Sequence-structure correlations in silk: Poly-Ala repeat of N. clavipes MaSp1 is naturally optimized at a critical length scale

Spider silk is a self-assembling biopolymer that outperforms many known materials in terms of its mechanical performance despite being constructed from simple and inferior building blocks. While experimental studies have shown that the molecular structure of silk has a direct influence on the stiffness, toughness, and failure strength of silk, few molecular-level analyses of the nanostructure of silk assemblies in particular under variations of genetic sequences have been reported. Here we report atomistic-level structures of the MaSp1 protein from the Nephila Clavipes spider dragline silk sequence, obtained using an in silico   approach based on replica exchange molecular dynamics (REMD) and explicit water molecular dynamics. We apply this method to study the effects of a systematic variation of the poly-alanine repeat lengths, a parameter controlled by the genetic makeup of silk, on the resulting molecular structure of silk at the nanoscale. Confirming earlier experimental and computational work, a structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly ββ-sheet crystal domains while disorderly regions are formed by glycine-rich repeats that consist of 310310-helix type structures and ββ-turns. Our predictions are directly validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots combined with an analysis of the secondary structure content. The key result of our study is our finding of a strong dependence of the resulting silk nanostructure depending on the poly-alanine length. We observe that the wildtype poly-alanine repeat length of six residues defines a critical minimum length that consistently results in clearly defined ββ-sheet nanocrystals. For poly-alanine lengths below six, the ββ-sheet nanocrystals are not well-defined or not visible at all, while for poly-alanine lengths at and above six, the characteristic nanocomposite structure of silk emerges with no significant improvement of the quality of the ββ-sheet nanocrystal geometry. We present a simple biophysical model that explains these computational observations based on the mechanistic insight gained from the molecular simulations. Our findings set the stage for understanding how variations in the spidroin sequence can be used to engineer the structure and thereby functional properties of this biological superfiber, and present a design strategy for the genetic optimization of spidroins for enhanced mechanical properties. The approach used here may also find application in the design of other self-assembled molecular structures and fibers and in particular biologically inspired or completely synthetic systems.

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► Spider silk is a self-assembling biopolymer constructed from simple building blocks.
► We report atomistic-level structures of N. Clavipes   spider dragline silk.
► Obtained using an in silico approach based on replica exchange molecular dynamics.
► Natural poly-alanine repeat length of six residues defines a minimum length to form clearly defined ββ-sheet.
► For poly-alanine lengths below six ββ-sheet nanocrystals are not well-defined.