A Terahertz Spectral Study Of Structure Changes For Solid-state B.mori Silk Fibroin

Silk from Bombyx mori silkworm is one of the most interesting biomaterials,consisting of two types of structural proteins,fibroin and sericin.The fibroin transforms from a gel to a sol state in the silk glands,finally result into solid filaments with impressive toughness and strength.It has been well established that similar to those of spider silks,the mechanical properties of silkworm silk is positively correlated with the β-sheet crystal structure in fibroin.The structure transition of fibroin occurring in the gland,may help us to gain insight into how spin filaments at low hydraulic pressures and temperatures without using any noxious chemicals as solvents.On the other hand,silk fibroin is widely used as a building block for biodegradable applications,including biomedical,optic,electronic,and optoelectronic devices,owing to its outstanding biological and optical properties.To fabricate such devises,degummed silk needs be dissolved by using cosolvents,e.g.CaCl2-EtOH-H₂O system and LiBr aqueous solution,and then reshaped into films,spongy-like membranes,fibers,or other shapes.While the reconstitution for silk will disrupt intermolecular forces and protein molecules,leading to significant reduction in the mechanical forces.Generally,reconstituted silk is much more fragile and stiffer in comparison with that of native silk thread.As a consequence,the knowledge on fibroin assembly is essential for both the understanding of silkworm spinning mechanism and the fabrication of silk-based devices.The mechanism of β-sheet crystallization in silk fibroin remains unclear,due to the incomplete information of protein assembly and structural state.The emerging terahertz（THz）spectroscopy（<10THz）has been taken as an important tool to detect new aspects of biomolecular structure and is used for the first time to analyze the structural changes of Bombyx mori silk fibroin.In Chapter 2,the room-temperature terahertz spectra of 18 kinds of amino acids consisting of Bombyx mori silk,are presented along with detailed solid-state density functional theory（DFT）analyses of the low-frequency vibrational modes and spectral features.At 294 K,glycine（Gly）exhibts two terahertz absorption features（1.59,and 2.47 THz）,alanine（Ala）exhibits one terahertz absorption feature（2.49 THz）,serine（Ser）have two terahertz absorption features（2.02,and 2.77 THz）,tyrosine（Tyr）have four terahertz absorption features（0.96,1.92,2.08,and 2.69 THz）.The origin of these absorption features for Gly,Ala,and Ser have been assiganed through DFT simulations to arise from the amidogen and carboxyl groups.While,the origin of the absorption features for Tyr have been assiganed to be related with the benzene ring.In Chapter 3,we studied the repeating fragments of Bombyx mori silk fibroin.The repeating fragments included dipeptide Gly-Ala,Gly-Ser,Gly-Tyr and their isomerides,Ala-Gly,Ser-Gly,and Tyr-Gly.Two kinds of hexapeptide Gly-Ala-Gly-Ala-Gly-Ser and Gly-Ala-Gly-Ala-Gly-Tyr are also studied by terahertz time-domain spectroscopy and Hartree-Fock（HF）simulation.In Chapter 4,we investigated the effects of sampling handling,laser system,temperature on terahertz absorption spectrum of silk fibroin,both from Bombyx mori silk fibroin and regenerated silk.In Chapter 5,we investigated the structural changes of Bombyx mori silk fibroin during silkworm spinning and reconstitution process.X-ray diffraction,Fourier transform infrared spectroscopy,polarized optical microscopy,and terahertz（THz）spectroscopy within 0.2 and 10 THz were applied to monitor the detailed structural features of silk fibroin from the posterior and middle divisions of silk gland,to fibers,and next to reconstituted fibroin.Results show that the fibroin from silk gland to fiber,experiences a significant transformation in crystal structure from a pure silk I state,to a silk I-rich mixed structure,and finally to silk II state,accompanied with a change in secondary structure from a-helix and random coil structures to β-sheets.The preferential orientation of crystal axes plays a key role in the formation of silk II state.The lack of both oriented crystallization and β-sheets conformation hinders the reconstituted silk fibroin from having mechanical properties compared with its natural counterparts.In 2-10 THz spectral region,two silk characteristic peaks is observed for the first time;their strength ratio is found to be correlated strongly with the β-sheets conformation in silk fibroin.In 0.2-2.0 THz region,the absorbance and the absorption coefficient also changes from natural fibroin to reconstituted fibroin.These differences in THz spectra cannot separate from the changes in the silk overall structure during spinning or reconstitution.THz analysis showed that in THz spectral region there are two characteristic peaks for silk,centered at 3.6-4.1 THz and 7.2-7.9 THz respectively.The absorbance strength ratio of Ipeak（7.2-7.9THz）/lpeak（3.6-4.1 THz）was positively correlated with the β-sheets conformation in silk.We further observed that the THz absorbance as well as the absorption coefficient properties ranging from 0.2 to 2.0 THz,was significant different between natural and reconstituted fibroins,indicating diverse conformational freedom in natural and reconstituted fibroins.In Chapter 6,terahertz（THz）spectroscopy（<10THz）has been taken as an important tool to detect the methanol-water（MeOH）induced structural changes of Bombyx mori silk fibroin.Mid-infrared spectroscopy（IR）and X-ray diffraction（XRD）results show that silk fibroin initially exists in a typical silk I form and reassemble into a predominant silk Ⅱ（antiparallelβ-sheet crystal）structure after MeOH treatment.The samples treated with MeOH-H₂O mixed solutions show a predominant silk I structure without any silk-Ⅱ-related peaks.As the MeOH concentration approaching to 40 vol%,the absorbance of the β-sheet-related IR bands and the XRD peaks gradually increase,indicating a formation of β-sheet crystal during this process.THz spectrum shows the absorption capacity below 3 THz as well as the absorbance at 5.1 THz and 7.9 THz is indeed affected by the MeOH-H₂O treatment,implying a MeOH-H₂O-dependent change of intermolecular H-bonds in silk fibroin.The THz spectrum for silk fibroin gives additional information to the existing studies on the β-sheet crystallization of silk fibroin,which may help us understanding the structural changes of nature silk.This study provides an important technique for the study of the effects of water and methanol on silk fibroin,as well as new information about the β-sheet crystallization in fibroin-based material.IR study reveals that theβ-sheet-related regions and the 1515 cm-1 band are both affected by MeOH-H₂O treatment.The silk fibroin THz spectra display the absorption capacity below 3 THz as well as the absorbance at 5.1 THz and 7.9 THz keep changes along with the change of MeOH concentration.This indicates the low-frequency THz absorption capacity and special THz band regions provide a reliable direct signal corresponding to the observed change from the MeOH-induced structure as well as the process of crystallization.This study provides a picture about the structural features of the silk fibroin from silkworm gland,to fibre,and next to artificial reconstituted film.This picture contains XRD,polarized light microscope,and spectral range between 4000 cm-1 and 6.6 cm-1（0.2 THz）.All of these observations will help in the study of overall structure in silk fibroin to understand more completely the fibroin assembly process in natural spinning and reconstitution process.This application will help in the study of macromolecular science to understand more deeply in silk fibroin-based materials.