Nanostructure and molecular mechanics of spider dragline silk protein assemblies

Abstract

Spider silk is a self-assembling biopolymer that outperforms most known materials in terms of its mechanical performance, despite its underlying weak chemical bonding based on H-bonds. While experimental studies have shown that the molecular structure of silk proteins has a direct influence on the stiffness, toughness and failure strength of silk, no molecular-level analysis of the nanostructure and associated mechanical properties of silk assemblies have been reported. Here, we report atomic-level structures of MaSp1 and MaSp2 proteins from the Nephila clavipes spider dragline silk sequence, obtained using replica exchange molecular dynamics, and subject these structures to mechanical loading for a detailed nanomechanical analysis. The structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly beta-sheet crystal domains, while disorderly regions are formed by glycine-rich repeats that consist of 3₁-helix type structures and beta-turns. Our structural predictions are validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots, alpha-carbon atomic distances, as well as secondary structure content. Mechanical shearing simulations on selected structures illustrate that the nanoscale behaviour of silk protein assemblies is controlled by the distinctly different secondary structure content and hydrogen bonding in the crystalline and semi-amorphous regions. Both structural and mechanical characterization results show excellent agreement with available experimental evidence. Our findings set the stage for extensive atomistic investigations of silk, which may contribute towards an improved understanding of the source of the strength and toughness of this biological superfibre.

Figure 1.

(a) Hierarchical structure of spider silk. The work reported here is focused on the scale of the nanocomposite structure, (b) where beta-sheet nanocrystals are immersed in a matrix of semi-amorphous protein. The focus of the present study is on a simple model system that contains a single beta-sheet nanocrystal embedded in the semi-amorphous matrix.

Figure 2.

Simulation protocol and representative molecular structure results. (a) Summary of the approach taken to identify the nanostructure of spider silk proteins, here focused on the MaSp1 and MaSp2 silk sequences from the N. clavipes spider. Monomers representing sections of the MaSp1 and MaSp2 proteins (containing a poly-alanine repeat in the centre) are used as the basic building block. Replica exchange simulations are carried out at multiple temperatures, and an ensemble of most likely, final low-temperature structures are compared with experimental evidence. (b) Illustration of the natural process of silk assembly (and fibre formation) during which silk proteins are subject to shear. The natural process of shearing and alignment of protein monomers motivates our choice of the initial geometry shown in (a). (c) The mechanical loading condition employed in our simulations in line with the shear topology of beta-sheet crystals in spider silk.

Figure 3.

Comparison of resulting structures for MaSp1 and MaSp2 with experimental data for validation. (a–d) For alanine residues, we observe that the most common φ–Ψ angle value is around (−150, 135), in excellent agreement with experimental findings that suggested (−135, 150), corresponding to a beta-sheet structure. For glycine residues, we observe a wide distribution around approximately (+/ − 75, − / + 75) with symmetry around the origin, in line with experimental findings around (+/60, − / + 135) that also show symmetric distribution. The wider distributions in MaSp2 may be due to a more amorphous structure caused by high proline content. (e) Dihedral angle distribution of proline residues cluster around (−60, −30) and (−60, 120), which correspond to type I and type II beta-turn conformations, respectively.

Figure 4.

Secondary structure distribution of selected protein assemblies. We select the most representative structures from the lowest temperature replica, and calculate the secondary structure distribution for (a) MaSp1 and (b) MaSp2 protein assemblies. (i–v) The five most likely structures selected for each sequence. The colouring is based on structural configuration, in which yellow represents beta-sheet and extended structures. (a) The selected structures for MaSp1. The majority of the structures are observed to be in beta-sheet or beta-turn conformation, and, for MaSp1 (a), the beta-sheet content is higher than for MaSp2 (b) owing to the lack of proline residues that reduce chain aggregation into sheets. The relative content of secondary structure controls the mechanical properties of protein assemblies, in which greater crystallinity typically means greater strength, whereas turn structures provide hidden length required for extensibility and toughness. (b) Representative structures for MaSp2. The results consistently show that poly-Ala regions form highly orderly beta-sheet crystals whereas the glycine-rich repeat units are less orderly, forming more amorphous domains.

Figure 5.

Force–displacement curves for selected structures. Figure shown illustrates the response of selected structures to shear forces applied to alternating strands. Force values shown are the loads applied per polypeptide strand. Illustration of the plots obtained from MaSp1 (a) and MaSp2 (b). The forces cause tensile stretching of the strands, in which a strain stiffening behaviour is evident once the chain reaches a certain length, independent of the chain's initial stretch state. The responses are similar for MaSp1 and MaSp2; however, it depends on the secondary structure content of the system. MaSp1 structures have a large variation on beta-sheet versus turn content, which leads to distinctly different mechanical responses (c). Solid lines indicate cases having the largest turn content, whereas dashed lines indicate structures with more beta-sheets. As the turn ratio increases, an initial stiff regime, followed by softening, followed by a stiff bond stretching regime is observed. For extended structures, the initial stiff regime disappears and the typical strain stiffening behaviour of polypeptide chains can be observed. The source of this difference is the existence of denser hydrogen bonding in amorphous regions owing to turn formation, which leads to higher stiffness and energy dissipation for structures containing more turns. The lower variation of turn and beta-sheet content in MaSp2 leads to the reduced variation of the mechanical response for this structure. As can be inferred from (d), the failure strength of both structures is more or less the same, as expected from the sequence similarity of the alanine-rich crystalline regions controlling the onset of failure.

Figure 6.

Stretching and structural transformation of the proteins. The figure illustrates the stretching behaviour of the amorphous domains and crystals under shear forces for (a) MaSp1 and (b) MaSp2. (i–v) The time sequence of events during the stretching simulations. As evident from the time sequence of snapshots (i–v), amorphous domains stretch significantly, and a transition from turn to beta-sheet structures is observed. The GGX repeats in MaSp1 are capable of forming beta-sheets during stretching, whereas this is observed to a lesser extent in MaSp2, in particular at low extension. A key observation is that failure of the system happens by sliding of strands with respect to each other upon breaking of the hydrogen bonds and side-chain contacts in the crystalline domain. This typically occurs at the interface region with solvent at the boundary of the crystal, leaving part of the crystal intact even after failure of the structure.

Figure 7.

Representative plots are shown from MaSp1 and MaSp2 structures with the highest turn content. Comparison of (a) and (b) shows the strain softening behaviour that occurs when the turn/beta-sheet ratio is high in the initial structures. Upon initial yielding, the number of hydrogen bonds in the system and also the turn content decreases; however, further stretching leads to a slight increase in the number of H-bonds and formation of beta-sheet structures in the amorphous region. This is followed by a final stiff regime dominated by the stretching of covalent bonds, which ultimately leads to rapid rupture of many hydrogen bonds (shown with a red arrow) in the non-crystalline domains. The system fails upon breaking of hydrogen bonds in the crystal, and sliding of beta-strands. The characteristic sigmoidal force–extension behaviour observed here for both structures shows resemblance to the macroscale response of spider silk. (a,b) Black line, turn; red line, beta-sheet.