar to those used by Fändrich and Dobson. In our earlier studies on acid-and-temperature-induced conformational transitions in PLGA, β1-aggregates were detected only as metastable transient forms occurring upon co–aggregation of PLGA and PDGA (poly-D-glutamic acid) [22]. This has led us to speculate that the amyloidogenic self-assembly of poly-Glu chains unperturbed by defects, or by polydispersity of the polymer, should produce higher order structures with β2-like infrared characteristics. This idea also resonates with the recent study from the Keiderling’s group demonstrating that synthetic (L-Glu)10 oligopeptide forms β2-fibrils [23].

The EDC-mediated modification of PLGA’s Glu side chains with NBA (Figure S1) has been carried out at the fixed PLGA:NBA ratio and at varying concentrations of added EDC (Materials and Methods). PLGA does not react with NBA in the absence of EDC, and neither the amine itself nor EDC itself affect formation of β2-aggregates (see control experiments in Figure S2). Thus in the presence of stoichiometric excess of NBA relative to Glu side chains, the number of carboxyl groups susceptible to reaction with amine is effectively controlled by the concentration of EDC. As the reaction leads to more hydrophobic and no longer ionizable side chains, it affects properties of PLGA even before polypeptide is converted into β-aggregates.

Unperturbed aggregation of defect-free PLGA chains ultimately leads to superstructures of amyloid-like fibrils with unusual infrared characteristics in the amide I′ band region, so-called β2-fibrils. With increasing number of structural defects introduced either by covalent modification of Glu side chains or through non-covalent co-binding of foreign peptides PLGA aggregates progressively depart from the β2-type towards less-ordered forms with β1-type spectral features that are commonly associated with intermolecular antiparallel β-sheet. Our study highlights the importance of thorough physicochemical characterization of polymerized-α-L-amino acids used in basic and applied research.

 

Source:

http://doi.org/10.1371/journal.pone.0105660