| DNA gyrase is one of the Toposomerase ⅱ.Indeed, we all know now that gyrase is the only enzyme able to introduce negative supercoils into DNA,using the energy of ATP hydrolysis.Gyrase are ubiquitous among bacterias,but until now there is no evidence show it exists in human cells.So it can be exploited as a target for many drugs,like the quinolone which is used common in the clinical application.There is no research on the exact domain where GyrA interact with GyrB.Our team had do some experiments to make sure the toprim is the domain that GyrA interact with GyrB, however,we aslo find the ATPase maybe mediate the interaction. So on this basis, we constructed several truncated mutants which was based on the structure of Mtb GyrB:gyrB79-714,gyrB183-714, gyrBA79-182, gyrB△294-465.Then we uesed Yeast two-hybrid system to prove the interaction between GyrA and GyrB mutants.Through SPR experiment we test the value of affinity and the rate constant. With the CD experiment we acquire the diffterence of the secondary structure between the GyrB and its mustants.This subject aims to understand the role of ATPase in the interaction between GyrA and GyrB, and how this domain affect the interaction. we hope our work can contrbute to exploit new drugs.The main results of our experiment are as following: First based on the sequence of the GyrB we construted several truncated gyrB mutants gyrB79-714,gyrB183-714, gyrBA79-182, gyrB△294-465. Purified proteins after ligasing these fragments to expression vector pET28a and pGADT7.Next the results of Yeast two-hybrid system and SPR expriment that the truncated mutants gyrB△79-182and gyrBA79-182loss majority interaction with GyrA. Finally, CD experiments aslo proved that after truncated the fragment79-182, the secondary structure of GyrB changes a lot. So we can conclud that ATPase domain in the GyrB really mediate the interaction between GyrA and GyrB,though we didnt know the exact mechanism.of course during the mediation, the Transcucer domain helps much. |
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