High-Resolution NMR Spectroscopy Reveals Structure of Lipoprotein flpp3

Abstract

Characterization of membrane proteins continues to be an enormous challenge in the field of structural biology compared to their soluble counterparts. Membrane proteins require the presence of a detergent micelle to protect the hydrophobic-exposed residues that are, under native conditions, inserted into a membrane. This provides challenging hurdles for expression, purification, and structural determination. However, for some lipoproteins and in the case of flpp3 from Francisella tularensis, the hydrophilic domain of the protein can be expressed and purified separately from the transmembrane domain. Flpp3 contains a single transmembrane helix at the N-terminus and therefore this technique allows for structural investigation of the majority of the protein without the necessity of a detergent micelle. In this study we report the results of expression, purification, biophysical characterization and NMR analysis of the soluble domain of flpp3 (flpp3-Sol). Sufficient quantities of pure flpp3-Sol are obtained via recombinant expression in E. coli cells followed by purification via Ni-NTA chromatography and gel filtration chromatography. Monodispersity and global secondary structure is investigated by dynamic light scattering (DLS) and circular dichroism (CD) respectively. NMR studies report local secondary structure via secondary chemical shifts and are used to generate a structure-model using CS-ROSETTA. Structures generated from NMR data are compared to structures in the Protein Data Bank using the Dali server and provide insight into possible protein function. The structure of flpp3-Sol is similar to the C-terminal domain of the lipoprotein bamC from E coli, a protein important for the insertion of beta-barrel proteins into the outer membrane of cells via the BAM complex; and as of yet unidentified in F tularensis.