Title :

Novel Protein Targets of Multi Drug Resistant Acinetobacter baumannii

Abstract :

In the current scenario, widespread multidrug resistivity in ESKAPE pathogens demands identification of novel drug targets to keep their infections at bay. For this purpose, we have identified histone acetyltransferase Hpa2 and DNA-repair enzyme TAG of A. baumannii. Correct sequence alignment and construction of 3D monomeric and dimeric models of proteins having optimal structural parameters is a troublesome task. To overcome these problems, we have designed an easy and optimized protocol for homology modeling. Improvement in the structural features of protein is an onerous process and generally achieved by paying time and computational cost. Herein, it is achieved by reconciliation of FoldX commands which takes less time in execution. Evaluations performed to validate structural parameters and stability of models attests to its quality.

Hpa2 and TAG protein genes were cloned and heterologously expressed in bacterial expression system. The expressed protein was purified up to 98% homogeneity by a two-step purification protocol involving affinity purification followed by the size exclusion chromatography step. The biophysical studies (CD, Fluorescence and ITC) were carried out to characterize the structural and substrate/inhibitors binding parameters.The oligomeric state of enzymes determined in presence of various substrates as well as in extreme physiological conditions using analytical size exclusion, native PAGE and glutaraldehyde crosslinking assay. The enzyme activity is checked using simplified HPLC based biochemical assay. The Hpa2 and TAG proteins were put onto crystallization trials using commercial Hampton screens. The outcomes of these studies will be presented.