Mechanical unfolding of alpha- and beta-helical protein motifs.

Alpha-helices and beta-sheets are the two most common secondary structure motifs in proteins. Beta-helical structures merge features of the two motifs, containing two or three beta-sheet faces connected by loops or turns in a single protein. Beta-helical structures form the basis of proteins with diverse mechanical functions such as bacterial adhesins, phage cell-puncture devices, antifreeze proteins, and extracellular matrices. Alpha-helices are commonly found in cellular and extracellular matrix components, whereas beta-helices such as curli fibrils are more common as bacterial and biofilm matrix components. It is currently not known whether it may be advantageous to use one helical motif over the other for different structural and mechanical functions. To better understand the mechanical implications of using different helix motifs in networks, here we use Steered Molecular Dynamics (SMD) simulations to mechanically unfold multiple alpha- and beta-helical proteins at constant velocity at the single molecule scale. We focus on the energy dissipated during unfolding as a means of comparison between proteins and work normalized by protein characteristics (initial and final length, # H-bonds, # residues, etc.). We find that although alpha-helices such as keratin and beta-helices CsgA and CsgB can require similar amounts of work to unfold, the normalized work per hydrogen bond, initial end to end length, and number of residues is greater for beta-helices at the same pulling rate. To explain this, we analyze the orientation of the backbone alpha carbons and backbone hydrogen bonds during unfolding. We find that the larger width and shorter height of beta-helices results in smaller angles between the protein backbone and the pulling direction during unfolding. As subsequent strands are separated from the beta-helix core, the angle between the backbone and the pulling direction diminishes. This marks a transition where beta-sheet hydrogen bonds become loaded predominantly in a collective shearing mode, which requires a larger rupture force. This finding underlines the importance of geometry in optimizing resistance to mechanical unfolding in proteins. The helix radius is identified here as an important parameter that governs how much sacrificial energy dissipation capacity can be stored in protein networks, where beta-helices offer unique properties.

Cohesive and adhesive properties of crosslinked semiflexible biopolymer networks.

Biomolecular semiflexible polymer networks with persistence lengths well above those of single polymeric chains serve important structural and adhesive roles in biology, biomaterials, food science and many other fields. While relationships between the structure and viscoelasticity of semiflexible polymer networks have been previously investigated, it remains challenging to systematically relate fibril and network properties to cohesive and adhesive properties that govern the function of these materials. To address this issue, here we utilize coarse-grained molecular dynamics simulations to thoroughly elucidate how the work of adhesion of a semiflexible polymer network to a surface depends on crosslink density and fibril persistence length. Two emergent characteristics of the network are its elasticity and its interfacial energy with the surface. Stiff networks that are either highly crosslinked or have high persistence length fibrils tend to have lower interfacial energy, and consequently, lower work of adhesion. For lightly crosslinked networks with flexible fibrils, considerable strain energy must be stored within the adhesive during detachment, which creates an additional penalty to detachment. Increasing persistence length while keeping crosslink density constant leads to porous, low density networks, leading to an optimal fibril persistence length at which maximum work of adhesion per mass density is attained for a given crosslink density. For any given fibril persistence length, increasing crosslink density has a slightly negative effect on network mass density and interfacial energy. A critical crosslink density is found, below which the networks have no significant load-bearing capacity. Lightly crosslinked networks above this threshold absorb more strain energy during desorption and consequently possess greater work of adhesion. The conflict between mass density and stiffness results in a non-monotonic trend between the ratio of work of adhesion to interfacial energy and persistence length. These findings provide physical insight into the adhesive mechanisms of biomaterials based on crosslinked semiflexible polymer networks, and reveal important design guidelines for bio-adhesives.

Biomimetic engineering of conductive curli protein films.

Bioelectronic systems derived from peptides and proteins are of particular interest for fabricating novel flexible, biocompatible and bioactive devices. These synthetic or recombinant systems designed for mediating electron transport often mimic the proteinaceous appendages of naturally occurring electroactive bacteria. Drawing inspiration from such conductive proteins with a high content of aromatic residues, we have engineered a fibrous protein scaffold, curli fibers produced by Escherichia coli bacteria, to enable long-range electron transport. We report the genetic engineering and characterization of curli fibers containing aromatic residues of different nature, with defined spatial positioning, and with varying content on single self-assembling CsgA curli subunits. Our results demonstrate the impressive versatility of the CsgA protein for genetically engineering protein-based materials with new functions. Through a scalable purification process, we show that macroscopic gels and films can be produced, with engineered thin films exhibiting a greater conductivity compared with wild-type curli films. We anticipate that this engineered conductive scaffold, and our approach that combines computational modeling, protein engineering, and biosynthetic manufacture will contribute to the improvement of a range of useful bio-hybrid technologies.

Bending energy penalty enhances the adhesive strength of functional amyloid curli to surfaces.

The functional amyloid curli fiber, a major proteinaceous component of biofilm extracellular matrices, plays an important role in biofilm formation and enterobacteriaceae adhesion. Curli nanofibers exhibit exceptional underwater adhesion to various surfaces, have high rigidity and strong tensile mechanical properties, and thus hold great promise in biomaterials. The mechanisms of how curli fibers strongly attach to surfaces and detach under force remain elusive. To investigate curli fiber adhesion to surfaces, we developed a coarse-grained curli fiber model, in which the protein subunit CsgA (curli specific gene A) self-assembles into the fiber. The coarse-grained model yields physiologically relevant and tunable bending rigidity and persistence length. The force-induced desorption of a single curli fiber is examined using coarse-grained modeling and theoretical analysis. We find that the bending energy penalty arising from high persistence length enhances the resistance of the curli fiber against desorption and thus strengthens the adhesion of the curli fiber to surfaces. The CsgA-surface adhesion energy and the curli fiber bending rigidity both play crucial roles in the resistance of curli fiber against desorption from surfaces. To enable the desorption process, the applied peeling force must overcome both the interfacial adhesion energy and the energy barrier for bending the curli fiber at the peeling front. We show that the energy barrier to desorption increases with the interfacial adhesion energy, however, the bending induced failure of a single curli fiber limits the work of adhesion if the proportion of the CsgA-surface adhesion energy to the CsgA-CsgA cohesive energy becomes large. These results illustrate that the optimal adhesion performance of nanofibers is dictated by the interplay between bending, surface energy and cohesive energy. Our model provides timely insight into enterobacteriaceae adhesion mechanisms as well as future designs of engineered curli fiber based adhesives.

Adhesion mechanisms of curli subunit CsgA to abiotic surfaces.

Curli fibers are functional amyloids that play a key role in biofilm structure and adhesion to various surfaces. Strong bioinspired adhesives comprising curli fibers have recently been created; however, the mechanisms curli uses to attach onto abiotic surfaces are still uncharacterized. Toward a materials-by-design approach for curli-based adhesives and multifunctional materials, we examine curli subunit adsorption onto graphene and silica surfaces through atomistic simulation. We find that both structural features and sequence influence adhesive strength, enabling the CsgA subunit to adhere strongly to both polar and nonpolar surfaces. Specifically, flexible regions facilitate adhesion to both surfaces, charged and polar residues (Arg, Lys, and Gln) enable strong interactions with silica, and six-carbon aromatic rings (Tyr and Phe) adsorb strongly to graphene. We find that adsorption not only lowers molecular mobility but also leads to loss of secondary structure, factors that must be well balanced for effective surface attachment. Both events appear to propagate through the CsgA structure as correlated motion between clusters of residues, often H-bonded between rows on adjacent ß strands. To quantify this, we present a correlation analysis approach to detecting collective motion between residue groups. We find that certain clusters of residues have a higher impact on the stability of the rest of the protein structure, often polar and bulky groups within the helix core. These findings lend insight into bacterial adhesion mechanisms and reveal strategies for theory-driven design of engineered curli fibers that harness point mutations and conjugates for stronger adhesion.

Design of polymer conjugated 3-helix micelles as nanocarriers with tunable shapes.

Amphiphilic peptide-polymer conjugates have the ability to form stable nanoscale micelles, which show great promise for drug delivery and other applications. A recent design has utilized the end-conjugation of alkyl chains to 3-helix coiled coils to achieve amphiphilicity, combined with the side-chain conjugation of polyethylene glycol (PEG) to tune micelle size through entropic confinement forces. Here we investigate this phenomenon in depth, using coarse-grained dissipative particle dynamics (DPD) simulations in an explicit solvent and micelle theory. We analyze the conformations of PEG chains conjugated to three different positions on 3-helix bundle peptides to ascertain the degree of confinement upon assembly, as well as the ordering of the subunits making up the micelle. We discover that the micelle size and stability is dictated by a competition between the entropy of PEG chain conformations in the assembled state, as well as intermolecular cross-interactions among PEG chains that promote cohesion between neighboring conjugates. Our analyses build on the role of PEG molecular weight and conjugation site and lead to computational phase diagrams that can be used to design 3-helix micelles. This work opens pathways for the design of multifunctional micelles with tunable size, shape and stability.

Mechanical Reinforcement of Proteins with Polymer Conjugation.

Conjugating poly(ethylene glycol) (PEG) to peptides, also known as PEGylation, is proven to increase the thermodynamical stability of peptides, and has been successfully applied to prolong the lifetime of peptide-based vaccines and therapeutic agents. While it is known that protein structure and function can be altered by mechanical stress, whether PEGylation can reinforce proteins against mechanical unfolding remains to be ascertained. Here, we illustrate that PEGylation prolongs the lifetime of α-helices subject to constant stress. PEGylation is found to increase the unfolding time through two mechanisms. We see that (1) the unfolding rate of a helical segment is decreased through prolonged plateau regimes where the peptide helical content remains constant, and (2) the proportion of refolding to unfolding is increased, primarily by shielding water molecules from replacing forcibly exposed backbone hydrogen bonds near the conjugation site. Our findings demonstrate the feasibility of improving peptide mechanical stability with polymer conjugation. This provides a basis for future studies on optimizing conjugation location and chemistry to build custom biomolecules with unforeseen mechanical functions and stability.