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Solid-state NMR spectroscopic studies on phospholamban and Saposin C proteins in phospholipid membranes [electronic resource] /

by Abu-Baker, Shadi.; Miami University (Oxford, Ohio). Dept.; Theses and, OhioLINK Electronic

Abstract (Summary)
Solid-state NMR spectroscopic techniques were used to investigate two significant membrane proteins, phospholamban (PLB) and Saposin C (Sap C). For the first protein, analysis of the 2H and 31P solid-state NMR data of chemically synthesized WT-PLB and its phophorylated form (P-PLB) in 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) multilamellar vesicles (MLVs) indicates that the interaction of P-PLB with POPC bilayers was less significant when compared to PLB. Moreover, the secondary structure using 13C=O site-specific isotopically labeled Ala15-PLB and Ala15-P-PLB in POPC bilayers suggests that this residue, located in the cytoplasmic domain, is a part of an [alpha]-helical structure for both PLB and P-PLB. Also, 2H NMR spectra of site-specific CD3-labeled WT-PLB and P-PLB at Ala15 exhibit one strong isotropic spectral component indicating the presence of additional motions as well as faster side-chain reorientations when compared with Leu51 and Ala24 representing the transmembrane domain. Conversely, the 15N Ala11 NMR spectrum of WT-PLB located on the cytoplasmic domain yields two dynamic components (powder pattern component and isotropic component) implying that the backbone dynamics of this residue exists in two populations: one that is immobile, and another which is motionally averaged on the NMR timescale. Upon phosphorylation, the 15N mobile component contribution increases. The POPC 15N NMR spectra indicate that the transmembrane domain has a tilt angle of 13 ± 6° with respect to the mechanically oriented POPC bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. For the second protein, 2H and 31P solid-state NMR data of Sap C in dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) mixed bilayers indicates that Sap C is not inserting deep into the bilayers and that it has no preference to DOPS over DOPG. Finally, several other solid-state NMR spectroscopic experiments indicate that protonated Sap C disturbs 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) lipid bilayers and not the neutral POPC lipids.
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School:Miami University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:phospholipids proteins nuclear magnetic resonance spectroscopy organic solid state chemistry peptides phospholamban saposin c nmr phase peptide synthesis protein membrane interaction multilamellar vesicles side chain and backbone dynamics structural topology

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