Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

4-POS Board 4 Structure Determination of Enzymes Involved in Mutagenesis in Mycobacterium Tuberculosis Simon Broadley 1 , Digby Warner 2,3 , Trevor Sewell 1,3 . 1 University of Cape Town, Cape Town, Western Cape, South Africa, 2 University of Cape Town, Cape Town, Western Cape, South Africa, 3 University of Cape Town, Cape Town, Western Cape, South Africa. Drug resistance in Mycobacterium tuberculosis (MTB) arises through the acquisition of spontaneous mutations in antibiotic target or related genes. This places enormous importance on the need to understand the DNA metabolic pathways in MTB, and identifies mutagenic repair mechanisms as compelling targets for novel anti-TB drugs. The DNA damage-inducible C family DNA polymerase, DnaE2, has been implicated in virulence and the emergence of antibiotic-resistant MTB mutants in vivo. DnaE2 operates as part of a three-component “mutagenic cassette” comprising ImuB - a pseudo Y-family polymerase - and ImuA’, a RecA-like protein of unknown function. The aim of this study is to obtain crystal structures of these proteins as well as the dnaE1 - encoded replicative polymerase in order to gain insight into the comparative geometries of the DnaE1 and DnaE2 active sites, and to elucidate potential interacting domains in DnaE2, ImuA’, and ImuB. To date, ImuA’ and DnaE1 have been solubly expressed in E. coli with maltose-binding protein tags. Thermostability studies (which provide an indication of proper protein folding and a measure of stability in crystallization buffers) have been inconclusive and indicate the need for further investigation. MTB proteins are notoriously difficult to express properly folded in large quantities; therefore, optimised codon usage for expression of MTB genes in E.coli has been explored to improve folding stability. Biophysics also suggests that slower expression increases the probability of proper folding; in addition, other expression vectors, strains and conditions have been tested. Provided purified, soluble protein is obtained in sufficient quantities, crystallisation conditions will be screened using a mosquito pipetting robot and hits will be optimised by exploring the crystallisation phase diagram. Crystal cryoprotectant conditions will be optimised using the diffractometer at UCT and the final, high-quality data sets obtained at a synchrotron.

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