Study Of Conformation Of PMP22-TM4 Based On Label-Free Spectroscopy

Different mutations in the peripheral myelin protein-22(PMP22)gene can lead to different hereditary peripheral neuropathy such as Charcot-Marie-Tooth(CMT),Dejerine-Sottas syndrome(DSS),and hereditary neuropathy with liability to pressure palsies(HNPP).Mutation of glycine(G)to asparticacid(D)at position 150 in the fourth transmembrane segment is thought to be associated with a severe DSS disease.In order to study the effect of mutations on the conformation of PMP22,we selected the fourth transmembrane segment of PMP22 as the study object and named as PMP22-TM4.This segment includes both mutational site asparticacid(150)and amino acid tryptophan(W)at position 140.As an autofluorescence probe,label-free protein conformation studies can be performed.Curcumin has recently been found to exhibit certain medicinal properties,in particular to prevent aggregation of A?(associated with Alzheimer's disease)and ?-Synuclein(associated with Parkinson's disease)proteins.Therefore,it is important to study the effects of curcumin on wild and mutant PMP22-TM4,clarify the conformational changes of PMP22-TM4 and its mutants,and promote the understanding of the related diseases caused by mutation of PMP22 gene at the molecular level.The main research contents of this article include:Using ultraviolet absorption spectroscopy,tryptophan fluorescence spectroscopy,and surface-enhanced Raman scattering(SERS)label-free spectroscopy techniques,we are studying the conformational differences of wild-type PMP22-TM4(WT)and mutant PMP22-TM4(G150D).Study results show that G150 D is more sensitive to pH value and denaturant concentration than WT and easier to loose native conformation in unfavorable conditions.The interaction between curcumin molecules and WT and G150 D is also studied,and curcumin is found to protect the hydrophobic core of tryptophan in PMP22-TM4.Using homology model simulation and molecular docking techniques,the differences in the properties of WT and G150 D are studied.It was concluded that after WT mutated into G150 D,the number of hydrogen bond reduces,the bond energy reduces,the conformation occurs tortuous,and the stability decreases.In addition,binding energy of WT is lower than that of G150 D,which is consistent with the experimental results of curcumin quenching WT fluorescence intensity.The protein folding experiment system based on the ultrarapid microfluidic mixer in our research group was developed and improved.The parameters of each index were tested and analyzed to verify the feasibility of this system.