Self-Assembly of Recombinant Hagfish Thread Keratins Amenable to a Strain-Induced α-Helix to β-Sheet Transition

Abstract

Hagfish slime threads are assembled from protein-based bundles of intermediate filaments (IFs) that undergo a strain-induced α-helical coiled-coil to β-sheet transition. Draw processing of native fibers enables the creation of mechanically tuned materials, and under optimized conditions this process results in mechanical properties similar to spider dragline silk. In this study, we develop the foundation for the engineering of biomimetic recombinant hagfish thread keratin (TK)-based materials. The two protein constituents from the hagfish Eptatretus stoutii thread, named EsTKα and EsTKγ, were expressed in Escherichia coli and purified. Individual (rec)EsTKs and mixtures thereof were subjected to stepwise dialysis to evaluate their protein solubility, folding, and self-assembly propensities. Conditions were identified that resulted in the self-assembly of coiled-coil rich IF-like filaments, as determined by circular dichroism (CD) and transmission electron microscopy (TEM). Rheology experiments indicated that the concentrated filaments assembled into gel-like networks exhibiting a rheological response reminiscent to that of IFs. Notably, the self-assembled filaments underwent an α-helical coiled-coil to β-sheet transition when subjected to oscillatory shear, thus mimicking the critical characteristic responsible for mechanical strengthening of native hagfish threads. We propose that our data establish the foundation to create robust and tunable recombinant TK-based materials whose mechanical properties are controlled by a strain-induced α-helical coiled-coil to β-sheet transition.

Homo‐ and heteropolymer self‐assembly of recombinant trichocytic keratins

“…These fibers do not have the typical IF morphology, but are relatively thicker and shorter. Similar heterogeneity in self‐assembled structures has been recently observed for homo‐ and hetero‐oligomeric hagfish thread keratins (TK) . Although the assembly mechanism of TKs remains to be fully established, this observation is interesting considering the low sequence identity between TKs and vertebrate keratins and the low cysteine content of TKs in comparison to hard keratins.…”

“…Recombinant keratins, as well as other intermediate filament proteins, have been previously produced and their self-assembly process has been extensively studied. [27,[63][64][65][66][67][68][69][70][71][72][73][74][75][76] It is known that the precursor to IF formation is the dimerization of one type I (acidic) and one type II (basic) keratin protein to create a heterodimer. [63] These obligate heterodimers then align in an antiparallel, staggered conformation to form a tetramer.…”

Designing Smart Materials with Recombinant Proteins

“…Mechanical stimulation may be used as an environmental trigger since some proteins undergo structural changes in response to mechanical forces. For example, proteins from egg capsules of marine snails and from hagfish slime threads undergo strain‐induced transitions in their secondary structures from α‐helices to β‐sheets. Electrical stimulation may affect cardiac cell growth, and using conductive scaffolds to grow electrically sensitive cells may improve their growth .…”

Yarının Yüksek Performanslı Liflerine Doğal Bir Yaklaşım: Balık Asalağı Salgısı Lifleri

“…Elastik davranıştan plastik davranışa geçiş, α-sarmallarının parçalanıp β-levhalar biçimine dönüşmesine tekâmül etmektedir [32]. β-levhalarının oluşumu ile birlikte hidrojen bağlarının sayısı artmakta ve yüksek kopma mukavemetine sahip salgı lifleri oluşmaktadır [9,10,27,31].…”

“…Balık asalaklarının kültür olarak üretimi ve salgılarının toplanması neredeyse imkânsız olduğundan bu liflerin ticari üretimi için ara filament proteinlerinin laboratuvar ortamında üretilip çoğaltılabilmesi üzerine araştırmalar halen devam etmektedir [46,47]. Kanada'da Guelph Üniversitesi tarafından yürütülen bir araştırmada Örümcek ipeğinin üretiminde başarılı sonuçlar veren genetik olarak işlenmiş bakterilerin kullanımı, balık asalağı salgısı liflerinin üretiminde de denemekte yani balık asalağı ara filament proteinlerini üreten DNA bakterilere aktarılarak, bu proteinin bakteriler tarafından üretilmesi amaçlanmaktadır [32,47,48]. Araştırmacılara göre, bu çalışmadan başarılı sonuçlar elde edilmesi halinde bu lifler; otomobillerin hava yastıklarından koruyucu giysilere birçok teknik tekstillerde kolaylıkla kullanılabilecek bir hammadde haline geleceği düşünülmektedir [46,48].…”