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Flexibility and specificity of the interaction of MCL-1 with BIM BH3

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dc.contributor.advisor Amy E. Keating. en_US
dc.contributor.author Fire, Emiko J. (Emiko June) en_US
dc.contributor.other Massachusetts Institute of Technology. Dept. of Biology. en_US
dc.date.accessioned 2009-09-24T20:49:06Z
dc.date.available 2009-09-24T20:49:06Z
dc.date.copyright 2009 en_US
dc.date.issued 2009 en_US
dc.identifier.uri http://hdl.handle.net/1721.1/46808
dc.description Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009. en_US
dc.description This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. en_US
dc.description Includes bibliographical references (p. 120-128). en_US
dc.description.abstract Interactions among proteins of the BCL-2 family regulate apoptosis - the process of programmed cell death. This thesis focuses on interactions between anti-apoptotic BCL-2 proteins and BH3 peptides derived from pro-apoptotic BCL-2 proteins, observed in earlier studies to occur with significant selectivity. In order to better understand determinants of specificity in anti-apoptotic/BH3 interactions, I have employed structural and binding studies of BIM BH3 point mutants with anti-apoptotic proteins. I report X-ray crystal structures of MCL-1 in complex with peptides of wild-type and three point mutant sequences of BIM BH3. Together, these structures exhibit a range of conformations within the complex. Structural change with respect to wild-type is most dramatic for an isoleucine-to-tyrosine mutant of BIM (I2dY). The larger residue is accommodated by helix [alpha]3 of MCL-1 and the BIM BH3 peptide helix shifting away from one another. In a phenylalanine-to-glutamate mutation (F4aE), the altered side chain is accommodated by a simple rotation of the side chain out of the hydrophobic pocket, a more modest structural change overall. I have also adapted SPOT array technology to qualitatively and simultaneously measure the interactions of anti-apoptotic BCL-XL and MCL-1 with a large number of surface-bound BH3 peptides. The best results were obtained when (i) longer BH3 peptides (26-mer vs. 20-mer) were employed, and (ii) antibodies were used to detect the binding of anti-apoptotic proteins. en_US
dc.description.abstract (cont.) The SPOT technique was used to perform substitution analyses, wherein each position of interest in a BH3 peptide is mutated to all possible amino acids (or a subset thereof). The results of these experiments identify several sites that, in the context of BIM, play a role in discriminating between MCL-1 and BCL-XL. Two of these sites are always occupied by small amino acids in known native BH3s, while two others are typically hydrophobic positions that become buried upon interaction with anti-apoptotic BCL-2 proteins. Together, these studies illustrate that the MCL-1 interaction with BIM BH3 is flexible enough to accommodate numerous point mutations, and that certain sites within BIM can be used to encode interaction specificity for both MCL-1 and BCL-XL. en_US
dc.description.statementofresponsibility by Emiko J. Fire. en_US
dc.format.extent 128 p. en_US
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. en_US
dc.rights.uri http://dspace.mit.edu/handle/1721.1/7582 en_US
dc.subject Biology. en_US
dc.title Flexibility and specificity of the interaction of MCL-1 with BIM BH3 en_US
dc.type Thesis en_US
dc.description.degree Ph.D. en_US
dc.contributor.department Massachusetts Institute of Technology. Dept. of Biology. en_US
dc.identifier.oclc 432671586 en_US


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