A novel technique for imaging discrete protein conformations and detecting protein-protein interactions was developed, known as bipartite tetracysteine display. Two pairs of cysteines appended at the N- and C-termini of avian pancreatic polypeptide (aPP) or at the N-terminus of the leucine zipper region of GCN4 were selectively bound over single point mutation variants by the profluorescent biarsenical dye, ReAsH•EDT2. Fluorescence intensity is shown to correlate with the thermodynamic stability of the protein fold or assembly. Studies in mammalian cells demonstrated a visible increase in fluorescence for cells expressing properly folded or associated proteins. The selectivity of binding was characterized by gel electrophoresis, which revealed fluorescence only for molecular weights corresponding to monomeric aPP or dimeric GCN4. Flow cytometry analysis measured a 400- or 300-fold increase, respectively, in ReAsH fluorescence over background for those cells expressing labeled wild-type (wt) variants.
At levels of imaging requiring the higher resolution of electron microscopy (EM), bipartite tetracysteine display was successfully combined with a new technique, complex-edited electron microscopy, to selectively image protein-protein interactions at high resolution. Since bipartite tetracysteine display introduces selectivity in unfixed cells it provides an image which more closely models the proteins' native state than previous methods using immuno-labeled gold antibodies. Dicysteine-GCN4 complexes localized to the nucleus revealed ReAsH fluorescence only in the nuclei, selectively excluding the nucleoli. Illumination of ReAsH in the presence of oxygenated 3,3'-diaminobenzidine tetrachloride (DAB) successfully produced a nuclear-localized DAB polymer which was then imaged via electron microscopy. EM images revealed a measurable increase in electron density in the nuclei of cells expressing wt variants of GCN4, with properly associated cells displaying 4-fold increases of electron density over cells not stained with ReAsH or DAB.
Bipartite tetracysteine display and complex-edited microscopy provide two novel methods for visualizing discrete conformational states and protein interactions at the fluorescent and electron microscopic levels.
|School Location:||United States -- Connecticut|
|Source:||DAI-B 71/07, Dissertation Abstracts International|
|Subjects:||Cellular biology, Biochemistry, Organic chemistry|
|Keywords:||Biarsenical, Protein-protein interactions, Tetracysteine|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be