Silkworm Spinning:the Programmed Self-assembly From Natural Silk Fibroin To Superfibre

Silk is one of the best natural protein fibers and has the advantages of lightweight,biodegradable and excellent textile properties that are incomparable for synthetic materials.Today,the role of silk has been transferred from a traditional textile raw material to one of the star biomaterials,and show great application potential in flexible electronics,tissue engineering,intelligent storage,food industry and other fields.Different from artificial spinning,silk is spun by the silkworm at ambient temperature and pressure using aqueous silk protein solution.It remains a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionics for the incomplete understanding of silkworm spinning mechanism,especially the structure and assembly of natural silk fibroin(NSF)in the silk gland.Here,with the assistance of amphiphilic molecules amphipol and digitonin,we studied the structure and assembly of NSF by means of analytical ultracentrifugation(AUC),circular dichroism(CD)spectrum,metal shadowing and dynamic light scatting(DLS)and measured the structural properties of NSF under different ions and pH conditions,and proposed the theory of programmed self-assembly of NSF nanofibrils induced by ions an pH.The major results are as follows:1.Digitonin/amphipol could stabilize NSF in vitroLarge-scale screening showed that membrane scaffold protein MSP1D1 could improve NSF stability from 144 h to 240 h in vitro in a concentration-dependent manner.In contrast,BSA decreased NSF stability from 144 h to 96 h.Inspired by the amphiphilicity of MSP1D1,we found that amphiphilic molecules amphipol and digitonin had better stabilization effect on NSF than MSP1D1,which can keep NSF stable for 336 h without precipitation.These results suggest that amphiphilic molecules digitonin and amphipol contribute to the structural stability of NSF in vitro.To verify whether amphipol and digitonin changed the initial structure of NSF,NSF before and after the addition of digitonin was examined by CD spectrum and metal shadowing.Digitonin did not change the secondary structure of NSF.Indeed,it stabilized the secondary structure of NSF without altering its state.In contrast,NSF transformed rapidly from random coil to β-sheet structure in the absence of digitonin in vitro,leading to protein precipitation.Amphipol was not suitable in CD study for high background absorption,although it could also stabilize NSF in vitro.These results indicated that digitonin and amphipol did not change the initial structure and molecular morphology of NSF,which laid an important foundation for further studies on the structure and assembly of NSF.2.NSFs are present as nanofibrils composed of random coils in the silk glandTo elucidate the structure and properties of NSF in silk glands,the composition of NSF was first identified.Bi-dimensional blue native/SDS-PAGE showed NSF had four subunits,which were further identified as fibroin heavy chain(Fib-H),fibroin light chain(Fib-L),P25 and P25-like by mass spectrometry.P25-like is a new subunit identified in NSF.Its content is slightly lower than that of P25.These results showed NSF was a macromolecular complex containing Fib-H,Fib-L,P25 and P25-like.With the aid of digitonin,NSF from anterior silk gland(ASG)was purified by ions exchange and size exclusion chromatography.CD spectra suggested the secondary structure of NSF consisted mainly of random coils and a small number of α-helices from posterior silk gland(PSG)to ASG.With the aid of amphipol,AUC showed that the sedimentation coefficients of NSF from PSG,posterior of MSG(PMSG),middle of MSG(MMSG)and anterior of MSG(AMSG)were 5.788 S,5.766 S,5.798 S and 5.862 S,and the corresponding friction ratios were 4.088,4.145,3.982 and 4.056,respectively.Friction ratios indicated that NSF has a large axial(length-to-width)ratio,implying it may be a fibrous protein.The sedimentation coefficients were very close,indicating NSFs are almost identical from PSG to AMSG.Metal shadowing showed that NSFs were present as nanofibrils without morphological differences from PSG to ASG,which was in line with AUC analysis.Meanwhile,cryogenic transmission electron microscopy(Cryo-TEM)further confirmed the formation of NSF nanofibrils.The above results showed that from PSG to ASG,the secondary structure and molecular morphology of NSF did not change significantly,and it present themselves as nanofibrils formed by random coil structure.3.Metal ions induce the formation of NSF nanofibrilsNSF is regarded as a rod-like structure formed by non-covalent aggregation of globular fibroin protein in previous related researches.However,even after incubation with 8 M Urea,2%Triton X-100 and 15 mM dithiothreitol(DTT)for 18 h at 25℃,NSFs were still present as nanofibrils without the appearance of globules,suggesting NSF are not aggregates of globular protein.To elucidate the assembly mechanism of NSF nanofibers,we studied the effect of metal ions on NSF by metal shadowing and DLS.Metal shadowing showed that NSF itself did not form nanofibrils in water.When in 50 mM NaCl,NSF tended to form nanofibrils which disappeared again after dialysis.Hence,we investigated the effects of different kinds of metal ions on NSF nanofibrils’formation.At 1 mM,Na+ and K+induced NSF to form immature fibrous-like structure,while Ca2+ and Mg2+ induced NSF to assemble into nanofibrils.At 2.5 mM,Na+ and K+further induced NSF to assemble into nanofibrils.Once the nanofibrils were formed,they did not change with metal ions concentration increasing,even up to 300 mM.The results suggested metal ions are necessary for the formation of NSF nanofibrils,and the induction effect of divalent ions is stronger than that of univalent ions.DLS showed that the hydrodynamic radius and polydispersion coefficient of NSF were 21 nm and 20.5%in 50 mM NaCl,respectively.After dialysis with water,the hydrodynamic radius slightly decreased to 18.4 nm,and the polydispersion coefficient was multimodal.No spherical NSF particles with uniform size and smaller hydrodynamic radius appeared after dialysis,suggesting that NSF nanofibrils may disassemble to form intertwined peptide chains after dialysis with water,rather than globules.4.The decrease of pH improves NSF hydrophobicityIt is known that the pH in the lumen of silk gland of both silkworms continuously decreases from PSG to ASG,which could be mimicked by continuously decreasing pH from 8.0 to 4.8 within 12 h in vitro.CD spectra showed that pH decreasing did not change the random coil structure of NSF,which was consistent with the random coil structure of NSF observed from different segments of the silk gland.ANS fluorescence spectra showed that pH decreasing induced a gradual blue-shift of the maximum emission peak(λmax,507 nm)of NSF,indicating a gradual exposure of NSF hydrophobic residues and an increase of NSF hydrophobicity.Further,we measured the stability of the secondary structure of NSF at different pH,and found that the random coil structure of NSF was stable in pH 4.8～5.6 within 160 h.However,it gradually transformed into β-sheet structure in pH 6.0～8.0.Increasing pH promoted the transition of random coil to β-sheet structure of NSF.The results suggested that the decrease of pH from PSG to ASG does not change the random coil structure of NSF,but results in the exposure of the hydrophobic residues,thus improving the hydrophobicity of NSF.5.NSFs self-assemble into herringbone patterns near the spinneretThe orientation of NSF was observed in solution and in the spinning dope by metal shadowing,respectively.The results showed NSFs were present as nanofibrils randomly arranged in solution.While the concentration was higher than 0.15 mg/mL,NSF nanofibrils were tightly stacked with random arrangement,and did not change with the increase of NSF concentration.From PSG to ASG-1,most of NSF nanofibrils were tightly stacked with isotropic orientation,which was consistent with the orientation of NSF nanofibrils in solution.However,a small number of NSF nanofibrils formed an ordered arrangement.Interestingly,the long-range ordered molecular alignment of NSF nanofibrils was not observed in ASG-1,but near the spinneret of ASG-2,where NSF nanofibrils self-assembled into herringbone patterns,with the long axes of adjacent molecules aligned parallel to each other.The herringbone patterns were further packed together to form the anisotropic liquid crystalline spinning dope ready for silkworm spinning.Our study reveals the mechanism by which silkworms cleverly utilize metal ions and pH gradient in the silk gland to drive the programmed self-assembly of NSF from disordered nanofibrils to anisotropic liquid crystalline spinning dope(herringbone patterns)for silkworm spinning,thus providing novel insights into silkworm/spider spinning mechanism and bionic creation of high-performance fibers.