The Kinetic Mechanism Of H~+ In Thermal Denaturation Of Lysozyme By Raman Spectroscopy

During the process of performing its specific functions,plenty of polypeptide folding and unfolding processes take place inside proteins.In this process,proteins are prone to the influence exerted by environmental factors and misfolded,leading to protein degeneration or amyloid fibrillation.In view of this,it is an absolute necessity to gain a precise understanding of how the secondary and tertiary structure changes and the effect of the local environment on protein denaturation.In this article,Raman spectra with vibration resolution was used to synchronously record the changes occurring to Raman shift,half height and full width(FWHM)of every single fingerprint group in the denaturation of lysozyme to perform analysis of the changes in its secondary structure and tertiary structure during denaturation.(1)lysozyme denaturation under heat and acidic conditionsIn this study,Raman spectroscopy was involved to perform study on the kinetic mechanism of lysozyme denaturation under high temperatures induced by H+.Over the course of denaturation,some of the Raman bands were synchronously recorded and then analyzed,which led to the discovery that as the pH value of lysozyme solution declined,the characteristic temperature of the secondary structure and tertiary structure dropped to varying degrees,suggesting that the denaturation of lysozyme was expeditedby the presence of H+.Throughout this process,H+ reduced the influence exerted byhydrogenbond,which resulted in the destruction of the stable tertiary structure and promoted unfolding of protein.Moreover,it was discovered that the Raman bands were largely arrributed to the side groups(Trp)which were distant to lysozyme molecular surface changed before those of side groups present on the surface(Tyr,Phe).This is clearly indicative of the inside-out mechanism caused by H+ in the process of lysozyme denaturation under the rising temperature.The promoting mechanism of H+that plays out during lysozyme thermal denaturation is observed by using the dynamic Raman spectroscopyin greater detail.(2)Amyloid fibrillation of lysozymeOne characteristic of neurodegenerative disease lies in the formation of amyloid fibrosis,which are accompanied by the appearance of ordered folding structure.This conformational transformation is believed to be in close association with the potential virulence of the pathogenic mechanism.Despite this,the definite kinetic mechanism of conformational transformation of lysozyme in amyloid fibrillation has yet to be illustrated.In this article,a study was performed of the structure changes to HEWL under thermal and acidic conditions(pH=2.0,65?)by using Raman spectroscopy and atomic force microscope(AFM)and ThT fluorescence,which led to a discovery that the conformational transformation of amyloid fibrillation conformed to the four-stage step-by-step mechanism.During the initial stage from 0 to 10 hours,by heating the lysozyme under acid conditions,the spherical three-dimensional structure unfolded immediately.Meanwhile,Trp groups characterizing the tertiary structure of lysozyme gradually were exposed to the surface of protein and accompanied by the destruction of the a-helix structure,which shaped the statistic coils and caused the assemble of lysozyme to be oligomer.In this process,?-sheet structure was yet to be formed and the size of oligomer was excessively small for detection by AFM.In the middle stage of 10-40 hours,a-helix structure was further damaged,which was followed by a larger number of statistic coils being shaped.In addition,there remained no ?-sheet structure observed to be formed.However,at the moment when the Trp groups on the side chain of lysozyme was further exposed to water,lysozyme was found to start to gather into spherical oligomers which were detectablefor AFM.There were no further significant changes occurring to the two processes until it reached 40 hours.With 40-90 hours later,the statistics coils structure started to be mostly transformed into the inter-molecular ?-sheet structure and the lysozyme molecules started to form fibrillar diametered about 6-12nm.During the final 90-160 hours,the formed fibrils gradually self-assembled into mature amyloid fibrosis and the process only continued until 160 hours later,which provides the requisite information to realize the artificial control of amyloid fibrillation in patients with neurodegenerative diseases.Although the effect of acids on protein thermal denaturation has been known,the mechanism of alkali on protein denaturation is not clear yet.We can choose proteins that can be stable under alkaline conditions to study the effect of alkali on protein thermal denaturation,which can improve the mechanism of protein denaturation.