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Native silk fibers exhibit strength and toughness that rival those of the best synthetic fibers. Despite significant research, further insight is still needed to understand the mechanisms by which silkworms are capable of spinning such tough fibers. Here we propose that π-π and π-OH group interactions of tyrosine side chains provide templating effects, such that the crystal-forming domains are in registration, thereby fostering the self-assembly of the spinning dope. Intrinsic fluorescence measurements, in conjunction with circular dichroism, showed that during self-assembly of regenerated silk solutions, the tyrosine residues were localized in a more hydrophobic local environment, suggesting preferential assembly. In situ Fourier transform infrared spectroscopy indicated that cross-linking of the tyrosine residues resulted in the development of extended β-sheet structure. Additionally, control of cross-link density directly influenced the degree of crystallinity upon drying. Molecular dynamics simulations were performed on silk mimetic peptides in order to more thoroughly understand the role of tyrosines. The results indicated that tyrosine residues tended to transiently colocate in solution due to π-π interactions and hydrogen bonds with adjacent residues and with the peptide backbone. These more stable tyrosine interactions resulted in reduced lateral chain fluctuations and increased incidence of coordinated intrachain association, while introduction of a dityrosine bond directly promoted the formation of β-sheet structures. In total, the experimental and modeling data support a critical role for tyrosine-tyrosine interactions as a key early feature in the self-assembly of regenerated silk protein chains and therefore are important in the robust and unusual mechanical properties ultimately achieved in the process. [ABSTRACT FROM AUTHOR]

Fibrous protein secondary structuraltransitions are affected bybound water. A family of specially designed recombinant spider silk-likeblock copolymers with a gradient of block length, hydrophobilicity,and degree of crystallinity was biosynthesized and characterized todemonstrate the effect of water on the protein structural transitions.These proteins were inspired by the genetic sequences found in thedragline silk of Nephila clavipes, comprising analanine-rich hydrophobic block, A, a glycine-rich hydrophilic block,B, and a C-terminus or a His-tag, H. Because the A-block is hydrophobicand the B-block is hydrophilic, the spider silk-like block copolymersbehave as amphiphilic molecules and self-assemble into various structuresin water solution. We employ time-resolved Fourier transform infrared(FTIR) spectroscopy to assign the origin of specific secondary structuraltransitions during heating. A transition from random coils to β-turnsdominates during a lower temperature glass transition (of plasticizedprotein) mediated by the removal of bound water. Once the proteinis in the dry solid state, further heating causes the now-dry proteinto undergo the glass transition to the liquid state through conversionof α-helices into β-turns. The structural transitionsduring protein glass transitions are intrinsic to the amorphous regionof protein and are hardly affected by protein hydrophobicity, blocklength, or crystallinity. The self-assembly morphology of the spidersilk-like block copolymers, investigated by scanning electron microscopy,indicates that the large-scale morphology is stable during heatingthrough both the lower and upper temperature glass transitions. [ABSTRACT FROM AUTHOR]

Substrate mechanical properties have remarkable influenceson cellbehavior and tissue regeneration. Although salt-leached silk scaffoldshave been used in tissue engineering, applications in softer tissueregeneration can be encumbered with excessive stiffness. In the presentstudy, silk-bound water interactions were regulated by controllingprocessing to allow the preparation of salt-leached porous scaffoldswith tunable mechanical properties. Increasing silk-bound water interactionsresulted in reduced silk II (β-sheet crystal) formation duringsalt-leaching, which resulted in a modulus decrease in the scaffolds.The microstructures as well as degradation behavior were also changed,implying that this water control and salt-leaching approach can beused to achieve tunable mechanical properties. Considering the utilityof silk in various fields of biomedicine, the results point to a newapproach to generate silk scaffolds with controllable properties tobetter mimic soft tissues by combining scaffold preparation methodsand silk self-assembly in aqueous solutions. [ABSTRACT FROM AUTHOR]

Silkworms and spiders generate fibers that exhibit highstrengthand extensibility. The underlying mechanisms involved in processingsilk proteins into fiber form remain incompletely understood, resultingin the failure to fully recapitulate the remarkable properties ofnative fibers in vitro from regenerated silk solutions. In the presentstudy, the extensibility and high strength of regenerated silks wereachieved by mimicking the natural spinning process. Conformationaltransitions inside micelles, followed by aggregation of micelles andtheir stabilization as they relate to the metastable structure ofsilk are described. Subsequently, the mechanisms to control the formationof nanofibrous structures were elucidated. The results clarify thatthe self-assembly of silk in aqueous solution is a thermodynamicallydriven process where kinetics also play a key role. Four key factors,molecular mobility, charge, hydrophilic interactions, and concentrationunderlie the process. Adjusting these factors can balance nanostructureand conformational composition, and be used to achieve silk-basedmaterials with properties comparable to native fibers. These mechanismssuggest new directions to design silk-based multifunctional materials. [ABSTRACT FROM AUTHOR]