Date of Completion

1-10-2017

Embargo Period

10-9-2017

Keywords

nanodisc, NMR, DMPC lipids, nanoscale bilayer system, TEM, membrane scaffold protein, beta-barrel, transmembrane helix, MOSP, TprC

Major Advisor

Dr. Olga Vinogradova

Associate Advisor

Dr. Nathan Alder

Associate Advisor

Dr. Andrew Wiemer

Associate Advisor

Dr. Arlene Albert

Associate Advisor

Dr. Debra Kendall

Field of Study

Structural Biology and Biophysics

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Several membrane proteins, comprising of either α-helical or β-barrel structures have been successfully studied in vitro using several membrane mimetic systems. Nanodiscs, a new class of discoidal nanoscale lipoprotein complex have been used extensively in the present study to investigate different membrane proteins. We begin by delineating the development of small (D7) nanodiscs through its rigorous characterization and demonstrate advantages in solution NMR applications. In addition, we show the development of an on-column method to generate nanodiscs through the selective use of variable protein tags. D7 discs have been further used to visualize the downstream phosphorylation events of activated integrin β3 through Src kinase. The C-terminal domains, TprC and MOSPC, two β-barrel membrane porins, were shown to trimerize when visualized through negatively stained transmission electron microscopy images in nanodiscs. The periplasmic and membrane conformers of native MOSP in Treponema denticola was established along with the partial identification of their multimeric complexes through a series of immuno-chemical experiments. Also discussed are two specific studies which revisit previously published papers where, (a) the direction of the cytoplasmic tail of integrin β3 bound to the SH3 domain of Src kinase was shown to be in the reverse orientation w.r.t the published crystal structure; and (b) PLIC proteins, [Protein Linking the cell membrane (IAP/CD47) to the Cytoskeleton] was shown to be involved in proteasomal degradation with no binding ability to the cytoplasmic tail of CD47. Small angle X-ray scattering studies of BTN3A1, an immune modulator, revealed the rearrangement of the intracellular cytoplasmic B30.2 domain with respect to the juxta membrane region in the presence of a phosphoantigen HMBPP. Lastly, a novel finding is briefly described involving orthlogs of TamB, a component of the transloaction and assembly module system, comprising of the TamA-TamB complex in proteobacteria, interacting with BamA (Beta-barrel assembly machinery protein A) in bacterial diderms where TamA is absent.

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