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The Effect of Styrene-Maleic Acid (SMA) Copolymers on Solubilizing Lipid Bilayers and Forming Nanodiscs

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The Effect of Styrene-Maleic Acid (SMA) Copolymers on Solubilizing Lipid Bilayers and Forming Nanodiscs Portland State University PDXScholar Dissertations and Theses Dissertations and Theses 12-20-2018 The Effect of Styrene-Maleic Acid (SMA) Copolymers on Solubilizing Lipid Bilayers and Forming Nanodiscs Ghada Alramadan Portland State University Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Biophysics Commons Let us know how access to this document benefits ou.y Recommended Citation Alramadan, Ghada, "The Effect of Styrene-Maleic Acid (SMA) Copolymers on Solubilizing Lipid Bilayers and Forming Nanodiscs" (2018). Dissertations and Theses. Paper 4843. https://doi.org/10.15760/etd.6719 This Thesis is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. The Effect of Styrene-Maleic Acid (SMA) Copolymers on Solubilizing Lipid Bilayers and Forming Nanodiscs by Ghada Alramadan A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Physics Thesis Committee: Drake C. Mitchell, Chair Erik J. Sanchez Shankar B. Rananavare Portland State University 2018 Abstract Cell membranes, or plasma membranes, play an essential role in the structure and the function of living cells. In 1972, the fluid mosaic membrane model was the first unifying paradigm of membrane structure. It is no longer considered adequate because evidence of many non-homogeneous lipid structures in both natural and model membranes have been discovered over the past thirty years. The field of membrane biophysics now uses updated versions of the mosaic model, which consists of the complex mixture of different lipid species. The lipid species found in natural membranes produce a range of dynamic, laterally segregated, non-homogeneous domains, which exist on time scales ranging from microseconds to minutes. The cell membrane is an enclosing or separating membrane that acts as a selectively permeable barrier within living things. It consists of the phospholipid bilayer with associated embedded proteins, integral (intrinsic) and peripheral (extrinsic) proteins used for various biological activities. Proteins, especially integral membrane proteins, perform a range of key functions vital to the cell, such as controlled movement of molecules across lipid bilayers, as well as participating in cell signaling and motility. The major obstacle to studying membrane proteins is the tendency for some of their properties to change and the proteins themselves may be denatured when extracted by detergents. One of the most significant approaches to solve this problem is the use of styrene–maleic acid copolymers (SMAs), which offers detergent-free solubilization of membrane, which allows studies of membrane proteins to be done in very small systems. i The main goal of this thesis is to examine the effects of these polymers on the interior of the lipid bilayer. With these, membrane proteins can be extracted from cell membranes while conserving a patch of near-native membrane around them. It has been suggested but not proven that proteins in nanodiscs reside in a hydrophobic environment that is identical to that found in the native cell membrane. Moreover, I also investigate the kinetics of membrane solubilization by SMA by using UV/visible spectrophotometer. In addition, I examine how lipid packing in the nanodiscs is affected by the presence of the polymers and how it depends on polymer composition by using SMA variants with different styrene-to-maleic acid ratios. ii Acknowledgements I would like to thank my advisor Dr. Drake C. Mitchell for his assistance, enthusiasm, and guidance over the two years throughout my time as a graduate student. I would like to thank my lab mate Min Schrader for her advice and helpful discussions and assistance. I acknowledge that this thesis would have been impossible without the help of Portland State University Biophysics Lab. iii Table of Contents Abstract ................................................................................................................................ i Acknowledgements ............................................................................................................ iii List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii 1 Introduction to Lipid Membranes ....................................................................................1 1.1 Molecular Structures of Plasma Membranes ..........................................................1 1.2 History of Membrane Models ................................................................................2 1.2.1 Historical Perspectives .....................................................................................2 1.2.2 Fluid Mosaic Model .........................................................................................3 1.2.3 Lipid Raft Hypothesis ......................................................................................4 1.3 Phospholipids .........................................................................................................9 1.3.1 Micelles, Liposomes, and Bilayers .................................................................11 1.4 Fatty Acids ...........................................................................................................12 1.4.1 Types of Fatty Acids ......................................................................................12 1.4.2 Monounsaturated and Polyunsaturated Fatty Acids .......................................14 1.5 Membrane Proteins ...............................................................................................14 1.5.1 Brief Introduction to Proteins and Membrane Proteins ..................................14 1.5.2 Studying Membrane Proteins Methods of Solubilizing .................................16 1.5.3 Approaches Used to Solubilize Membrane Proteins ......................................17 1.5.4 Kinetics of Membrane Solubilization by SMA .............................................24 1.5.5 Model for the Mode of Action of SMA Copolymers .....................................25 2 Fluorescence Spectroscopy and Fluorescent Probes .....................................................28 2.1 Background of Fluorescence ................................................................................28 2.2 DPH ......................................................................................................................29 3 Materials ........................................................................................................................31 3.1 Material Sources ...................................................................................................31 3.2 Sample Preparation ...............................................................................................31 3.2.1 Vesicles ..........................................................................................................31 4 Methods ........................................................................................................................33 4.1 Background: Jablonski Energy Diagram ..............................................................33 4.2 Definition of Absorbance and Optical Density ....................................................34 4.3 Ultraviolet-Visible Measurements .......................................................................35 4.4 Fluorescence Spectroscopy ..................................................................................36 4.4.1 Light Scattering ..............................................................................................37 5 Frequency-Domain Lifetime and Anisotropy Measurement .........................................39 5.1 Time-resolved Fluorescence (Intensity Decays) ..................................................39 5.2 Time-resolved Anisotropy Decays .......................................................................41 5.3 Rotational Diffusion Model ..................................................................................44 5.4 Least-Squares Analysis of Frequency-Domain Intensity Decays ........................46 6 Instrumentation ..............................................................................................................48 6.1 Agilent 8453 UV-Visible Spectrophotometer ......................................................48 6.1.1 Fluorescence Spectra ......................................................................................50 6.2 ISS Chronos Spectrometer ...................................................................................52 iv 7 Results and Discussion ..................................................................................................57 7.1 Kinetics of Membrane Solubilization by SMA ....................................................57 7.2 Fluorescence Lifetime ..........................................................................................59 7.3 Dynamic Fluorescence Anisotropy ......................................................................61 8 Conclusions ...................................................................................................................68 References ..........................................................................................................................69
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