Raman Spectroscopy Study On The Fibrosis Mechanism Of Hen Egg White Lysozyme

Misfolding of natural proteins always leads to fibralation generation of?-fold-rich structures,triggering many neurodegenerative diseases,such as Alzheimer's disease.Since this fibrosis process is a complex and multi-step process and its dynamic behavior is sensitive to surrounding conditions,its mechanism is still very controversial.Thus,a great deal of studies have been conducted to investigate the corresponding mechanism and kinetics,in order to uncover potential interactions and provide crucial clues for developing miracle drugs to alleviate the progression of such diseases.In comparison to those conventional analytical approaches for morphologies and structures,we have predominantly applied Raman spectroscopy to approximately in situ investigate influence of succinimide and and silver nanoparticles on amyloid fibriliation kinetics of hen egg-white lysozyme,uncovering their interaction mechanisms at the molecular level.The main results are listed as followings:(1)Promotion Effect of Succinimide on Amyloid Fibrillation of Hen Egg-White Lysozyme.According to that the intermediate soluble oligomers and protofibrils are more toxic,reducing their concentrations in protein solutions by accelerating fibrillation is believed as a feasible strategy for treatment or remission of the diseases.Using hen egg-white lysozyme(HEWL)as a model protein,the promotion effect of succinimide was revealed from a series of experiments,e.g.atomic force microscopy(AFM),thioflavin T(ThT)fluorescence assay,Far UV circular dichroism(CD)and Raman spectroscopy,modeling the effect of succinimide-like derivative intermediates of intramolecular deamidation of backbone during amyloid fibrillation.The AFM measurement confirmed that succinimide effectively accelerated the morphological changes of HEWL,whilst,at the molecular level the accelerative transformation of protein secondary structures was also clarified by ThT fluorescence assay and Far UV CD spectroscopy.The incubation time-dependent Raman spectroscopy further revealed that the direct transformation from ?-helix to organized ?-sheets occurred with skipping the intermediate random coils under the action of succinimide.This "bridge" effect of succinimide was attributed to its special influence on disulfide bonds.In the presence of succinimide in protein solutions,the native disulfide bonds of lysozyme could be broken more efficiently and quickly within hydrolysis,resulting in exposure of the buried hydrophobic residues and accelerating the formation of cross ?-sheet structures.The present investigation provides very useful information for understanding the effect of intramolecular deamidation on the whole amyloid fibrillation.(2)Concentration-dependent influence of silver nanoparticles on amyloid fibrillation kinetics of hen egg-white lysozyme.Understanding the influence of nanoparticles on the formation of protein amyloid fibrillation is crucial to extend their application in related biological diagnosis and nanomedicines.In this work,Raman spec-troscopy was used to probe the amyloid fibrillation of hen egg-white lysozyme(HEWL)in the presence of silver nanoparticles(AgNPs)at different concentrations,combined with atomic force microscopy(AFM)and Thioflavin T(ThT)fluorescence assays.Four representative Raman indicators were utilized to monitor transformation of the protein tertiary and secondary structures at the molecular level:the Trp doublet bands at 1340 and 1360 cm-1,the disulfide stretching vibrational peak at 507 cm-1,the N-Ca-C stretching vibration at 933 cm-1,and the amide I band.All experimental results confirmed the concentra-tion-dependent influence of AgNPs on the HEWL amyloid fibrillation kinetics.In the presence of AgNPs at low concentration(17?g/ml),electrostatic interaction of the nanoparticles stabilizes disulfide bonds,and protect the Trp residues from exposure to hydrophilic environment,thus leading to formation of amorphous aggregates rather than fibrils.However,with the action of AgNPs at high concentration(1700 ?g/ml),the native disulfide bonds of HEWL are broken to form Ag-S bonds owing to the competition of electrostatic interaction from a great deal of nanoparticles.According to providing functional surfaces for protein to interact with,AgNPs play a bridge role on direct transformation from a-helices to organized ?-sheets.The present investigation sheds light on the controversial effects of AgNPs on the kinetics of HEWL amyloid fibrillation.(3)Raman spectroscopy characterizes intramolecular hydrogen bonds of protein tyrosines.Intramolecular hydrogen bonds of proteins assume a pivotal role in the stability and changes of protein structures.We basically confirmed that the characteristic peak of the Raman spectrum of lysozyme at 1197 cm-1 is contributed by the intramolecular hydrogen bonding of the tyrosine by comparing the aqueous lysozyme,phenylalanine and tyrosine variable temperature experiments.By analyzing and comparing the kinetic changes of this peak with incubation time,we were able to observe clearly the kinetic changes of local intramolecular hydrogen bonding in lysozyme.The characteristic peak at 1197 cm-1 during protein fibrillation is basically consistent with the kinetic of other tertiary structures,indicating that the dynamics of the tertiary structure changes during protein fibrillation will be well visualized by monitoring the changes of the intensity of this peak.