Primary, Secondary, Tertiary and Quaternary Structure Levels in Linear Polysaccharides: From Random Coil, to Single Helix to Supramolecular Assembly.

Polysaccharides are ubiquitous in nature and represent an essential class of biopolymers with multiple levels of conformation and structural hierarchy. However, a standardized structural nomenclature, as in the case of proteins, is still lacking due to uncertainty on their hierarchical organization. In this work we use carrageenans as model polysaccharides to demonstrate that several structural levels exist and can be unambiguously resolved by statistical analysis on high resolution Atomic Force Microscopy images, supported by spectroscopic, X-ray scattering and rheological techniques. In direct analogy with proteins, we identify primary, secondary, tertiary and quaternary structures. The structure-property relationship induced by monovalent ions for κ-, ι- and the non-gelling control λ-carrageenan is established from the single chain regime to the occurrence of hydrogels at higher concentrations. For κ-carrageenan in the presence of potassium, a disorder-order transition from random coil to single helix is first observed (secondary structure), followed by intrachain supercoiling events (tertiary structure) and macroscopic anisotropic domains which are parts of a network (quaternary structure) with tunable elasticity up to ∼103 Pa. In contrast, κ-carrageenan in the presence of sodium only produces changes in secondary structure without supercoiling events, prior to formation of gels, highlighting the ion-specificity of the process. Loosely intertwined single helices are observed for ι-carrageenan in the presence of sodium and potassium chloride, providing an elastic mesh with many junction zones, while λ-carrageenan does not undergo any structural change. A generality of the observed behavior may be inferred by extending these observations to a distinct class of polysaccharides, the weak carboxylic polyelectrolyte Gellan gum. These results advance our understanding of ion-specific structural changes of polysaccharides and the physical mechanisms responsible for their gelation.

Anomalous stiffening and ion-induced coil-helix transition of carrageenans under monovalent salt conditions.

The macromolecular conformations of anionic polysaccharides with decreasing linear charge densities­lambda, iota, and kappa carrageenan­, at varying NaCl concentrations, are studied by single-chain statistical analysis of high-resolution atomic force microscopy (AFM) images. Lambda remains in the random coil conformation, whereas iota and kappa undergo ion-induced coil-helix transitions, with a 2-3-fold increase in chain rigidity. At low ionic strengths, I, the polymer chains sequester Na⁺, leading to a greater flexibility, and beyond a critical I to the formation of an intramolecular single helix. The persistence length exhibits a sublinear dependence on the Debye screening length, κ⁻¹, L(p)(e) ∼ κ(-y) (with 0 < y < 1), deviating from the classical polyelectrolyte behavior expressed by Odijk-Skolnick-Fixman or Barrat-Joanny models. Above a certain I, the L(p) shows an upturn, resulting in polymer stiffening and nonmonotonic behavior. This phenomenon is inferred from specific ion-polymer interactions and/or nonlinear electrostatic physics involving ion-ion correlations.

Magnetic Control of Macromolecular Conformations in Supramolecular Anionic Polysaccharide-Iron Complexes.

The anionic iota carrageenan polysaccharide is enriched with Fe(II) and Fe(III) by ion exchange against FeSO4 and FeCl3 . With divalent iron, portions of polymer chains undergo a secondary structure transition from random coils to single helices. The single-chain macromolecular conformations can be manipulated by an external magnetic field: upon exposure to 1.1 T, the helical portions exhibit 1.5-fold stiffening and 1.1-fold stretching, whereas the coil conformations respond much less as a result of lower contents of condensed iron ions. Along with the coil-helix transition, the trivalent iron triggers the formation of superstructures. The applicability of iron-enriched iota carrageenan as functional ingredient for food fortification is tested by free Fe(2+) and Fe(3+) contents, respectively, with the most promising iota-Fe(III) yielding 53% of bound iron, which is due to the superstructures, where the ferric ions are chelated by the supramolecularly self-assembled polymer host.

Unravelling secondary structure changes on individual anionic polysaccharide chains by atomic force microscopy.

The structural conformations of the anionic carrageenan polysaccharides in the presence of monovalent salt close to physiological conditions are studied by atomic force microscopy. Iota-carrageenan undergoes a coil-helix transition at high ionic strength, whereas lambda-carrageenan remains in the coiled state. Polymer statistical analysis reveals an increase in persistence length from 22.6±0.2 nm in the random coil, to 26.4±0.2 nm in the ordered helical conformation, indicating an increased rigidity of the helical iota-carrageenan chains. The many decades-long debated issue on whether the ordered state can exist as single or double helix, is conclusively resolved by demonstrating the existence of a unimeric helix formed intramolecularly by a single polymer chain.

Resolving self-assembly of bile acids at the molecular length scale.

The self-assembly behavior of the naturally occurring steroidal bile compounds cholic, deoxycholic, ursodeoxycholic, and lithocholic acid was studied by combining atomic force microscopy (AFM), polarized optical microscopy (POM), Fourier-transform infrared spectroscopy (FTIR), absorption spectroscopy (UV-vis), circular dichroism (CD), and wide-angle X-ray scattering (WAXS). Molecular solutions of these mono-, di-, and trihydroxyl substituted bile acids spontaneously evolved into supramolecular aggregates upon the incremental addition of H(2)O as a poor solvent. Highly crystalline nanostructured multilayered assemblies were formed, which revealed a very rich polymorphism of micro- and macro-structures depending on the chemical structure of the bile acid and the properties of the cosolvent (EtOH or DMSO) used. In particular, AFM allowed resolving the crystalline structure to an unprecedented level. It was thus possible to establish that bile acids associate into H-bonded chiral dimer building blocks, which organize in 2D layers of nanostructured lamellar surface topologies with unique facial amphiphilicity. The detailed understanding of the hierarchical organization in bile acid assemblies may contribute to develop strategies to design bioinspired materials with tailor-made nanostructured surface topologies.

Supramolecular chiral self-assembly and supercoiling behavior of carrageenans at varying salt conditions.

The self-assembly of anionic kappa and iota carrageenan polysaccharides in the presence of NaCl, KCl and CaCl2 is studied by high-resolution atomic force microscopy (AFM). A hierarchical supramolecular chirality amplification over various length scales is observed upon the addition of KCl, whereas in the presence of NaCl and CaCl2 the chains undergo solely a coil-helix transition with stiff kappa carrageenan and more flexible iota carrageenan helical conformations.

Biomimetic self-assembly of recombinant marine snail egg capsule proteins into structural coiled-coil units.

The egg capsules of the marine snails from the Melongenidea family feature unique biomechanical properties, including large reversible elasticity combined with a relatively high stiffness and outstanding strain energy absorption, making it an attractive biomimetic model system for restorative and tissue engineering applications. The capsules' building blocks are proteins called egg capsule proteins (ECPs), which we recently sequenced. ECPs are predicted to contain relatively large coiled-coil domains, which are directly responsible for the high elasticity arising from the extension of α-helical coiled-coil domains into extended ß-sheet domains. In this work, de novo synthesized ECPs genes were cloned and expressed in a bacterial expression system. Following purification under denaturing conditions by strong ion-exchange chromatography, individual and paired mixtures of ECPs were self-assembled using a controlled dialysis protocol, resulting in the folding of ECPs into soluble coiled-coil units. Circular Dichroism (CD) spectroscopy of the fibrils suggested that ECPs self-assembled into heteromeric coiled-coil units. The enhancement of the α-helical coiled-coil content during pair assembly was confirmed by Fourier Transform Infrared (FTIR) spectroscopy. Transmission Electron Microscopy (TEM) imaging of covalently-fixed self-assembled units corroborated the formation of elongated intermediate filaments-like structures. Polymer statistical analysis of Atomic Force Microscopy (AFM) images of unfixed self-assembled fibrils suggested that the observed coiled-coils were made of dimeric subunits. This study establishes the key protein engineering and physicochemical parameters needed to assemble ECPs into building blocks that can be processed into biomaterials that mimic the unique biomechanical properties of marine snail egg capsules.