Fourier Infrared Spectroscopy Characterization On The Secondary Structure Of Silk Fibroin

Silk fibroin based materials possess many attractive properties that make them assuitable candidates for various potential applications.Understanding the secondarystructure of silk fibroin is vital if we are ever to truly capitalize upon its superiormechanical properties.Fourier transform infrared spectroscopy is very sensitive tomolecular structure and has been extensively used in the study of protein structure aswell as conformation transition.Hereby near-infrared (NIR) and attenuated totalreflectance (ATR) spectroscopy were employed in our study to investigate the effectof external disturbance,namely water content,thermal and chemical treatment,on thestructural modification of silk fibroin.Additionally,spectral results revealed that theconformation of peptide chains might be contributive to the rheological characteristicof silk solution.The dynamic spectra were analyzed by 2D correlation method whichcan not only provide better resolution,but also distinguish the motion sequential orderof different functional groups of materials.Thus progress had been made regardingthe relationship between molecular mechanisms and spectral signals.The ability to control the processing of silk protein based materials is the key tosuccessful applications of this important and high performance biopolymer.Rheological measurements were applied to regenerated silk fibroin solution withdifferent molecular weight cut-offs and concentrations,observations revealed theexistence of critical concentration Cg below which an extended gel network can form.This concentration dependence was explained as the preponderance for reconstitutedsilk molecules to form either inter- or intra- molecular bonds depending on theirconcentration.It showed that a change in rheological properties can be linked to achange in fibroin structures present in solution.The results may have importantimplications for future successful artificial processing of silk.Based on our comprehension on silk fibroin solution,NIR spectra of various silkfibroin films cast from its aqueous solution were collected for the characterization ontheir secondary structure.Clear band assignment was made and the peak at 4530 cm^-1 was assigned to the combination mode of amide A and amideⅢcharacteristic ofβ-sheet.In the NIR spectrum of silk fibroin,analysis on the combination modes ofamide groups could lead to a profile of conformation ratio,which was confirmed bythe ATR and NMR observation.Therefore,NIR spectroscopy was proved to be arapid and effective tool to investigate qualitatively or even quantitatively theconformation of the RSF (regenerated silk fibroin) film with different thicknessesregardless of the disturbance effect of water.Furthermore,NIR spectroscopy was used to monitor dynamic changes in thehydration and secondary structure of silk fibroin induced by thermal and chemicaltreatment.Inspired by differential scanning calorimetry (DSC) observations wesuggested that the formation ofβ-sheets in wet film should be strongly related to thedissociation of hydrogen bonds between water and peptide residues,and the freezingwater might facilitate the movement of peptide chains and thereby contribute to theconformational transition at 60℃.In addition,structural changes of fibroin inducedby higher temperature and ethanol aqueous solution were also analyzed respectively.By monitoring the dynamic variations of the spectral components,an NIR procedurefor tracking the conformational transition of silk fibroin was established.At last,time-resolved ATR infrared spectroscopy was used to monitor thestructural change of silk fibroin in water and chemical vapor respectively in situ,thereafter the mechanism of the conformation transition as well as water absorptionand de-association in silk fibroin at room temperature was discussed.The ATRobservations showed that acetic acid was contributive to the highly dominatingcontent ofβ-sheet conformation in silk fibroin film.The kinetic curve of solventdiffusion was similar to theβ-sheet growth,which indicated that the access of aceticacid into silk fibroin architecture was a necessary step to break the original hydrogenbond of silk fibroin and to force the re-alignment of peptide chains.Meanwhile,threetypes of water were distinguished in this dynamic system,whose motion sequentialorder was illustrated by the two-dimensional correlation analysis.