Award Date

12-2010

Degree Type

Thesis

Degree Name

Master of Science in Biological Sciences

Department

Life Sciences

First Committee Member

Eduardo Robleto, Co-chair

Second Committee Member

Tarmo Roosild, Co-chair

Third Committee Member

Helen Wing

Fourth Committee Member

Andrew Andres

Graduate Faculty Representative

Ernesto Abel-Santos

Number of Pages

67

Abstract

Apoptosis, or programmed cell death, is a vital process intimately involved in the embryonic development and homeostatic maintenance of all multicellular organisms. The committing step to apoptosis is regulated by a key protein, Bax, and its ability to integrate and form a pore structure at the outer mitochondrial membrane.


Unfortunately, the molecular details of apoptosis remain largely unclear due to the lack of structural data of integral membrane (IM) Bax. Experimental limitations of membrane protein production have slowed the pursuit of an IM-Bax structure simply because standard protocols for producing recombinant IM-Bax are inefficient in producing adequate quantities of IM-Bax protein. This complication makes it difficult to study Bax in its activated oligomeric state and essentially impossible to develop a protein crystal for x-ray structure analysis.


In this thesis, we have developed solutions to these substantial problems by utilizing the membrane affinity properties of the protein Mistic to promote the membrane integration and efficient protein production of Bax in an E. coli expression system. In addition, we have discovered detergent conditions optimized for high protein yield and stability of an activated trimeric Bax. The activated trimer complex was further characterized by size-exclusion chromatography and multi-angle light scattering techniques to detail the molecular contributions of protein in the detergent-protein complex. Most importantly, the effectiveness of our protocols has produced the first xray diffracting crystal of an activated Bax multimer, an invaluable tool for the solution structure of activated Bax complex. This hallmark in apoptosis research will further complete the understanding of apoptosis mechanism and ultimately lead to the development of a new realm of effective pharmaceuticals.

Keywords

Apoptosis; Bax; Bcl-2; Cell death; Multimer; Oligome; Proteins

Disciplines

Biochemistry | Cell Biology | Molecular Biology

Language

English


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