Membrane proteins constitute more than half of all drug targets in the pharmaceutical industry today, which is not surprising given that a third of human genome codes for membrane proteins, and that they perform a large number and variety of cellular functions. However, membrane proteins to date have been structurally under-characterized as a consequence of the technical difficulties encountered by solution NMR and x-ray crystallography in solving atomic-resolution structures of these proteins. Work presented here focused on development of the Nanodisc platform for structural studies of membrane proteins using solid-state NMR (SSNMR).
SSNMR has emerged in the recent years as a rapidly developing technique complementary to solution NMR and x-ray crystallography for atomic-resolution studies of membrane proteins. To obtain the most structurally and functionally relevant information, membrane proteins should be studied under conditions most resembling their native state. Nanodiscs, the reconstituted nascent high-density lipoprotein particles have been developed, optimized, and successfully utilized as a native-like membrane mimetic for biophysical and biochemical characterization of a variety of membrane proteins. This work aimed to develop and optimize Nanodisc preparations for SSNMR studies. SSNMR was used to obtain structural information on membrane scaffold protein (MSP), the main protein component of nascent high-density lipoprotein particles, and distinguish between different proposed models of the lipid-bound state of this protein. Within this work, SSNMR was also used to study Nanodisc-embedded human cytochrome P450 3A4, a drug-metabolizing enzyme, in the active state. Cytochrome P450 3A4 is responsible for metabolism of almost half of commercial drugs by oxidizing a large number of structurally diverse substrates, yet many aspects of how it performs its function with such a wide range of substrates remain unknown. Additionally, lipid-protein correlations in the Nanodisc were observed by SSNMR, and these studies were initiated with the MSP and cytochrome P450 3A4.
Work presented in this thesis exemplifies the potential of SSNMR in combination with Nanodiscs for obtaining structural information on membrane proteins embedded in native-like lipid bilayers that preserve activity and function of the protein. Furthermore, it shows that SSNMR can provide information about the interaction between the lipid membrane and the membrane protein in the Nanodisc, including the location and depth of insertion of membrane protein in the lipid bilayer.
|Advisor:||Sligar, Stephen G., Rienstra, Chad M.|
|School:||University of Illinois at Urbana-Champaign|
|School Location:||United States -- Illinois|
|Source:||DAI-B 71/01, Dissertation Abstracts International|
|Keywords:||Apolipoprotein, Lipid binding, Membrane proteins, Nanodiscs|
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