Towards the Backbone Structure Determination of the Membrane Protein, LspA, from Mycobacterium Tuberculosis using Solid State NMR

Abstract

Lipoprotein Signal Peptidase A (LspA) is an essential enzyme in one of the three distinct steps in lipoprotein maturation. After targeting, the signal peptide of a prolipoprotein, the peptide is cleaved by LspA to result in 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 is 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 uniformly aligned samples is being used to determine the structure of the protein backbone. Anisotropic 15N-1H dipolar couplings and 15N chemical shifts are obtained from separated local field NMR spectra of the protein in aligned bilayer preparations. The resulting orientational restraints can be used to generate a high resolution structure as well as a determination of how the helices are oriented with respect to their environment.In this study, LspA is reconstituted into synthetic lipids (DMPC/DMPG and POPC/POPG) to mimic the membrane environment and it is aligned uniaxially either mechanically (using glass slides) or magnetically (using bicelles).