Structural studies of the influenza and HIV viral fusion proteins and bacterial inclusion bodies

The infection of a cell by enveloped viruses, such as influenza and HIV, begins with the fusion of the viral and cellular membranes, which is mediated by proteins in the membrane of the virus referred to as fusion proteins. For influenza this is the HA2 protein and for HIV the gp4 1 protein. The structure of several constructs of these proteins have been studied, focusing on the ectodomain regions. Expression in E. coli has been successfully optimized to produce large quantities of fusion protein with amino acid specific 13C and 15N labeling. Analysis of the protein conformation at specific residues was done by solid state NMR. Detection of specific 13C in the membrane associated and inclusion body protein was achieved by use of a filtering REDOR pulse sequence. The measured 13C chemical shifts are correlated with local conformation. Initial studies focused on determining the conformation of specific residues in the membrane associated ectodomain of the HA2 protein.;After method development regarding purified membrane associated protein, the focus shifted to the structural study of protein while still within the bacterial cell. Recombinant protein expression is typically plagued by the production of insoluble aggregates of the expressed protein, known as inclusion bodies. Little it known about the structure of proteins in this form, but theories range from an amyloid-like beta sheet structure to aggregates of fully folded, functional protein. Previous structural studies of inclusion bodies have mostly used IR, which only gives overall structural information.;We were able to develop a solid-state NMR method in which site-specific structural information can be obtained of the inclusion bodies both in the whole-cell and in the insoluble fraction of the cell without dehydrating the samples. Using amino acid specific labeling and the REDOR pulse sequence, detection of specific carbonyl signal was achieved for multiple positions throughout the constructs of both the HA2 and gp41 fusion proteins. Comparing the inclusion body results to structural studies we previously conducted on the membrane associated protein, our results indicate that the FHA2 inclusion bodies maintain their native structure and there is no indication of beta sheet formation. This is a simple, efficient method to study the structure of inclusion bodies in their native form, while still in the cell, and that should be easily transferable to any other inclusion body producing protein.