Structural Study on Antitoxin protein from Mycobacterium tuberculosis H37 Rv by NMR spectroscopy

Abstract

Mycobacterium tuberculosis is a pathogenic bacterial species and the causative factor of tuberculosis. Tuberculosis is one of the most oldest disease which has human afflicted. Despite of people in the world use a live attenuated vaccine and several antibiotics, this disease make lots of death until now. They have already some kind of antibiotics and therapeutic tactic, but upcoming of multi drug resistance so we need to make new antibiotics. Toxin – Antitoxin system which is found in most of the bacteria and fungi was originally created for adapt extremely environmental conditions. Toxin – Antitoxin system constitute relatively stable toxin and labile antitoxin. Depend on type of antitoxin, they divide all of 3 types Toxin – Antitoxin system. Basically Toxin has lots of toxic effect such as Rnase, interrupt DNA gyrase and then cause of these toxic effect they killed cell or they interrupt cell growth. In Toxin – Antitoxin system, antitoxin bind to toxin tightly to inhibit toxin activities. Rv0599c, antitoxin protein included in Type2 Toxin - Antitoxin system, and it inhibit toxin`s toxic effect by binding to toxin. Under the several stress conditions, the protease which induced by stress will degrade antitoxin from the toxin, and then the free toxin has activity such as cell growth halt, and cell death. Type 2 Toxin – Antitoxin system composed of toxin protein and labile antitoxin protein. Type2 Toxin – Antitoxin system include VapBC, MazEF, RelAB and so on. Rv0599c is VapB antitoxin. Be known this protein has DNA binding function. Rv0599c has 78 amino acid and pI value is 5.0365. In order to determine tertiary structure of Rv0599c protein, we used E. coli expression system and purified hexa-histidine tagged Rv0599c by IMAC purification system. To get a more purified fractions, we used Ion – Exchange purification system, finally we used Size Exclusion purification system. And we also used Circular Dichroism to get a proper buffer condition for NMR spectroscopy. Using the buffer condition, We performed HSQC at various temperature condition, and we decided certain temperature. Finally, using buffer condition and temperature I finished backbone assignment. The secondary structure and tertiary structure of Rv0599c were predicted by TALOS server and CS23D server respectively. we found homologues by DALI server. Actually, Toxin – Antitoxin complex and antitoxin protein was known bind the certain DNA sequence. Also Rv0599c antitoxin protein, we made certain DNA sequence, and we performed NMR titration experiment to confirm antitoxin protein binding function. I think if we inhibit binding function of antitoxin to toxin, new antibiotic can be proper to M. tuberculosis by toxin activities.