Interplay between glutamine-containing amino acid sequence motifs and hydration in the secondary structure and liquid-liquid phase separation of spider dragline silk

Spider dragline silk has several characteristic properties including high toughness and supercontraction. However, key motifs for the supercontraction remain unclear. The supercontraction of spider draglines is related to hydration of spider silk as well as the QQ motifs according to previous reports, and hence we designed and synthesized a novel MaSp1 variant containing an AGQG motif and investigated its hydration behavior, focusing on the role of Q or QQ-containing motifs. On the basis of molecular dynamics (MD) simulations and Fourier transform infrared (FT–IR) spectroscopy, we analyzed the secondary structural changes during hydration and their relationship with hydration. At relative humidities (RHs) > 80%, the water uptake of the AGQG variant was greater than that of AGAG, indicating increased hydration around Q residues. Simulations and hydration free energy calculations confirmed that AGQG is more readily hydrated, with stable water association near Q. Increasing the Q content, particularly in the QQ motif, induced intramolecular hydrogen bonding during hydration, resulting in main-chain twisting and bending. FT-IR measurements and liquid-liquid phase separation (LLPS) experiments indicate that the hydration-driven structural changes do not affect LLPS significantly. These results highlight the critical role of Q-containing motifs in linking molecular hydration. This paper uses molecular dynamics simulations and Fourier transform infrared spectroscopy to analyze secondary structural changes during hydration of spider dragline silk. At relative humidities <80%, water uptake of the AGQG variant is greater than that of AGAG, indicating increased hydration around Q residues.