Studies On Environmental-variation Induced Conformational Transition Of Silk Fibroin With FTIR And The Properties Of Silk Fibroin Blend Film

The conformational change of proteins has long been a fundamental issue in science.Studies on silk and silk proteins show that the excellent mechanical properties of silk fibers are closely related to their conformation.Theβ-sheet conformation is related to the strength of the silk fibers,and the content of theα-helix and the random coil structures is related to the elasticity of the silk fibers.Under the variation of environmental conditions,such as organic solvent,temperature,metal ion,pH change and shear force,the conformations of silk fibroin may change accordingly.Therefore,studies on the conformational transition process of silk fibroin contributes to an in-depth understanding of the superior mechanical properties of silk.Two-dimensional correlation spectroscopy（2D-COS）has been widely used in the study of protein conformational changes.These studies were essentially based on the two so-called major advantages of two-dimensional correlation spectroscopy:（1）band resolution enhancement;（2）“sequence order”derivation of spectral signal changes under external perturbation.As a result,almost all studies on protein conformational changes using generalized 2D correlation spectroscopy have concluded that the conformational change of a protein is a process with sequential elements.However,our past theoretical simulations and experimental results showed that the sequence order results obtained by the“sequence order”rule in two-dimensional correlation spectroscopy are not universally applicable.At the same time,the first advantage of the two-dimensional correlation analysis technique also has large uncertainty.In response to the above problems,combining in situ FT-IR analysis methods（second derivative,Fourier self-deconvolution,curve fitting）and two-dimensional infrared correlation spectroscopy,we systematically studied the secondary structure changes of silk fibroin under different environmental perturbations,hoping to answer a fundamental scientific issue:Whether the secondary structure change of silk fibroin induced by environmental perturbations is completely synergistic or has a certain sequence order.In addition,based on the principle of conformational transition of silk fibroin induced by chemical factors,we intend to improve the properties of the silk fibroin-modified polyvinyl alcohol composite film by regulating the silk fibroin conformation,thus finding an application background for this purely basic research.The main research contents are as follows.In Part 1,the infrared spectra of silk fibroin films in the temperature range of 130^oC to 220℃ were first collected.Then,combined with two-dimensional correlation spectroscopy,Fourier self-deconvolution,second-order derivative,and curve fitting techniques were used to analyze the secondary structure changes of regenerated silk fibroin during the temperature rise from 130 to 220℃.Finally,the“sequence order”results from in-situ spectral analysis were compared with those obtained by two-dimensional correlation analysis.It was found that the sequential order derived from generalized 2D correlation spectroscopy is apparently doubtful;β-sheet was the first one to change at low temperature,then the random coil,followed by the nonamide C=O and,finally,theα-helix.A detailed in situ infrared spectral analysis showed that the main secondary structures of silk fibroin,includingα-helix,β-turn,random coil andβ-sheet（high-wavenumber component）,all changed with a fully cooperative manner at a relatively low temperature of 150℃.But the low-wavenumber component ofβ-sheet started to change at a higher temperature of 180^oC.Besides,it has also been found that,before 200℃,the loss ofα-helix and random coil was transformed intoβ-turn,β-sheet,and nonamide C=O.After 200℃,someβ-turn structure was also disruptured and transformed intoβ-sheet and nonamide C=O.At the same time,the conformational transitions of silk fibroin in the temperature range（30-80℃）under different conditions were compared.The results showed that water plays an important role in the conformational transition of silk fibroin.Finally,the thermal degradation behavior of silk fibroin was investigated by combining thermogravi-infrared spectroscopy,solid-state nuclear magnetic resonance and infrared spectroscopy.When the silk fibroin film is treated at high temperature,it will degrade and produce small molecules such as CO₂ and NH₃,and contains small amounts of non-amide C=O.The conformation of silk fibroin changes from a random coil/α-helical structure to aβ-sheet conformation.In Part 2,we studied the conformation transition of silk fibroin induced by alcoholic organic solvents,different pH values and different calcium ion contents.1)The secondary structure transformation of silk fibroin under the induction of methanol,ethanol and different contents of ethanol was simultaneous.There was no sequence order.The secondary structure of silk fibroin induced by methanol,ethanol and n-propanol was transformed from random coil,α-helix,andβ-turn toβ-sheet conformation.The conversion rate of silk fibroin in three alcohol solvents was methanol>ethanol>n-propanol,in which methanol could induce the fastest and most abundantβ-sheet conformation of silk fibroin,and the conformation conversion rate of fibroin in n-propanol is slowest.2)The conformation of silk fibroin induced by different pH values was changed simultaneously,and there was no sequence order.When pH was increased,the secondary structure of silk fibroin gradually changed fromβ-sheet andβ-turn toα-helix and random coil,i.e.,it is more favorable for the formation ofβ-sheet under weakly acidic conditions,and the stability ofα-helix and random coils is more favorable under weakly basic conditions.3)After the addition of different amounts of calcium ions to the ethanol-induced silk fibroin dispersion in 1),the dispersion becomes clear.In the presence of calcium ions,it is more favorable for the formation ofα-helix and random coil in the secondary structure of silk fibroin.As the concentration of calcium ions increases,theβ-sheet andβ-turn conformations gradually change toα-helix and random coil conformation.This study is helpful to understand that calcium chloride solution can dissolve silk to prepare an aqueous solution of regenerated silk fibroin,thereby preparing a series of silk fibroin-based materials.In Part 3,time-resolved spectroscopy was used to study the effect of pressure on the conformation of silk fibroin in its wet film.Pressure does not induce a conformational change in the dry film of silk fibroin;however,when the silk fibroin film contains some heavy water,pressure can induce a transition from random coil,α-helix,andβ-turn conformations toβ-sheet structures.At a constant pressure and constant heavy water content,the time-resolved process of the conformational transition of silk fibroin in the silk fibroin film can be divided into three stages.In the initial stage,the random coil,α-helix andβ-turn conformation are converted toβ-sheet conformation;in the second stage,the random coil andβ-turn conformation are converted toβ-sheet(1618 cm^-1)andα-helix(1647 cm^-1,⁵%);In the final stage,there was no significant change in the contents of secondary structure of silk fibroin and it was in the equilibrium stage.In Part 4,we prepared the silk fibroin/polyvinyl alcohol cross-linked composite film based on the principle of ethanol and acidic solution induced the conformational transition of silk fibroin,in order to regulate the performance of the composite film by regulating the silk fibroin conformation.To reduce the phase separation between silk fibroin and polyvinyl alcohol,glutaraldehyde was used as a cross-linker.The changes of the conformation of silk fibroin,the mechanical properties of the composite film,the degree of phase separation and the maximum temperature of weight loss after the composite film was treated with alcohol solution and acidic solution were compared.The degree of phase separation of SF/PVA composite film after glutaraldehyde cross-linking was significantly lower,a complete and uniform composite film can be obtained.In the composite films treated with ethanol solution,the silk fibroin molecular chains were converted intoβ-sheet conformations,which resulted in closer molecular arrangement and more regular conformation,thereby further improving the mechanical properties of SF/PVA cross-linked composite films.In the acidic solution-treated composite films,the silk fibroin molecular chains can also be rearranged to form part of theβ-sheet structure,but the relative content is lower than the ethanol-inducedβ-sheet conformation.Therefore,the mechanical properties of the composite film treated by the acidic solution is lower than those treated by ethanol.