Preliminary Study On The Structure Of Treponema Pallidum Outer Membrane Protein TprK

Part 1:Biological information prediction of the outer membrane protein TprK of Treponema pallidumObjective:Prediction of Treponema pallidum TprK protein bioinformatics by an online platform.Methods:Biochemical information such as molecular weight,isoelectric point,absorbance,and cysteine content were predicted by ProtParam online tool.PSORT predicted TprK protein localization and PRED-TMBB predicted β-barrel outer membrane protein transmembrane region.SianalP 5.0 online server was used to predict TprK protein Signal peptide.TprK protein homology analysis by BLAST program.Phyre2 online tool to predict protein secondary structure and domain composition.SWISS-MODEL,I-TASSER and AlphaFold online platform was used to predict TprK protein tertiary structure.Results:The prediction results of the ProtParam online tool show that TprK contains 503 amino acid residues,its molecular weight is 55391.99 Dalton,the theoretical isoelectric point is 9.11,and the absorption coefficient is 2.089,with a stability coefficient of 24.17.The subcellular localization of TprK was analyzed by the PSORT online tool,and the localization score of the outer membrane was 9.49,which was much higher than the scores of other localizations(cytoplasmic:0.03;cytoplasmic membrane:0.01;periplasmic:0.09;extracellular:0.38).The results of PRED-TMBB showed that most of the TprK protein sequence constituted the transmembrane region,the-NH2 and-COOH terminal were both on the cytoplasmic side,and the extracellular part contained some peptide chains of varying lengths.In addition,SianalP 5.0 online software for signal peptide analysis showed that there is a signal peptide sequence at the-NH2 terminus of TprK protein,which also supports the possibility that TprK is an outer membrane protein.Phyre prediction results show that the secondary structure of TprK protein contains a large number of β-sheets and less a helices.The two tertiary structure online platforms of SWISS-MODEL and I-TASSER did not provide a highly reliable tertiary structure of TprK protein.AlphaFold prediction results show that TprK protein is a β-barrel structure composed of 20 anti-parallel β-sheets.The-NH2 terminus andCOOH terminus of the protein are both located on the cytoplasmic side,and the amino acids 1-60 of the-NH2 terminus are irregular Loop regions.The extracellular side of TprK contains abundant Loop regions.Conclusions:TprK is an outer membrane protein of Treponema pallidum with a molecular weight of 55391.99 Dalton and contains a large number of β-sheet structures.The predicted three-dimensional protein structure is a β-barrel composed of 20 antiparallel β-sheets,with abundant Loop regions on the extracellular side.Part 2:Expression,purification,structural analysis of Treponema pallidum outer membrane protein TprKObjective:The purpose of this study was to express the full-length TprK recombinant protein using the Escherichia coli prokaryotic expression system,and to screen out a suitable lipid-like environment to maintain the stable structure of the TprK protein,so as to provide a basis for the high-resolution structure analysis of TprK.Methods:The prokaryotic expression vectors of pET28a-6×His-tprK and pET28a3×FLAG-tprK were constructed by recombinant gene technology.The expression vector was transferred into Escherichia coli BL21(DE3)strain for overexpression,and the expression of the target protein was identified by polyacrylamide gel electrophoresis,Western blotting and mass spectrometry.After Cell membranes collected,detergent was added to the homogenized cell membrane solution to dissolve the membrane.Then affinity chromatography and gel chromatography were performed to purify the TprK protein.In order to obtain a TprK protein sample with stable properties,the eluted protein sample from affinity chromatography was concentrated,then dropped into a buffer containing new detergent(DDM,DM,OG,LADO,CYMAL7,LMNG).The detergent exchange sample was re-concentrated and then through gel filtration chromatography.The peak fraction of gel filtration chromatography was subsequently assessed using negative-stain electron microscopy.The peak fraction of LMNG-TprK gel filtration chromatography was concentrated to 10 mg/ml and then screened for crystallization.In addition,the TprK protein was reconstituted by Nanodiscs,and the peak samples of the reconstituted samples were observed by negative staining electron microscope.Then,the TprK protein samples reconstituted by Nanodiscs with better homogeneity and dispersion were selected for the preparation and observation of frozen samples.Results:Sequencing proved that the homologous recombination vector was consistent with the theoretical sequence.SDS-PAGE and Western blot showed that the ultracentrifugation pellet(containing Escherichia coli membrane)had a significant band at about 55 kDa,which was consistent with the expected molecular size of TprK.Mass spectrometry analysis showed that the SDS-PAGE strips contained a large number of peptides of the TprK protein of Treponema pallidum Nichols strain.Foscholine-12,LDAO,LMNG,DDM,DM,CYMAL7,OG,commonly used detergents for membrane protein purification,were used to dissolve membranes.Fos-choline-12 can dissolve TprK protein from bacterial lipid bilayers.Other Detergents(LDAO,LMNG,DDM,DM,CYMAL7,OG)have poor solubilization effect.TprK protein with higher purity could not be obtained by Ni-NTA affinity chromatography.After changing the Flag-tag,the purity was greatly improved.While gel chromatography of Foscholine-12 showed more than one peak.The Gel filtration chromatography in which Fos-choline-12 was replaced by LMNG showed one peak,and better than other detergent exchanging.Negative staining electron microscope observed that the TprK protein particles encapsulated by LMNG were uniform,and most of them were dispersed single particles.Only two crystallization conditions were obtained from protein crystallization screening,and the crystals were judged to be salt crystals by Xray diffraction.Gel chromatography of purified TprK protein-Nanodiscs reconstituted samples showed a narrow peak at an elution volume of about 16.5 ml.The negative staining electron microscope observation of the peak sample showed that the protein samples had uniform particles and no aggregation.Negative-staining electron microscope observed LMNG-encapsulated TprK protein samples and Nanodiscreconstituted TprK protein samples.The results showed that light-colored circular spots were scattered in the dark gray background,and a black dot was distributed in the center of the circular spot.The TprK protein samples reconstituted by Nanodiscs were observed to have poor homogeneity and particle contrast under transmission electron microscopy,and the samples could not be recovered for cryo-EM data collection.Conclusions:The Treponema pallidum outer membrane protein TprK was successfully expressed on the bacterial membrane using the Escherichia coli expression system.Membrane localization showed that most of the target proteins were overexpressed in Escherichia coli cell membrane fractions.The overexpressed TprK can be correctly applied to the membrane,suggesting that the three-dimensional structure is close to the native conformation of Treponema pallidum TprK protein.The detergent Fos-choline12 with the highest TprK dissolution efficiency was obtained by detergent screening.After extracting the overexpressed TprK protein from the cell membrane of Escherichia coli by using detergent,and a sufficient amount of high-purity full-length TprK recombinant protein was obtained by affinity purification and gel chromatography.In order to obtain a more homogeneous protein sample for the high-resolution structure analysis of TprK,the method of detergent replacement in solution,combined with negative-stain electron microscopy to evaluate the protein sample and grope for preliminary structural analysis,constructed two membrane lipid bilayer mimic system(detergent LMNG and Nanodiscs),these two systems can well maintain the protein conformation and stable state of TprK in solution.In addition,crystal screening and protein frozen sample preparation were also carried out,and tentative attempts were made to resolve the high-resolution structure of TprK protein by X-ray cry stallography and single-particle cryo-electron microscopy.The pore-like structure in the center of TprK protein was observed by negative-stain electron microscope,which was consistent with the structural characteristics of the outer membrane protein β-barrel,indicating that the purified TprK protein in vitro maintained its natural conformation.It is speculated that TprK protein has the structure and function of outer membrane porin.