Study On The Dynamics And Structure Of Rv3882c Protein Transmembrane Domains In Mycobacterium Tuberculosis

Tuberculosis is a contagious disease caused by infection with Mycobacterium tuberculosis.It is a serious hazard to humans and has become the second most deadly infectious disease,killing about 2 million people every year.The M.tuberculosis secretion system has been found to have four systems:SecA1,SecA2,Tat,and ESX.The ESX secretion system was originally discovered during the secretory process of the secreted proteins ESAT-6 and CFP-10,and its general transport lacks traditional signals.Proteins of peptide sequences are closely related to virulence and pathogenicity.The ESX secretion system consists of five separate subsystems,ESX-1,ESX-2,ESX-3,ESX-4 and ESX-5.The ESX-1 system is involved in the defense process of bacterial invasion of host cells and the body,and is the most studied sub-system.Among the constituent proteins of this secretion system,the MtRv3882c protein(EccE protein)encoded by the rv3882c gene is a conserved component of the ESX-1 secretion system.However,there is no structural and functional homologous protein tertiary structure,so it is impossible to understand the tertiary structure of the M/Rv3882c protein,and it is impossible to understand its role and function in the body.At present,yeast two-hybrid assay confirmed that M/Rv3882c interacts with cytoplasmic protein MtRv3614c to form a hetero complex to promote the secretion of related secreted proteins.It is also found that MtRv3882c protein is combined with several other ESX system conserved proteins(EccB,EccC,EccD).Together,they form a large membrane protein complex(about 1.5 MDa)that constitutes a substrate transport pathway that promotes secretion of the substance.In this study,we used a heterologous expression system of Escherichia coli to express and purify the single mutant and double mutant protein of MtRv3882c-TM,and obtain the target protein with uniform state and high purity.Then,combined with Site-Directed Spin Labelling and Electron Paramagnetic Resonance technology,the structural characteristics of the transmembrane domain of MtRv3882c protein were explored.In the environment of detergent micelle carrier,the information of the motion,accessibility and distance measurement of the labeled transmembrane domain was obtained by EPR,and the start and stop sites,secondary structure features and transmembrane helix were judged.The orientation information is combined to derive the structural model of MtRv3882c-TM.Through the CW-EPR experiment,we obtained the kinetic parameters of AH-1 and<H2>-1 and the spectral width Azz value of the side chain of the transmembrane region,and the kinetics of the side chain of the transmembrane region in the detergent micelle.The parameters are higher than the value of random coil,which indicates that the transmembrane helix region is less mobile,the mobility of the random coil is higher,and the Mt0082 protein is measured in the detergent micelle by the power saturation method.The aptitude information indicates that the ?NiEDDA values at positions 35 to 54 are maintained at a lower level,ie the regions in which these sites are located are more hydrophobic;the results of?o2 values indicate values at positions 35 to 54 maintaining in a very high range,and then combined with the change of ? value,the transmembrane region and its two sides of the sequence generally exhibit a "hydrophilic-hydrophobic-hydrophilic-hydrophobic-hydrophilic"alternating model.Through our research,we will provide a basis for the structure and function of MtRv3882c protein.Besides,this topic also provides ideas and methods for expanding the application of SDSL-EPR technology in membrane protein research.