This laboratory aims to elucidate the regulatory mechanisms that govern the assembly of supramolecular complexes and the folding of macromolecules, as well as those that underlie the synthesis of organelles, cells, and tissues. In the past year, we have discovered that three-dimensional reconstruction from cryo-electron micrographs of antibody-labelled virus particles affords a method of mapping epitopes with remarkable, and unexpectedly high, precision. Previous forms of immuno-electron microscopy are indirect, detecting an electron-dense label (ferritin or colloidal gold) which may be 15-25nm from the epitope of interest: In contrast, our method directly visualizes the interaction between the Fab fragment and the underlying epitope, and may distinguish between epitopes as close as 1nm apart. It has been applied to three different monoclonal antibodies which bind to the outer surface of the capsid of herpes simplex virus. Two Mabs bind to distinct sites on the hexons, but not to pentons: the third binds to the protruding tips of pentons, but not to hexons. Taking into account our recent biochemical evidence that hexons and pentons are most likely composed of the same viral protein (VP5; 148 kda), these results indicate that there are major conformational differences between the same protein as deployed in pentons (at the capsid's vertices) and hexons (which form the rest of its shell). We have also devised techniques to quantitate the protein compositions of the cell envelopes of cornified epidermal keratinocytes, these structures are covalently cross-linked, rendering them inaccessible to conventional quantitation by gel electrophoresis. Thus we have found that in native epidermal envelopes, the primary constituent is loricrin (70-80%), whereas cultured cell envelopes contain little or no loricrin, but are mainly composed of involucrin, cystatin A and a cystein-rich protein. We infer that only the early stages of native cornification are induced under these in vitro conditions.
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