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Solid-state NMR studies of phospholipid model membranes and membrane-associated macromolecules

Lu, Junxia
http://rave.ohiolink.edu/etdc/view?acc_num=miami1184090235

Abstract Details

2007, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
Cholesterol is commonly found in eukaryotic cell membranes and has implicated cellular functions. The synthetic peptide KIGAKI is a peripheral membrane protein that has greater antimicrobial activity than many natural-occurring antimicrobial peptides. Phospholamban (PLB) is an integral membrane protein that regulates the contraction and relaxation cycle of cardiac muscle cells in the heart by binding to Ca-ATPase. The three membrane-associated macromolecules and their interactions with the phospholipid membranes were studied. The topology of magnetically oriented phospholipid bilayer model membranes (bicelles) was probed using a high-resolution 1H-13C heteronuclear dipolar solid-state NMR spectroscopy (SAMMY). Three different membrane-associated molecules (cholesterol, KIGAKI and PLB) were incorporated into the bicelles. The effects of these molecules on the membrane topology were compared and the SAMMY technique proved to be a fast and promising way to detect the different modes of interaction of these membrane-associated molecules with the bicelle membrane lipids. The effect of cholesterol on the bicelles was further studied utilizing both solid-state 2H NMR and the EPR spin labeling approach. The results indicate a higher molecular ordering of the lipids and a higher alignment transition temperature of the bicelles in the presence of cholesterol. Antimicrobial peptides can interact directly with the bacterial membranes and disrupt the membranes without causing any harm to the host mammalian membranes. The interaction mechanism between KIGAKI and different membrane mimics with various lipid compositions was analyzed using solid-state 2H, 31P NMR and relaxation studies from the membrane perspective. The results suggested a carpet interaction mechanism for KIGAKI. Uniform 15N-labeled PLB was expressed as a maltose fusion protein in E. coli. BL21 (RIL) (DE3) cells and purified with a combination of affinity chromatography and reverse phase C4 HPLC. Solid-state 15N NMR studies concluded that PLB has two populations with different dynamic motions upon reconstitution into POPC bilayers and the difference is in the cytosolic segment of PLB. 15N NMR studies of PLB on oriented membranes also indicated that the transmembrane segment of PLB aligns close to the bilayer normal, whereas the cytosolic segment aligns nearly perpendicular to the bilayer normal and closer to the membrane surface.
Gary Lorigan (Advisor)
POPG/POPC; bilayer; NMR; POPC; KIGAKI; DMPC/DHPC; bicelles

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Citations

  • Lu, J. (2007). Solid-state NMR studies of phospholipid model membranes and membrane-associated macromolecules [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1184090235

    APA Style (7th edition)

  • Lu, Junxia. Solid-state NMR studies of phospholipid model membranes and membrane-associated macromolecules. 2007. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1184090235.

    MLA Style (8th edition)

  • Lu, Junxia. "Solid-state NMR studies of phospholipid model membranes and membrane-associated macromolecules." Doctoral dissertation, Miami University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=miami1184090235

    Chicago Manual of Style (17th edition)

miami1184090235
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© 2007, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.