Title Loaded From File Escalating concentrations of Tween 20 showed a direct correlationNovember 2021 Title Loaded From File volume 203 Challenge 21 e0019621 jb.asm.orgLyman et al.Journal of BacteriologyFIG two HbHp binding to C. Representative experiments are shown.with decreased protein size, suggesting that the detergent is able to partially dissociate the big HbpAFL protein complex a similar observation was noted in the native gels Fig. 2B. The predicted size of HbpAFL based on its amino acid sequence is 35.four kDa, although it migrates at an approximate size of 53 kDa in SDS gels Fig. 2A.November 2021 Volume 203 Situation 21 e0019621 jb.asm.orgFunctional Evaluation of HbpAJournal of BacteriologyFIG three SEC analysis of C. diphtheriae hemin transport protein HbpA. A SEC traces showing purified recombinant HbpAS, HbpAFL, and HbpAFL preincubated with growing amounts of Tween 20 Tw. Higher retention occasions indicate smallermolecularweight species. Retention instances of molecular weight requirements BioRad gel filtration standards are indicated by dotted lines. B SEC molecular weight standards and purified HbpAFL protein were analyzed employing a Superose 6 increase 3.2300 chromatography program. The elution trace shows the column retention times of HbpAFL and three highmolecularweight standards blue dextran BD, thyroglobulin Thy, and ferritin Fer. All graphs show a representative sample from no less than three replicates.The SEC profile of HtaAS showed a single peak constant with all the monomer form of the protein Fig. S4. HtaAFL showed 3 distinct regions a diffuse peak that eluted in the column void volume, indicating the presence of a big aggregate, a second peak consistent with all the monomer form comparable to HtaAS, and a smaller sized breakdown item. The addition of Tween 20 to HtaAFL resulted in longer retention instances consistent with smaller sized sized proteins. The SEC profile of ChtAFL and ChtCFL showed a number of bands, which includes peaks inside the column void volume, consistent using the presence of substantial protein aggregates Fig. S4.Sulted in diffuse peaks with longer retention occasions, indicating a smaller sized protein size Fig. 3A. Escalating concentrations of Tween 20 showed a direct correlationNovember 2021 Volume 203 Issue 21 e0019621 jb.asm.orgLyman et al.Journal of BacteriologyFIG 2 HbHp binding to C. diphtheriae hemin transport proteins. A The FL and S derivatives of different recombinant proteins were assessed for binding to HbHp. Proteins had been separated by SDSPAGE four to 15 gradient gel and have been stained with Coomassie blue left panel or examined for binding to HbHp using an in situ system ideal panel. The volume of protein loaded for every lane is indicated below the gel. ChtA Term was employed as a negative binding handle. Note lowered quantities micrograms loaded of HbpAFL. B The exact same proteins utilised in panel A were separated utilizing four to 15 gradient native gels. The presence 1 or absence 2 of 0.1 Tween 20 is indicated for FL proteins. C Comparison of supernatant Sup from C. diphtheriae 1737D3 grown in lowiron 0.25 m M FeCl3 mPGT medium and purified recombinant HbpAFL separated on a native 4 to 15 gel. Left panel, antiHbpA Western blot appropriate panel, HbHp in situ binding detected with antiHp antibodies. Plus and minus indicate the presence and absence, respectively, of 0.1 Tween 20. D Comparison of Hb and HbHp binding among the indicated proteins.