Secondary Structure and Topology of the Transmembrane Domain of LspA, from Mycobacterium Tuberculosis, using Solid State NMR Spectroscopy: Initial Study

Abstract

Lipoprotein Signal Peptidase A (LspA) belongs to type II signal peptidases and it is an essential enzyme in one of the three distinct steps in lipoprotein maturation. After targeting the prolipoprotein, LspA cleaves the signal peptide to result in the signal peptide cleaved lipoprotein, which is the precursor for the mature lipoprotein.In Mycobacterium tuberculosis (Mtb), LspA is encoded by the gene Rv1539 resulting in a 202 amino acid residue peptide with a molecular weight of 21 kDa that is predicted to possess four transmembrane helices. It has been recognized in vivo that LspA is an essential protein for Mtb virulence and that it may also contribute to antibiotic resistance. As a result LspA has been identified as a potential drug target for the treatment of tuberculosis. Globomycin, a cyclic-peptide is known to inhibit LspA in a non-competitive manner.Solid-state NMR enables studies of membrane proteins in a lipid bilayer environment. Since the native-like environment is important for studying the structure and function of membrane proteins, ssNMR is increasingly useful. In Oriented Sample ssNMR used here, the protein is uniformly aligned with respect to the magnetic field and 15N spectra of isotopically labeled protein have provided initial structural insights.Here, all sample preparation steps and initial spectroscopy will be emphasized. Expression of LspA in E.coli, purification using IMAC, reconstitution of LspA into proteoliposomes via dialysis and the preparation of bicelles as an alignment media with different types of lipids and detergents will be presented. 1D NMR 31P and 15N spectra and 2D separated local field NMR spectra of uniformly and selectively labeled LspA will be shown.