Denaturation And Fibrillation Of Protein Lysozyme By Raman Spectroscopy

Protein unfolding and misfolding commonly exist in the biological processes[1-4]and are related to some serious deseases like Parkinson's disease or Alzheimer's disease[5-10].The previous investigations mainly focuses on the macroscopic characteristics of amyloid growth during protein misfolding(e.g.morphology changes,etc.),but the studies of protein denaturation processes,especially the nucleation process,in molecular level is very limited.Therefore,although there are many literatures discussing the protein fibrosis,the accurate mechanism of amyloid formation is still debatable due to the lack of direct spectroscopic and dynamic evidences,especially,the lack of a detailed microstructure description of the oligomer and fiber molecules.The change of protein secondary or tertiary structures and the microenvironment variation play a vital role in understanding the mechanism of protein degeneration.In this dissertation,we utilize the lysozyme as an example protein to study the change of secondary and tertiary structures during its thermal or chemical denaturation and fibrillation by Raman spectroscopy.The vibrational peaks of S-S,N-C?-C functional groups and amide I band are selected as the spectral fingerprints to reflect the secondary structure change of lysozyme,especially the populations of ?-helix and ?-sheet structrues in fibrillation.In addition,the qualitative and quantitative analyses of amino acid side chains,tryptophan,tyrosine,phenylalanine and C-H groups,are employed to study the molecular interactions and the microenvironment change,in order to understand the tertiary structure change.(1)Thermal and chemical denaturation of lysozymeThe thermal or chemical denaturation mechanism of hen egg white lysozyme has been controversial until now.Two different models were employed to describe the unfolding process of proteinll[11,12].One is the single-stage model[13-15,and the other is the multi-stage model.In this article,Raman spectroscopy are applied to study the thermal and chemical denaturation mechanism of lysozyme.All the Raman bands are recorded and analyzed during the denaturation process.It is found that the Raman bands of the side groups are changed prior to those of the skeleton groups.This directly confirms the two-stage mechanism of thermal denaturation of lysozyme.In the first stage,the tertiary structures are changed with a midpoint transition temperature of about 74 ?.In the next stage,the secondary structures are changed at about 76.5 ?.The intermediate with an incompact tertiary structure and intact secondary structure is verified in the thermal denaturation of lysozyme,so-called the molten globle intermediate.This preferential changes of the side groups are also found in the chemical denaturation of lysozyme by guanidine hydrochloride.In the first stage,the tertiary structures are changed at about 31%(m/m)of guanidine hydrochloride.Subsequently,the secondary structures are changed at about 32.5%(m/m)of guanidine hydrochloride.Moreover,the "outside-in" denaturation behavior is confirmed,in which the chemical denaturants prefer to interact with the surface amino acids of protein than those in the core.The chemical denaturation of lysozyme is also conformed as the multi-stage process instead of a simple two-stage mechanism.Many intermediates are found in the chemical denaturation of lysozyme,and the molten globle intermediate is just one of them.In summary,the Raman spectroscopy not only show the direct experimental evidences of the multi-stage mechanism of lysozyme denaturation process,but also clearly suggest the characteristic dynamics of the tertiary and secondary structure changes in denaturation.(2)Amyloid fibrillation of lysozymeProtein fibrosis is a hallmark of neurodegenerative disease,accompanied with the formation of an ordered cross-? core.The conformational transform of secondary structures is considered to be related to the pathogenic toxicity.However,the protein structure change during the amyloid formation is still in questions.The present study is mainly focused on the amyloid formation of lysozyme under heat-acid condition using Raman spectroscopy.We not only find a novel in situ Raman spectral fingerprint to trace the succinimide derivative intermediate during amyloid formation,but also provide a clear overall dynamics of protein conformal change with the aid of the three spectral fingerprints,e.g.Raman intensity of N-C?-C(900 cm-1),Raman shift of Amide I(1650cm-1)and the Raman intensity ratio of Trp(I1340/I1360).At last,a four-stage step-by-step mechanism is proposed to describe the lysozyme fibrillation process under thermal/acid treatment(pH 2.0 and 65?).In the initial stage(0-10 hours)of being heated in acid solution,the convolutional 3-D structure of the native protein immediately starts to unfold and rearrange the packing of a-helixes,?-sheets and random coils.The a-helixes gradually uncoil into the random coils,but no new P-sheets are formed.As the result of unfolding,the side chains of amino-acid residues like Trp and Asp,are exposed outside.At this stage,HEWL starts to aggregate but their sizes are too small to be readily observed by AFM.During the second stage(10-40 hours),a-helixes continue to uncoil and the most of them are exhausted finally.No considerable amount of ?-sheets are yet formed as shown by the blue-shift of Amide I band and the ThT fluorescence experiment.The growing lysozyme aggregates can be observed,with an average size of ca.8 nm.In the third stage(40-90 hours),the new P-sheets are formed and stabilized by border side chains.The combined percentage of random coils and P-sheets increases rapidly to the maximum.However,the formed ?-sheets are not in the highly-ordered form and well-packed as suggested by the ThT fluorescence intensity.The lysozyme aggregates are grown into the unbranched prefibrils with a diameter of 6-12 nm.In the mature stage(90-160 hours),the prefibrils slowly assemble into fibrils.The aforementioned formed ?-sheets are rearranged to become more orderly packed,and the intermolecular ?-sheets are drawn closer and stacked.After 160 hours,the amyloid fibrils are abundant with the orderly-packed P-sheets and become strong,stiff,and mature.Metal ions such as Al(III)were considered to play a remarkable role on this fibril process.However,it remains a few questions about the influence of Al(III)ions on proteins' structure during amyloid formation.To illuminate its effect on amyloid formation of lysozyme under thermal incubation,Raman spectroscopy,atomic force microscope(AFM)images and ThT fluorescence are performed respectively.At the early stage of incubation under thermal/Al(?)conditions,the intensity ratio I1340/I1360 of Trp doublet peak is gradually increased,indicating that the more Trp indole rings exposes to water with unfolding of the lysozyme tertiary structure.About several hours later,a blue-shift of the N-Ca-C stretching frequency at ca.933 cm-1 is apparently observed,which suggestes the destruction of the ?-helix structure of lysozyme.Almost simultaneously,the blue-shifts of the Amide ?(1620-1700 cm-1)and Amide ?(1220-1300 cm-1)vibrations are also found.Thus,all spectral evidences suggest that the more intermolecular ?-sheets are formed during thermal incubation.The morphology and spectral features of the mature fibrils are almost identical to those produced in thermal/acid conditions.The specific role of Al(?)ion on amyloid fibrillation of lysozyme has been discussed through comparing the related kinetics with that with another frequently-used fibrillation condition of thermal/acid.In the heated solution with Al(?)ions,the lysozyme amyloid fibrils are formed with a slower rate than those under thermal/acid conditions,implying that Al(?)ions is not as effective as pH on inducing the formation of lysozyme amyloid fibrillation.In a certain extent,Al(?)ions postpone the destruction of ?-helix through bonding to the carbonyl oxygen atom of peptide backbone.However,the ionic electric force can attract the N-H groups in the backbone of the surrounding peptide strands and accelerate the formation of hydrogen bond network of the intermediate ?-sheets.As a result,Al(?)ions promote the transform from ?-helix to ?-sheets,and make its delay time shortened.Therefore,the mysterious influence of Al(?)ion on amyloid fibrillation of lysozyme is clarified as a double-edged role.(3)Prediction of protein structure by Raman spectroscopyAchieving the real structures of proteins is very important to understand the proteins' function.Raman spectroscopy is commonly used to detect the secondary structure of proteins.Here,a simple and rapid method has been proposed for quantitative analysis of the secondary structure of proteins with Raman spectroscopy.The Raman shift of Amide I(1600-1700cm-1)band is used as a parameter,to obtain the relationships between the frequency of Amide I band and the percentages of a-helix,P-sheet,random coil(derived from X-ray)in nearly 13 proteins.It is found that the a-helix and ?-sheet have the nearly linear relationship with the frequency of Amide I band,and their correlation coefficient reaches 0.88.This method also has been used to successfully predict the other protein or protein mixtures.