Our lab uses bacteriophages as model systems to learn how proteins assemble into complex biological structures. The structural components of phages are particularly appropriate because they are large enough that we can visualize them using electron microscopy and detect them using simple gel assays, but simple enough that we know all of the genes required to produce them and can easily make mutations and study their effects. Three easily studied parts of phages are tails, tail fibers, and capsids. My research focuses mainly on capsids and in particular the capsid of E. coli phage HK97 and some close relatives.
Viral capsids are superficially simple: they are made from a small number of proteins that make up the capsid shell as hexons on the capsid faces and pentons at the corners. In many cases there are proteins or domains required for assembly, but missing from the final structure - these serve as chaperones or scaffolding proteins.
(above) The Bacteriophage HK97 Capsid Assembly Pathway
As a model system, the HK97 capsid has many advantages, 1) it is simple - only three genes are required to build it, 2) it is efficiently made in the laboratory using plasmid expression of cloned genes, 3) much of the pathway of its assembly and maturation are well understood, and 4) there is a large repertoire of structural information from both electron microscopy and x-ray crystallography.
Research topics in the lab:
How does the same protein assemble into hexamers and pentamers?
How is capsid size determined?
How is the capsid maturation protease incorporated and regulated?
How is the portal incorporated into one and only one vertex? - The portal forms the site where DNA goes in and out of the capsid and where the tail is attached.
What are the functions of the HK97 scaffolding-protein-like domain called the delta domain? - This domain is comprised of the first ~100 residues of the major capsid protein and is required for assembly, but removed by proteolysis after assembly is complete.
Comparative capsidomics: We are also studying two larger phages, called D3 and phi1026b in collaboration with James Conway (here in Pittsburgh). These two phages use proteins which are very similar to make capsids that have ~540 subunits in an arrangement called T=9 vs the 420 subunits used by HK97 to make its smaller T=7 capsids. We are hoping that sub-nanometer electron density maps of these larger capsids will help us build models that suggest how capsid size is determined.
(above) Cryo-EM Density Map of D3 Prohead fit with Models based on a HK97 Prohead II x-ray structure
Opportunities for undergraduates: In the past few years we have developed new "mini" capsid production methods that have made it easier for undergraduates to participate in our research program. Several dedicated undergraduate researchers have made significant contributions to our work by making capsid gene mutations on plasmids and rapidly characterizing how the mutations affect capsid assembly.
Collaboration: My main collaborators are Roger Hendrix in Biological Sciences (with whom I share a large, well-equipped lab), James Conway in the Structural Biology Dept. at the University of Pittsburgh Medical School, Alasdair Steven and Naiqian Cheng in the Laboratory of Structural Biology at the NIH and Jack Johnson at The Scripps Research Institute in La Jolla, CA.
- Joshua Maurer, Research Specialist
- Nassima Bouhenni, Undergraduate researcher
- Huixin Wu, Undergraduate researcher
- Victor Morell, Undergraduate researcher
- Alexis Huet, Research Assistant Professor