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Miami University – the Graduate School MIAMI UNIVERSITY – THE GRADUATE SCHOOL CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation Of Elvis K. Tiburu Candidate for the Degree: Doctor of Philosophy Dr. Gary A. Lorigan, Director Dr. Christopher A. Makaroff, Reader Dr. Robert E. Minto, Reader Dr. Richard T. Taylor, Reader Dr. David G. Pennock Graduate School Representative ABSTRACT DEVELOPMENT OF NEW METHODS FOR THE ALIGNMENT OF LONGER CHAIN PHOSPHOLIPIDS IN BICELLES AND SOLID-STATE NMR STUDIES OF PHOSPHOLAMBAN by Elvis K. Tiburu Magnetically aligned phospholipid bilayers or bicelles are model systems that mimic biological membranes for magnetic resonance studies. A long chain phospholipid bilayer system that spontaneously aligns in a static magnetic field was characterized utilizing solid-state NMR spectroscopy. The oriented membrane system was composed of a mixture of the bilayer-forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short-chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated. Traditionally, the shorter 14-carbon chain phospholipid dimyristoyl- phosphatidylcholine (DMPC) has been utilized as the bilayer-forming phospholipid in bicelle studies. The effect of cholesterol in bicelles containing chain perdeuterated 2 DMPC, a partially deuterated (a-[2,2,3,4,4,6- H6]) cholesterol, and stearic acid-d35 has been reported as a function of temperature using 2H solid-state NMR spectroscopy. The order parameters of the labeled probes were calculated and compared with values obtained from unoriented samples in the literature. In addition, 2H solid-state NMR spectroscopy was used to investigate the orientation and side chain dynamics of specific- labeled methyl groups of leucines in PLB in unoriented as well as in magnetically and mechanically aligned phospholipids bilayers. Phospholamban (PLB), is a 52 amino-acid protein that assembles into a pentamer in cardiac sarcoplasmic reticulum membranes. The protein is a key regulator of the calcium ATPase through an inhibitory association that can be reversed by phosphorylation. From the 2H NMR studies, the exhibited line shapes were characteristic of either methyl group reorientation about the Cg-Cd bond axis, 15 or by additional wobbling motion about the Ca-Cb and Cb-Cg bond axes. Using N NMR spectroscopy, the backbone dynamics of PLB as well as the orientation of PLB in the model membranes were also studied. A comparative study of PLB in shorter chain DMPC as well as in longer chain DOPC bilayers was conducted to determine the effect of hydrophobic length on peptide orientation. Taken together, the results are discussed in terms of the structure of PLB in phospholipid bilayers previously proposed on the basis of mutational and molecular modeling studies, thus, providing an understanding of the structure of PLB in model membranes. DEVELOPMENT OF NEW METHODS FOR THE ALIGNMENT OF LONGER CHAIN PHOSPHOLIPIDS IN BICELLES AND SOLID-STATE NMR STUDIES OF PHOSPHOLAMBAN A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry By Elvis K. Tiburu Miami University Oxford, Ohio 2004 Dissertation Director: Gary A. Lorigan Table of Contents List of Figures vii List of Tables vi Chapter 1: Development of Magnetically Aligned Phospholipid Bilayers in Mixtures of Palmitoylstearoylphosphatidylcholine and Dihexanoylphosphatidylcholine by Solid-State NMR Spectroscopy 1 1.0 Introduction 3 1.1 Phospholipid bilayers as model systems for protein studies 3 1.2 Materials 4 1.3 Methods 5 1.3.1 Sample preparation 5 1.3.2 NMR spectroscopy 6 1.4 Results 6 1.4.1 Magnetic alignment of phospholipid bilayers with 2H NMR spectroscopy 6 1.4.2 Magnetic alignment of phospholipid bilayers with 31P NMR spectroscopy 12 1.5 Discussion 12 1.6 References 18 Chapter 2: Solid-State 2H NMR Studies of the Effects of Cholesterol on Acyl Chain Dynamics of Magnetically Aligned Phospholipid Bilayers 21 2.0 Introduction 23 2.1 The effects of cholesterol and stearic acid on phospholipid bilayers 23 2.2 Materials 26 2.3 Methods 26 2.3.1 Sample preparation 26 2.3.2 NMR spectroscopy 27 2.3.3 Order parameter calculations 28 ii 2.4 Results and discussion 29 2.4.1 Cholesterol dynamics in phospholipid bilayers 29 2.4.2 Magnetic aligned DMPC/DHPC bicelles investigated by stearic acid-d35 36 2.4.3 Calculating order parameters for stearic acid-d35 inserted into magnetically aligned phospholipid bilayers 39 2.5 Conclusion 41 2.6 References 44 Chapter 3: An Improved Synthetic and Purification Procedure for the Hydrophobic Segment of the Peptide Phospholamban 49 3.0 Introduction 51 3.1 The role of phospholamban in Ca2+ transport 51 3.2 Materials 54 3.3 Methods 55 3.3.1 Peptide synthesis 55 3.3.2 Peptide purification 56 3.4 Results and discussion 56 3.5 References 63 Chapter 4: Investigating Structural Changes in the Lipid Bilayer upon Insertion of the Transmembrane Domain of the Membrane-Bound Protein Phospholamban Utilizing 31P and 2H Solid-State NMR Spectroscopy 68 4.0 Introduction 70 4.1 Protein-lipid interaction 70 4.2 Materials 71 4.3 Methods 72 4.3.1 Synthesis and purification of PLB 72 4.3.2 NMR Sample preparation 72 iii 4.3.3 NMR spectroscopy 72 4.3.4 NMR data analysis 73 4.4 Results and discussion 74 4.4.1 31P NMR study of PLB incorporated into POPC bilayer 74 4.4.2 2H NMR study of PLB incorporated into POPC bilayer 80 4.5 Conclusion 84 4.6 References 86 Chapter 5: Investigating the Dynamic Properties of the Transmembrane Segment of Phospholamban Incorporated into Phospholipid Bilayers Utilizing 2H and 15N Solid-State NMR Spectroscopy 92 5.0 Introduction 94 5.1 Dynamics of PLB in phospholipid bilayers 94 5.2 Materials 98 5.3 Methods 99 5.3.1 Peptide synthesis 99 5.3.2 Peptide purification 99 5.3.3 Solid-state NMR sample preparation 100 5.3.4 2H solid-state NMR spectroscopy 100 5.3.5 15N soilid-state NMR spectroscopy 101 5.3.6 NMR data analysis 101 5.4 Results 102 5.4.1 2H side chain dynamics 102 5.4.2 15N backbone dynamics 105 5.4.3 Helical orientation of PLB with respect to the lipid bilayer 107 5.5 Discussion 109 5.5.1 Dynamics of PLB in phospholipid bilayers 109 5.5.2 Structural implications of the leucine side-chain motions 109 5.5.3 Structural model of TM-PLB in lipid bilayers 115 5.6 Conclusion 116 5.7 References 118 iv Chapter 6: 2H and 15N Solid-State NMR Spectroscopic Studies of the Helical Tilt of the Transmembrane Segment of Phospholamban Incorporated into Magnetically Aligned and Mechanically Aligned Phospholipid Bilayers 125 6.0 Introduction 127 6.1 Incorporation of PLB into lipids of different chain lengths 127 6.2 Materials 130 6.3 Methods 131 6.3.1 Peptide synthesis 131 6.3.2 Peptide purification 131 6.3.3 2H and 15N aligned solid-state NMR sample preparation 132 6.3.4 2H solid-state NMR spectroscopy 133 6.3.5 15N soilid-state NMR spectroscopy 133 6.3.6 2H tilt angle calculations 133 6.3.7 15N tilt angle calculations 135 6.4 Results 136 6.5 Discussion 141 6.6 References 150 v List of Tables Table 5.1 CSA values measured from 15N-labeled PLB 114 Table 6.1 5N chemical shifts from 15N-labeled PLB 143 vi List of Figures Figure 1.1 2H NMR of a 25% (W/W) q=3.5 DMPC/DHPC 7 Figure 1.2 2H NMR of a 25% (W/W) q=1.8 PSPC/DHPC 8 Figure 1.3 2H NMR of PSPC/DHPC as a function of temperature 9 Figure 1.4 2H NMR of a 25% (W/W) q=2.0 PSPC/DHPC/Yb3+ 10 Figure 1.5 31P NMR of a 25% (W/W) q=1.8 PSPC/DHPC 14 Figure 2.1 Structure of stearic acid-d35 29 Figure 2.2 2H NMR of deuterated cholesterol in DMPC/DHPC 30 Figure 2.3 2H NMR of DMPC/DHPC with cholesterol 32 Figure 2.4 2H NMR of a 25% (W/W) q=3.5 DMPC/DHPC/Yb3+ 33 1 Figure 2.5 Temperature-dependent SCD order parameter profile 35 2 3+ Figure 2.6 H NMR of stearic acid-d35 in DMPC/DHPC/Yb 37 2 3+ Figure 2.7 H NMR of stearic acid-d35 in DMPC/DHPC/ Chol/ Yb 38 1 3+ Figure 2.8 Temperature-dependent SCD with Yb 40 2 3+ O Figure 2.9 H NMR of stearic acid-d35 in DMPC/DHPC/Yb at 40 C 42 Figure 3.1 Two models of PLB 53 Figure 3.2 Fmoc deprotection of amino acids 58 Figure 3.3 Reverse-phase HPLC profile of PLB 59 Figure 3.4 MALDI-TOF MS of PLB 61 Figure 4.1 31P NMR of POPC bilayers as a function of temperature 75 Figure 4.2 31P NMR of POPC bilayers with 4 mol% PLB 76 Figure 4.3 31P NMR of POPC bilayers with 6 mol% PLB 78 Figure 4.4 2H NMR powder pattern spectra of PLB in POPC bilayers 81 Figure 4.5 2H NMR powder pattern spectra of PLB with temperature 82 1 Figure 4.6 Temperature-dependent SCD order parameter profile 84 vii Figure 5.1 The two models of PLB in lipid bilayers 97 Figure 5.2 The amino acid sequence of PLB 102 Figure 5.3 2H NMR powder pattern spectra of PLB in POPC 103 Figure 5.4 15N NMR powder pattern spectra of PLB in POPC 106 Figure 5.5 15N solid-state NMR of PLB in oriented DOPC bilayers 108 2 Figure 5.6 Simulations of CD3-LeuPLB NMR lineshapes from H spectra 111 Figure 5.7 A model of pentameric PLB in phospholipid bilayer 116 Figure 6.1 2H NMR spectra of a DMPC/DHPC/2H-labeled PLB bicelle Sample 138 Figure 6.2 2H NMR spectra of DOPC/DOPE/2H-labeled PLB samples 139 Figure 6.3 One-dimensional solid-state 15N NMR spectra of specifically 15N-labeled PLB in oriented DMPC/DHPC bicelles with q ratio 3.5 and DOPC/DOPE phospholipid bilayers 142 Figure 6.4 Contour plots representing the chemical shifts calculated for the rotation, r from 00 to 3600 and for tilt angle, t from 00 to 900 144 Figure 6.5 The two models showing the orientation of monomeric PLB incorporated into phospholipid bilayers 148 viii Dedication This work is dedicated to Jesus Christ, whom I am privileged to know as Lord, Savior and Friend.
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