Mycobacterium Tuberculosis Protein Rv1861: Structural Insights into GTP Hydrolysis in a Native-Like Membrane Environment

Abstract

1/3 of the world's population is infected with Mycobacterium tuberculosis 10% of whom will become sick from the bacilli. Multidrug resistant strains resistant to the leading tuberculosis antibiotics, isoniazid and rifampicin have emerged necessitating new treatments. Integral membrane proteins are an excellent source of novel drug targets. Structural biology can provide rich information regarding the influence of lead compounds on protein function. Solid state nuclear magnetic resonance is increasingly being used to understand membrane protein structure and dynamics in lipid membrane environments. The structural information can be accompanied with other biophysical techniques to make conclusions regarding membrane protein function and the influence of regulatory compounds. Rv1861 is an integral membrane protein from Mycobacterium tuberculosis. It contains the signature GTP binding domain motif AXXXXGKT near the N-terminus of the protein and is predicted to contain three transmembrane alpha helices from hydrophobicity analysis. Oriented sample solid state magnetic resonance accurately measures peptide plane orientations through PISEMA experiments on protein in liquid crystalline lipid bilayers aligned between glass slides. The structural data can be correlated to functional information from experiments such as isothermal titration calorimetry on the protein in liposomes allowing protein structure and function to be understood in a native-like environment. Here we present the initial structural characterization of the Rv1861 protein in lipid bilayers. PISEMA experiments on uniformly and amino acid type specifically labeled samples are used to determine helix orientation. Light scattering, size exclusion chromotography and electrophoresis experiments on the protein in micellar environments will provide the oligomeric state and compliment the secondary structure characterization. The data provide a firm foundation for further structure determination and functional characterization of the protein in a native-like environment.