October 23, Tue 2012
1:00 pm, MRB 200 Conference Room
Dr. David Weliky
Department of Chemistry, Michigan State University
Structure/function of membrane-associated viral fusion proteins and structure of recombinant proteins in bacterial inclusion bodies
Enveloped viruses infect cells by joining their membrane with that of the target host cell. This process is catalyzed by a viral fusion protein and in particular by the ~20-residue N-terminal fusion peptide (FP) region which binds to the host cell membrane. Solid-state NMR (SSNMR) has been used to determine high-resolution structures of the HIV and influenza virus FPs in membranes. SSNMR has also been used to measure distances between nuclei in the FP and nuclei in the lipid molecules in the membrane and it has been observed that there is a strong correlation between fusogenicity and depth of FP membrane insertion. Finally, SSNMR was used to quantitatively determine the population distribution of beta sheet registries for the membrane-associated HIV FP. There was a broad distribution of antiparallel registries and a good correlation between individual registry populations and their free energies of membrane insertion. A very different registry distribution was detected for the non-functional V2E mutant which binds to membranes but is not membrane-inserted.
There has also been significant progress in SSNMR of large domains of the membrane-associated influenza and HIV fusion proteins that contain FPs. In one approach, the FP was chemically synthesized, the large C-terminal region of the fusion protein was produced recombinantly in bacteria, and the large FP-containing protein was produced by native chemical ligation. Initial chemical synthesis of the FP allows for controlled isotopic labeling. In a second approach, most of the fusion protein including FP was produced recombinantly in bacteria. Purified protein yields from the bacterial lysates were initially unacceptably low and it was unclear whether the recombinant protein wasn’t being produced in the bacteria or whether solubilization and/or purification was ineffective. SSNMR was carried out on the insoluble fraction of the cell lysate and it was shown that >100 mg recombinant protein was being produced per L of culture. Most protein was in inclusion bodies and subsequent effort was therefore put into increasing solubilization of inclusion body protein. Similar results were obtained for several other proteins produced recombinantly in bacteria and point to solubilization rather than expression as the major problem in recombinant protein purified yield. The SSNMR method for detecting quantity of recombinant protein in inclusion bodies is general and straightforward and sample preparation is rapid and inexpensive. One other interesting SSNMR result is strong evidence for predominantly folded protein in inclusion bodies.