The Dynamic Conformation Of BamA During The Assembly Process Of Bacterial Outer Membrane Protein

Research Background:Theβ-barrel assembly machine（Bam）is an outer membrane protein assembly machine for gram-negative bacteria.Bam is closely related to bacterial survival and pathogenicity.As the core of the Bam complex,BamA is crucial in the functioning of the Bam complex.Disruption of BamA functional activity prevents normal assembly of outer membrane proteins,leading bacterial death.The mechanisms involved in how the Bam complex mediates the folding and insertion of outer membrane proteins into the outer membrane remain unclear,and a unified view of the mechanisms of outer membrane protein integration has not yet been reached.In-depth research on the conformational changes of BamA during the integration of outer membrane proteins will help to reveal the mechanism of the outer membrane of gram-negative bacteria,which is of great significance for the search for drug targets and the development of new antibiotic drugs.In this study,electron paramagnetic resonance technology was used to explore the dynamic changes of BamA analyzing the mechanism of BamA when it exerted its function.Research Purposes:To detect the dynamic conformational changes of BamA when it functions,analyze the role of BamA in the integration and assembly of outer membrane proteins,and provide insights for the development of new antibiotics and drugs targeting BamA.Materials and Methods:（1）According to the three-dimensional structure and functional characteristics of BamA,cysteine（Cys）mutation sites were designed in the BamA barrel region,extracellular segment Loo6,and periplasmic polypeptide translocation-associated（POTRA）domain for spin labeling.Using overlap extension PCR（sequence overlap extension PCR,SOE-PCR）,the key amino acid sites were mutated to Cys,and the PCR product was cloned into p ET22b Plasmid,the prokaryotic expression vector p ET22b-Strep-BamA containing the BamA protein mutation was constructed and sequenced for identification.（2）The p ET22b-Strep-BamA plasmid identified by sequencing was transferred into Escherichia coli BL21（DE3）,isopropy-β-D-thiogalactopyranoside（IPTG）was used to induce the expression of the mutant protein,cells were disrupted and the outer membranes were collected by ultracentrifugation.Outer membrane protein was purified by Strep-Tactin Sepharose column affinity chromatography.The lipoprotein expression method was the same as that of the BamA and was purified by Ni-column affinity chromatography.（3）The in vitro construction system and method of BamA liposome were established and optimized,and BamA mutant protein liposome,BamA mutant protein+Bam D liposome were constructed respectively,and the construction of liposome was tested by SDS-PAGE.（4）Methanethiosulfonic acid（MTSL）was used to label BamA protein solution,proteoliposome,and in vivo state.Direct labeling of E.coli cells induced to express BamA mutants in vivo state,Electron paramagnetic resonance（EPR）spectroscopy was performed after elution of free label.Based on Spectrum simulation-Fourier deconvolution distance measurement（SS-FDDM）software,the EPR spectrum curve of the single mutation site at room temperature（297k）was fitted to extract the spectral mobility parameters,including the rotation correlation timeτ_c,the strong and weak immobilization ratio S/W,and the overall dynamic parameters of the spectrum,Intermediate Spectral Peaks First-Order Momentum（35）H₀^-1;After curve fitting of the low-temperature（130k）EPR spectra of the single/double mutation sites,the distance between the double Cys-labeled sites was calculated.（5）Based on the analysis of EPR spectroscopy,the movement characteristics and spacing changes of amino acid sites in different states were compared,and the conformational characteristics of BamA protein were analyzed,including the spectral comparison between solutions,BamA liposome,BamA+Bam D liposome and in vivo.Research Results:（1）The SOE-PCR product and sequencing results showed that all designed sites were mutated to Cys.（2）After the p ET22b-Strep-BamA vector was induced by IPTG and the purification by Strep-Tactin Sepharose column affinity chromatography,a high concentration and purity of BamA mutant protein was obtained.The lipoprotein was induced by IPTG and purified by Ni column affinity chromatography to obtain Bam B of40 KD,Bam C of 34 KD,Bam D of 26 KD,and Bam E of 11 KD.（3）A stable proteoliposome construction system was established,and different BamA mutant proteins and BamA mutant protein+lipoprotein liposomes were constructed.The membrane state of BamA without and with outer membrane protein integration function was simulated in vitro.The results of SDS-PAGE showed that the precipitate contained the folded BamA protein,which proved that the BamA protein was successfully integrated into the liposome.（4）The in situ spin labeling technology of BamA protein in vivo was optimized by studying the effects of label concentration,labeling time,and spectral detection time on the spectrum.The results show that when the concentration of MTSL is 10μM,the in situ spin labeling of living cells can be achieved effectively and there are fewer free labels in the system;the spectral signal intensity shows different degrees of attenuation with the increase of time,and the spectral detection needs to be controlled and standardized;marking time has no effect on the marking effect.（5）The conformational motility characteristics of different domains（barrel region,extracellular segment,and POTRA domain）of BamA protein in different states（solution,BamA liposome,BamA+lipoprotein liposome,In vivo state）were obtained.EPR spectra showed that there was no difference in the motility of mutants I29C（at POTRA1）,D560C（at L4）,and S690C（at L6）in vivo and solution;In vivo,mutants G263C（at POTRA3）,G313C（at POTRA4）,and Y649C（at L6）had more restricted movement.T434C（atβ1 chain）added a motility-limited component;G807C（atβ16chain）was dominated by weakly immobilized components in the solution state,and strongly immobilized components in the bulk state,and the transition of motility may mean its transition from non-functional to functional shift.The EPR spectra of the mutants G136C（at POTRA2）and T255C（at POTRA3）were obtained in solution,BamA liposome,and in vivo state.There was no significant difference in the spectra of the mutants in different states.EPR spectra showed that the motility of mutants F440C（atβ2）and D498C（at L3）was gradually restricted in the above four states,indicating that the function of BamA requires the site to maintain a restricted movement state,and the surrounding area of the site needs to maintain a conserved conformation;About N666C（at L6）motility,The change rule was consistent but the change range was small,indicating that its conformation tends to be conserved when BamA functions;Mutant F804C（atβ16）has gradually increased motility in three states:solution,BamA liposome,and BamA+Bam D liposome.（6）The distance changes of two sets of double mutation points of F440C-F804C（atβ2-β16）and D498C-N666C（at L3-L6）were obtained based on EPR low-temperature spectrum in solution,BamA liposome alone,BamA+Bam D liposome and in vivo state.The site spacing of mutant F440C-F804C in the solution state is consistent with the site spacing shown by the crystal structure when the side gate of BamA barrel region is opened.The site spacing decreased sequentially in liposomes and in vivo.The site spacing of mutant D498C-N666C in solution is consistent with the site spacing shown by the crystal structure when the side gate of the BamA barrel region is closed,the site spacing decreases sequentially in solution,BamA alone,and BamA+Bam D liposomes;The site spacing increased in vivo,consistent with the site spacing shown by the crystal structure when the side gates of the BamA barrel region were opened.The above results show that the structure of the protein is restricted by the membrane in vitro.Research Conclusions:The conformational mobility of POTRA1-2 domain in vitro and in vivo has less difference,and the conformational mobility of POTRA3-5domain,which is closer to the barrel region and also closes the barrel region channel,is more restricted,indicating that BamA function may require related adjustment of domain conformation.The in vitro and in vivo conformational motility of BamA extracellular segment sites was less different,indicating that its contribution to BamA function was small.The position of the lateral portal opening and the Loop6-related site in the extracellular loop6 are relatively highly motility when BamA is inactive,but restricted when BamA is functional,and the side door was closed with Loo6 moving in the extracellular direction.It indicates that BamA may have a conformation that keeps the lateral door of the barrel region closed while opening the lumen channel when it functions.