(Supported in part by DOE 61225-00-110 to J. Trent) One of the fundamental problems in biology today is how proteins fold into their functioning form in the crowded confines of living cells. Recent studied suggest that this process is mediated by a class of proteins called molecular chaperones. One of the most important chaperones in this process is a multi-subunit complex known a chaperonin that is a double-ring structure believed to fold proteins inside its hollow core. We have evidence from purified chaperonins that they aggregate in vitro to form ordered filaments, and observations in cells that suggest similar filaments exist in vivo. We propose to use the BMIRR facilities to 1. Look inside minimally treated frozen hydrated cells or thick cryosections by HVEM or IVEM to improve the images of the internal structures seen in cells by conventional EM techniques; 2. Collect high quality images of negatively stained and frozen hydrated chaperonin filaments to compare to the filaments seen in cells to clarify their structural relationship and to gain insights into the basis for chaperonin filament formation; and 3. Use the 3D tomographic reconstruction facilities to image the filaments themselves in an attempt to understand the nature of this structure as a site for attaching other cell components. During Dr. Trent's visit, we examined cryosectioned (Tokuyasu method) S. shibatae cells with the HVEM, and made fresh-prepared whole-mount preparations of cells growing in culture. Many stereo pairs were recorded, and a tomographic reconstruction was made from a section. The reconstruction revealed that the S-layer is intact and that uranyl acetate stain did not get inside the cell. In the whole mounts, we could see through the whole cell and visualize the stained S-layers, but we could not see the internal structures. We also looked at the cultured cells with video microscopy. The irregular, bean-bag, shape seen may be due to an internal cytoskeleton or to features of the s-layer and the cell's turgor pressure. Dr Trent also recorded several stereo pairs of cells that had the surface layer removed in hopes of seeing the structure of chaperonin as well as its architecture. These images were very difficult to interpret. Samples of E-coli (L-forms), brought by Dr. Trent, as well as live cells at both ambient and high-temperature conditions, were high pressure-frozen for a totoal of 30 runs. The cells were freeze-substituted and embedded. The blocks were sent to Dr. Trent for sectioning.
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