Structure Determination of HIV-1 Tat/Fluid Phase Membranes and DMPC Ripple Phase Using X-Ray Scattering (Springer Theses)

This Thesis in biological physics has two components, describing the use of X-ray scattering techniques to study the structure of two different stacked lipid membrane systems. The first part focuses on the interaction between a short 11-mer peptide, Tat, which is part of the Tat protein in the HIV-1 virus. Although highly positively charged, the Tat protein has been shown to translocate through hydrocarbon lipid bilayers easily, without requiring the cell's energy, which is counter to its Born self-energy. In this work Tat's location in the headgroup region was demonstrated using a combined X-ray scattering and molecular dynamics approach. Bilayer thinning was observed as well as softening of different membrane mimics due to Tat. It was concluded that Tat's headgroup location, which increases the area/lipid, and its bilayer softening likely reduce the energy barrier for passive translocation. The second part is a rigorous investigation of an enigmatic phase in the phase diagram of the lipid dimyristoylphosphatidylcholine (DMPC). The ripple phase has fascinated many researchers in condensed matter physics and physical chemistry as an example of periodically modulated phases, with many theoretical and simulation papers published. Despite systematic studies over the past three decades, molecular details of the structure were still lacking. By obtaining the highest resolution X-ray data so far, this work revealed the complex nature of the chain packing, as well as confirming that the major side is thicker than the minor side of the saw-tooth ripple structure. The new model shows that the chains in the major arm are tilted with respect to the bilayer normal and that the chains in the minor arm are slightly more disordered than all-trans gel-phase chains, i.e., the chains in the minor arm are more fluid-like. This work provides the highest resolution X-ray structure of the ripple phase to-date Supervisor's Foreword 6 Abstract 8 Acknowledgments 10 Contents 12 Notations 16 Acronyms 20 1 Introduction 22 1.1 Lipid Bilayers 22 1.2 Tat Peptide 24 1.3 Pβ Ripple Phase 26 Bibliography 27 2 Structural and Material Perturbations of Lipid Bilayers Due to HIV-1 Tat Peptide 30 2.1 Introduction 30 2.2 Materials and Methods 33 2.2.1 Stock Solutions 33 2.2.2 Thin Film Samples 33 2.2.3 Volume Measurements 33 2.2.4 X-Ray Setup 35 2.2.5 Analysis of Diffuse Scattering 36 2.2.6 Modeling the Bilayer Structure 37 2.2.6.1 Functional Forms 38 2.2.6.2 Constraints 41 2.2.6.3 Fits with Lower Bounds 42 2.2.7 Molecular Dynamics Simulation 44 2.3 Analysis of Molecular Dynamics Simulation Data 45 2.3.1 SIMtoEXP Program 45 2.3.2 Local Thinning of Membranes 46 2.3.3 Lateral Decay Length of Membrane Thinning 47 2.4 Results 49 2.4.1 Bending and Bulk Modulus 49 2.4.2 Form Factors 49 2.4.3 Volume 50 2.4.4 Electron Density Profile Modeling 51 2.4.5 Molecular Dynamics Simulations 67 2.5 Discussion 76 2.6 Conclusions 80 Bibliography 80 3 Ripple Phase 85 3.1 Introduction 85 3.1.1 Some Historical Detail 85 3.1.2 Purpose of This Study 88 3.2 Materials and Methods 89 3.2.1 Sample Preparation 89 3.2.2 Instrumental Resolution 91 3.2.2.1 Divergence 92 3.2.2.2 Energy Dispersion 92 3.2.2.3 Geometric Broadening 92 3.2.2.4 Final Instrumental Resolution 98 3.2.3 Low Angle X-Ray Scattering (LAXS) 99 3.2.4 Near Grazing Incidence Wide Angle X-Ray Scattering (nGIWAXS) 101 3.2.5 Transmission Wide Angle X-Ray Scattering (tWAXS) 101 3.2.6 Sample q-Space 103 3.3 LAXS Data Reduction 105 3.3.1 Lattice Structure: Unit Cell 105 3.3.2 Lorentz Correction 106 3.3.3 Absorption Correction for LAXS 111 3.3.4 Correction Due to Mosaic Spread 114 3.3.5 Synopsis of Intensity Corrections 116 3.4 Results for |Fhk| Form Factors 117 3.5 Models to Fit the |Fhk| and Obtain the Phase Factors 121 3.5.1 Contour Part of the Form Factor 123 3.5.2 Transbilayer Part of the Form Factor 123 3.5.3 Some Results of Model Fitting 125 3.5.4 Results for the Phase Factors 127 3.6 Electron Density Profiles and Coarse Grained Bilayer Structure 127 3.7 nGIWAXS: Results 138 3.7.1 Fluid and Gel Phase 138 3.7.2 Ripple Phase 141 3.8 tWAXS: Results 145 3.9 Thin Rod Model 149 3.9.1 Gel Phase Model 150 3.9.2 Ripple Model 158 3.10 Combining WAXS and LAXS Results for the Major Arm 160 3.11 Discussion 162 3.11.1 Major Arm 162 3.11.2 Minor Arm 166 3.12 Conclusion 169 Bibliography 170 Appendices 174 Appendix A 175 A.1 Mosaic Spread for NFIT Analysis 175 A.1.1 Mosaic Spread: Calculation 175 A.1.2 Mosaic Spread: Near Equivalence of Two Methods 179 A.1.3 NFIT 181 A.2 Derivation of the Contour Part of the Form Factor 182 A.3 Derivation of the Transbilayer Part of the Form Factor in the 2G Hybrid Model 184 A.4 Correction Due to Refractive Index 185 Bibliography 186 This Thesis in biological physics has two components, describing℗lthe use of X-ray scattering techniques℗lto study the structure of two different stacked lipid membrane systems.℗l The first part focuses on the interaction between a short 11-mer peptide, Tat, which is part of the Tat protein in the HIV-1 virus.℗l Although highly positively charged, the Tat protein has been shown to translocate through hydrocarbon lipid bilayers easily, without requiring the cellĺls energy, which is counter to its Born self-energy.℗l In this work Tatĺls location in the headgroup region was demonstrated using a combined X-ray scattering and molecular dynamics approach.℗l Bilayer thinning was observed as well as softening of different membrane mimics due to Tat.℗l It was concluded that Tatĺls headgroup location, which increases the area/lipid, and its bilayer softening likely reduce the energy barrier for passive translocation. The second part is a rigorous investigation of an enigmatic phase in the phase diagram of the lipid dimyristoylphosphatidylcholine (DMPC).℗l The ripple phase has fascinated many researchers in condensed matter physics and physical chemistry as an example of periodically modulated phases, with many theoretical and simulation papers published.℗l Despite systematic studies over the past three decades, molecular details of the structure were still lacking.℗l By obtaining the highest resolution X-ray data so far, this work revealed the complex nature of the chain packing, as well as confirming that the major side is thicker than the minor side of the saw-tooth ripple structure.℗l The new model shows that the chains in the major arm are tilted with respect to the bilayer normal and that the chains in the minor arm are slightly more disordered than all-trans gel-phase chains, i.e., the chains in the minor arm are more fluid-like.℗l This work provides the highest resolution X-ray structure of the rippl Front Matter....Pages i-xx Introduction....Pages 1-8 Structural and Material Perturbations of Lipid Bilayers Due to HIV-1 Tat Peptide....Pages 9-63 Ripple Phase....Pages 65-153 Back Matter....Pages 155-168