Seeking Solvation: Exploring the Role of Protein Hydration in Silk Gelation

Abstract

The mechanism by which arthropods (e.g., spiders and many insects) can produce silk fibres from an aqueous protein (fibroin) solution has remained elusive, despite much scientific investigation. In this work, we used several techniques to explore the role of a hydration shell bound to the fibroin in native silk feedstock (NSF) from Bombyx mori silkworms. Small angle X-ray and dynamic light scattering (SAXS and DLS) revealed a coil size (radius of gyration or hydrodynamic radius) around 12 nm, providing considerable scope for hydration. Aggregation in dilute aqueous solution was observed above 65 °C, matching the gelation temperature of more concentrated solutions and suggesting that the strength of interaction with the solvent (i.e., water) was the dominant factor. Infrared (IR) spectroscopy indicated decreasing hydration as the temperature was raised, with similar changes in hydration following gelation by freezing or heating. It was found that the solubility of fibroin in water or aqueous salt solutions could be described well by a relatively simple thermodynamic model for the stability of the protein hydration shell, which suggests that the affected water is enthalpically favoured but entropically penalised, due to its reduced (vibrational or translational) dynamics. Moreover, while the majority of this investigation used fibroin from B. mori, comparisons with published work on silk proteins from other silkworms and spiders, globular proteins and peptide model systems suggest that our findings may be of much wider significance.

Broadband Multidimensional Spectroscopy Identifies the Amide II Vibrations in Silkworm Films

“…The use of linear infrared spectroscopy to study the molecular structure of protein in unpurified biological materials like silks is very common [ 1 , 2 , 3 , 4 ]. Among all vibrations, the most studied is the amide I vibration, which consists mostly of the stretching of the carbonyl.…”

In Situ Identification of Secondary Structures in Unpurified Bombyx mori Silk Fibrils Using Polarized Two-Dimensional Infrared Spectroscopy

“…The β-sheet appears to be the most stable form and dominates the crystalline content of natural silk fibers, while helical and random structures can be observed in the noncrystalline material . Less stable helical and random coil structures dominate films cast from water by evaporation under mild conditions, assumed to reflect the prevalence of random coil conformations in aqueous solution. − Upon exposure to high humidity, the increased water content causes glass-transition-induced softening and an increase in extensibility, as helical structures can be converted to swollen random coil structures, while the β-sheet content can increase . Multiple techniques have been applied to study the silk–protein structure under different humidity conditions, providing a clear understanding of the adopted conformation in the nanocrystals.…”

Flow-Induced Protein Chain Deformation, Segmental Orientation, and Phase Separation in Native Silk Feedstock

“…All experiments were performed using NSF specimens freshly taken from the middle-posterior sections of silk glands dissected from commercially bred B. mori silkworms in their 5th instar, at the earliest stages of cocoon construction, as described previously. ,,− …”

“…A closer examination of the entropically penalized solvation shell hypothesis has been reported recently, which suggested that the loss of independent motion caused a significant decrease in both the heat capacity ( C p ) and entropy (Δ S ) of the bound water relative to free water. Thus, it appears that a thermodynamic explanation can be given for fibroin gelation in NSF at elevated temperatures or following freezing.…”