Genomic sequencing efforts have provided the amino acid sequences of thousands of membrane proteins, but even with all this information, we still understand little about the relationships between sequence, structure, and function in this important class of molecules. Membrane proteins reside in an apolar and anisotropic environment, so it is likely the relationships will differ significantly from those in soluble proteins. In addition to high resolution structural data, a thorough consideration of membrane protein energetics will be required to fully elucidate the molecular determinants of membrane protein stability.
In this thesis, we have focused on the thermodynamics of membrane protein self-association and have explored two systems: the transmembrane (TM) domains of the epidermal growth factor receptors (EGFRs) and the transmembrane β-barrel outer membrane phospholipase A (OMPLA). We find the EGFR TM domains have little propensity to self-associate, despite containing a sequence motif once thought to be sufficient for strong helix-helix interaction. These results suggest the domains do not play a significant role in specifying or stabilizing the EGF receptor complexes.
Our sedimentation equilibrium analytical ultracentrifugation studies of OMPLA represent the first quantitative study of the self-association of a transmembrane β-barrel and provide a new framework in which to examine the concepts that have emerged from work on helix-helix interactions. We find that interactions with the substrate acyl chain stabilize OMPLA dimerization more significantly than binding of calcium (a required co-factor), consistent with a model in which access to substrate regulates OMPLA activity. Using a series of sulfonyl fluoride inhibitors, we also find an unexpected sigmoidal response in dimer stability as a function of chain length. This result is in contrast to the linear correlation between stability and contact surface found in several helix-helix studies. Further highlighting the context dependence of molecular interactions, we find that an intermolecular hydrogen bond at a highly-conserved glutamine residue contributes surprisingly little to dimer stability. While the crystal structure of an OMPLA dimer was known, many of our results were unanticipated, emphasizing the need for thermodynamic data if we are to identify the molecular interactions and forces that govern membrane protein stability.
|Advisor:||Fleming, Karen G.|
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 68/04, Dissertation Abstracts International|
|Keywords:||EGFR, Membrane proteins, Phospholipase A, Self-association, Transmembrane domains|
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