Thermostabilization And Biochemical Characterization Of The Membrane-bound Sterol ?8,?7-isomerase

Cholesterol is an essential part of lipid membrane in human cells.As such,lack of cholesterol owing to inborn diseases is detrimental to human health or even fatal.On the other hand,metabolic disorder that causes too much cholesterol in blood is also harmful.Because both the substrates and the enzymes are insoluble,the pathway from lanosterol to cholesterol has been lagged behind.In this pathway,?8,?7-isomerase is the key enzyme responsible for the isomerization of C8-9 hydrogen bond to C7-8hydrogen bond without NADPH.Dysfuncion of this enzyme causes CDPX2 and MEND disease in human.Human?8,?7-isomerase is also known as h EBP due to its binding to calcium channel inhibitor emopamil.In budding yeast,the equivalent enzyme is sc ERG2 which has no sequence homology to the human enzyme.As noted,h EBP has five transmembrane and only once of sc ERG2.Although the two protein only 22?26 k Da,but could binds with many ligands,even same ligands.This thesis focuses on the thermostability engineering of two human-type isomerases(h EBP and tt EBP).Because of the difficulity of expression,purification and crystallization of h EBP.Thermostability engineering was carried out to increase the stability and the possibility of crystallization.A high-throughput scanning method was developed based on TGP(thermostable green fluorescent protein)to evaluate and screen the stability of mutants.Employing this method,amounts of the mutants including truncation,Ala,Leu,Phe and Val mutagenesis,and fusion constructs were screened.A functional construct toleranced heating at 85?for 20 min was obtained(h EBP-5m1-BRIL)compared to the WT(31.2?).For tt EBP(Thermothelomyces thermophilu EBP),based on homologous sequences from thermophilic bacteria,the apparent T_m was raised from 35.9?to 69.9?(con EBP,consensus EBP)in one-step by appling the consensus design into membrane protein structure biology but loss 80%activity.The activity was recovered by quick chimeric and back-mutation which furtherly increased the apparent T_m to 88.8?(N4-M5 mutant,which has in vivo function in yeast cells).Almost two years was cost to obtain the thermostable mutants by scanning method.However,a more thermostable mutant was obtained by consensus method costing many weeks.Comparising this two methods,the consensus method was more efficiency and time-saving.This will encourage more useage of the consensus method in the thermostable engineering of membrane protein.To evaluate the activity of mutants come from the protein engineering,a rescue assay in ERG2-Fen1-KO yeast strain was estabilished by homologous recombination,verified,and used for characterization of mutants.At the same time,GC/MS based assay was established to detect the substract and product for?8,?7-isomerase.It is worth note that,h EBP has isomerase function in vivo,but lost in vitro,suggesting the isomerase may need a chaperone protein or lipids.The thermostable ERG2,an isoenzyme of h EBP in yeast was also expressed and purified,and used for crystallization screening.The thermostable isomerases of h EBP-5m1-BRIL,N4-M5 and ERG2 obtained in this thesis will be useful for future structural biology studies by solving crystal-clear structure of EBP with substrate/product to uncover its catalytic mechanism,as well as the structural basis for its broad activity for ligand binding.