The protein complex composed of the cytoplasmic domain of band 3 (cdb3) and ankyrin forms one of the two major contact sites between the spectrin-based membrane skeleton and the lipid bilayer in human erythrocytes. This linkage is critical for maintaining the shape and viscoelastic properties of the membrane. Among the known membrane skeleton protein mutations, band 3 (anion exchanger 1 or AE1) variants account for about 20% of hereditary spherocytosis cases among Caucasians. The structure and function of cdb3 and ankyrin repeat domain 3-4 have been studied via site directed labeling methods in combination with conventional electron paramagnetic resonance (EPR), double electron electron resonance (DEER), and fluorescence spectroscopy. The central compact region (55-356) of the cdb3 dimer under physiological pH is indistinguishable from the crystal structure determined at pH 4.8. The N terminus (1-54) of cdb3 is dynamically disordered and capable of docking various cytoplasmic proteins. The similar disorder in the C terminus (357-359) of cdb3 is consistent with the weak motional coupling between the cytoplasmic domain and the transmembrane domain of band 3. The surface of the cdb3 peripheral domain that is opposite the dimerization arm interacts with the ankyrin groove. The á-helix 2, á-helix 3 and â strands 6, 7 (hairpin) of cdb3 may be directly involved in ankyrin binding. The band 3 Tuscaloosa mutation (P327R), which is located at a loop turning point of the dimerization arm, does not dissociate the cdb3 dimer but does perturb the region adjacent to position 327 and the downstream C terminus, thereby slightly destabilizing the dimer structure. These spectroscopic studies establish a structural model for cdb3 and its binding partner ankyrin in solution at neutral pH, which provides an important platform to further characterize protein-protein interactions that stabilize the membrane and naturally occurring mutations that cause human diseases.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-04032006-101559 |
| Date | 06 April 2006 |
| Creators | Zhou, Zheng |
| Contributors | Hassane S. Mchaourab, P. Anthony Weil, Phoebe Stewart, Terry P. Lybrand, Charles R. Sanders II |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-04032006-101559/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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