Crystallography and Detergent Use in CMC
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Questions and Answers

Which of the following statements about critical micelle concentration (CMC) is TRUE?

  • Above CMC, adding more detergent leads to an increase in free detergent molecules.
  • The CMC is the concentration at which all detergent molecules are associated with micelles, with minimal free detergent molecules. (correct)
  • CMC is not relevant for biological applications as it only relates to detergent behavior in solutions.
  • CMC is the concentration of detergents below which micelles are spontaneously formed.

    • What is the primary role of detergent in the process of solubilizing proteins in crystallography?

  • To facilitate the formation of micelle-encapsulated proteins (correct)
  • To reduce the overall volume of protein present
  • To enhance the stability of the protein structure
  • To increase the concentration of protein in the solution

    • What typically happens to the concentration of detergent during the purification of micelle-encapsulated proteins?

  • It decreases to below critical micelle concentration (CMC)
  • It increases to levels exceeding the critical micelle concentration (CMC) (correct)
  • It remains constant at the critical micelle concentration (CMC)
  • It fluctuates unpredictably during purification

    • Why is the exchange of detergent to a high-CMC detergent important in the process outlined?

    <p>It reduces the likelihood of protein aggregation (B)</p> Signup and view all the answers

    What is the main goal after transferring to a nanodisc in the process of crystallography preparation?

    <p>To achieve a stable and monodisperse protein solution (B)</p> Signup and view all the answers

    In the context of this crystallography method, what does 'lyse cells' refer to?

    <p>To break open cells to release internal components (B)</p> Signup and view all the answers

    In the HI phase, what structure is formed by the hydrophobic tails?

    <p>They face inward towards the center of the cylinder. (D)</p> Signup and view all the answers

    Why are cubic phases considered a good approximation for membrane environments?

    <p>They provide a stable, ordered arrangement. (C)</p> Signup and view all the answers

    What type of detergent is characterized as having a positive, zero net charge?

    <p>Zwitterionic detergent (A)</p> Signup and view all the answers

    What physical property makes extended bilayers energetically unfavorable?

    <p>High surface area to volume ratio (B)</p> Signup and view all the answers

    What is the significance of spherical structures in cellular compartment generation?

    <p>They are the most efficient shape for enclosing space. (D)</p> Signup and view all the answers

    Which cubic phase is based on the mathematical minimal surface G?

    <p>Fd3m (A)</p> Signup and view all the answers

    Which compound is classified as a non-ionic detergent?

    <p>CHAPS (A)</p> Signup and view all the answers

    What is the role of hydrophilic head groups in the HII phase?

    <p>They form the outer layer of the structure. (B)</p> Signup and view all the answers

    What do cholesterol and sphingomyelin primarily contribute to in cell membranes?

    <p>Formation of lipid rafts (B)</p> Signup and view all the answers

    Which type of molecules are mainly attracted by lipid rafts within the Golgi membranes?

    <p>Cargo proteins (A)</p> Signup and view all the answers

    What is the primary role of lipid rafts regarding signaling proteins?

    <p>Clustering and amplifying signaling activity (A)</p> Signup and view all the answers

    How do sphingomyelin molecules affect the thickness of lipid patches?

    <p>Their long and saturated nature leads to thicker patches. (C)</p> Signup and view all the answers

    What phenomenon is described by 'interleaflet coupling' in membrane interactions?

    <p>Coupling of acyl chains in upper and lower leaflets (D)</p> Signup and view all the answers

    Which factor is crucial for the formation of mesophases in water-lipid mixtures?

    <p>Temperature and composition of the solvent (A)</p> Signup and view all the answers

    What characteristic is attributed to the lipid fluid lamellar phase?

    <p>It resembles the physiologically relevant state of biomembranes. (A)</p> Signup and view all the answers

    What is a major factor influencing phase transitions in lipid membranes?

    <p>Cholesterol concentration (B)</p> Signup and view all the answers

    What effect does cholesterol have on the packing of lipids in the membrane?

    <p>It tightens lipid packing and increases membrane rigidity. (A)</p> Signup and view all the answers

    How does cholesterol behave at low temperatures regarding membrane fluidity?

    <p>It decreases interactions between lipids, increasing fluidity. (D)</p> Signup and view all the answers

    What happens to lipid bilayer permeability when cholesterol is added?

    <p>Permeability is increased in the fluid phase and decreased in the gel phase. (B)</p> Signup and view all the answers

    In what manner does cholesterol act as a buffer for membrane fluidity?

    <p>It decreases fluidity at low temperatures and increases it at high temperatures. (C)</p> Signup and view all the answers

    What is the role of cholesterol in phase transitions of lipid membranes?

    <p>It eliminates the transition phase of the lipid bilayer. (C)</p> Signup and view all the answers

    What characterizes the 'liquid ordered phase' (Lo) induced by cholesterol?

    <p>It has interactions similar to both gel and fluid phases. (D)</p> Signup and view all the answers

    What consequence does cholesterol have on the temperature transition of lipid membranes?

    <p>It narrows the usable temperature range of pure phospholipid membranes. (B)</p> Signup and view all the answers

    Which phrase accurately describes the interactions cholesterol has with phospholipid molecules in a membrane?

    <p>Cholesterol orients with its polar head group near the polar groups of phospholipids. (D)</p> Signup and view all the answers

    In a P/T phase diagram, what happens to a substance at temperatures and pressures away from the phase equilibrium curves?

    <p>The substance will exist solely in one phase. (B)</p> Signup and view all the answers

    What is the state of matter referred to as a "supercritical fluid?"

    <p>A state where a substance exhibits properties of both liquid and gas. (A)</p> Signup and view all the answers

    What is a distinctive feature of the lamellar phase in biological lipid systems?

    <p>The arrangement of lipid molecules into sheets of bilayers separated by bulk liquid. (A)</p> Signup and view all the answers

    What is the primary characteristic that distinguishes the liquid disordered (Lα) phase from the gel phase in lamellar structures?

    <p>The higher fluidity and lateral diffusion within the Lα phase. (A)</p> Signup and view all the answers

    Which of the following statements accurately describes the transition between the gel and fluid lamellar phases?

    <p>The transition is primarily driven by changes in temperature, typically with higher temperatures favoring the fluid phase. (D)</p> Signup and view all the answers

    What is the significance of the lamellar fluid crystal phase being a mesophase?

    <p>It signifies that the phase exhibits properties intermediate between a true crystal and a liquid. (B)</p> Signup and view all the answers

    Based on the provided content, which of the following is NOT a property of the gel phase in lamellar structures?

    <p>High permeability to water (B)</p> Signup and view all the answers

    What is the primary reason why the "lamellar fluid crystal phase" is considered the closest to the functional biomembrane state under physiological conditions?

    <p>It provides a balance between structural stability and fluidity, enabling membrane function. (C)</p> Signup and view all the answers

    Flashcards

    Cholesterol in Eukaryotes

    1 cholesterol molecule per phospholipid enhances membrane stability.

    Cholesterol Orientation

    Cholesterol aligns with polar head groups of phospholipids, impacting membrane structure.

    Membrane Packing

    Cholesterol tightens packing of lipids, increasing membrane rigidity.

    Permeability Change

    Cholesterol decreases membrane permeability at high concentrations.

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    Phase Transitions

    Cholesterol inhibits phase transitions in lipid bilayers at high concentrations.

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    Membrane Fluidity Buffer

    Cholesterol acts as a buffer for membrane fluidity across temperature changes.

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    Lβ and Lα Phases

    Cholesterol influences Lβ (gel) and Lα (disordered) lipid phases to maintain function.

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    Crystalline State

    Cholesterol eliminates phase transition to crystalline state in membranes.

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    P/T Phase Diagram

    A diagram showing phase equilibrium at varying pressure and temperature.

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    Phase Equilibrium Curves

    Lines on a P/T diagram indicating coexistence of phases.

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    Critical Point

    The end point of a phase equilibrium curve, marking conditions of phase coexistence.

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    Supercritical Fluid

    A state above the critical point where distinct liquid and vapor phases do not exist.

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    Lamellar Phase

    A structure formed by polar molecules in sheets separated by liquid.

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    Fluid Lamellar Phase

    The liquid-crystalline state closest to a functional biomembrane.

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    Gel Phase

    A tightly packed state with reduced mobility and impermeability.

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    Gel-to-Fluid Phase Transition

    A temperature-controlled change from a gel to a fluid phase.

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    HI Phase

    Phase where hydrophobic tails are at the center of the cylinder.

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    HII Phase

    Phase where hydrophobic tails face outward, and hydrophilic heads comprise the core.

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    Cubic Phases

    Ordered structures resembling membrane environments, useful for films.

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    Non-lamellar Lipid Self-Assemblies

    Complex structures like hexagonal and cubic phases formed by lipids.

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    Energetic Favorability

    The concept of structural configurations that minimize energy states.

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    Surface to Volume Ratio

    A measure of efficiency in shapes for minimizing cell membrane area per volume.

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    Types of Detergents

    Categories include anionic, zwitterionic, and non-ionic based on charge.

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    Sodium Dodecyl Sulfate (SDS)

    An anionic detergent commonly used in biochemistry labs.

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    Role of Detergents

    Detergents are used to solubilize membrane proteins for purification in crystallography.

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    Solubilize Protein

    Detergents solubilize proteins at high concentrations, exceeding critical micelle concentration (CMC).

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    Critical Micelle Concentration (CMC)

    The CMC is the minimum detergent concentration needed to form micelles and solubilize proteins.

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    Micelle-Encapsulated Protein

    Proteins can be purified in a micelle by using detergents to encapsulate them for stability.

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    Detergent Exchange

    Detergent exchange involves replacing the initial detergent with a higher-CMC one during purification.

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    Triton X-100

    A detergent used to solubilize membrane proteins by forming micelles.

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    Micelles

    Structures formed by detergents in solution, encapsulating lipophilic substances.

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    Detergent Function

    Detergents solubilize proteins by enclosing them in micelles.

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    Solubilization Needs

    To extract membrane proteins, concentrations must exceed 2x CMC.

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    Effect of NaCl on CMC

    CMC can decrease significantly in the presence of NaCl, affecting detergent efficiency.

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    Low CMC Detergents

    Detergents that foam easily and are less effective for solubilization.

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    Cationic Detergents

    Detergents that can have low CMC and may not efficiently solubilize proteins.

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    Lipid Rafts

    Microdomains in membranes rich in cholesterol and sphingomyelin.

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    Role of Lipid Rafts

    Segregate proteins and attract signaling molecules for signal amplification.

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    Sphingomyelin

    A lipid that contributes to raft formation, characterized by long, saturated tails.

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    Cholesterol's Role

    Modifies membrane phase transitions and stabilizes bilayers.

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    Self-assembly of Phospholipids

    Phospholipids spontaneously organize into bilayers in polar solvents.

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    Phases of Lipid Bilayers

    Bilayers exist in various phases influenced by temperature and composition.

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    Asymmetric Distribution

    Different lipid distributions in the inner and outer leaflets of membranes.

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    Hydrophobic Mismatch

    Occurs when lipid tails and protein segments do not align, affecting function.

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    Study Notes

    Lecture 2: Membrane Fluidity, Detergents, and Lipid Rafts

    • Membrane Fluidity: Lipids and proteins diffuse laterally within the membrane bilayer via Brownian motion. Lateral diffusion can be measured by Fluorescence Recovery After Photobleaching (FRAP).
    • FRAP Technique: Lipids are fluorescently labeled and a small area bleached by a laser. The rate of fluorescence recovery in the bleached area tracks lipid diffusion.
    • Lipid Diffusion Speed: A molecule in an erythrocyte outer leaflet can traverse the entire cell in seconds. Typical phospholipids move at ~1µm/second.
    • Phases and Phase Diagrams: A phase is a region of space within a thermodynamic system, where physical properties are uniform. A thermodynamic system is described by parameters like temperature, volume, and pressure (though not always independent). The number of parameters defines the dimension of the state space of the system.
    • Intensive vs. Extensive Properties: Intensive properties are independent of the system's size (e.g., temperature, pressure, specific volume, chemical potential, density). Extensive properties depend on the system's size (e.g., mass, total volume, energy, Gibbs energy, enthalpy, entropy).
    • Phase Diagrams: Given a system's composition, only specific phases are possible at given temperature and pressure. Phase diagrams show these relationships, and phase equilibrium curves signify points where multiple phases coexist. A critical point defines the end of a phase equilibrium curve, notably the liquid-vapor critical point.
    • Lamellar Phases – Temperature Transition: The gel-to-fluid phase transition of lamellar phases is controlled by temperature. This is especially relevant to biological lipids where the transition is frequently observed in fats (e.g., butter).
    • Bilayer Composition and Phase Transition Temperature: Shorter hydrocarbon chains, or unsaturated hydrocarbon chains, lower the phase transition temperature. This is because shorter chains, or chains with a double bond, have reduced interaction, resulting in greater mobility and fluidity at lower temperatures.
    • Cholesterol in Membranes: Cholesterol modulates membrane fluidity. It tightens the packing of lipids, stiffens the membrane, decreases permeability, and inhibits phase transition into a crystalline state. It can be viewed as an "ordering agent" as it lowers the temperature for gel-to-fluid phase transition.
    • Detergents: These are surfactant molecules with both hydrophobic and hydrophilic components essential for membrane protein extraction.
    • Critical Micelle Concentration (CMC): The concentration defining the spontaneous formation of micelles (globular structures that sequester detergents) from individual detergent molecules.
    • Detergents and Membrane Proteins: Detergents solubilize membrane proteins at concentrations exceeding the CMC. The effective concentration is often higher than the CMC for proper solubilization, particularly with respect to extraction from the membrane.
    • Model Membranes and Techniques: These include supported lipid bilayers, black lipid membranes (BLMs), and liposomes, used for electrophysiological studies, including measurement of membrane permeability and channels’ properties and characterizing protein function (e.g. via patch-clamping).

    Lipid, Cubic, and Mesophases

    • Lipid Mesophases: These are phases in water-lipid mixtures that are neither a normal crystalline nor fluid state. Monoolein provides an example of how the composition and temperature result in a specific phase when in water.
    • Cubic Phases: Cubic phases present good ordered approximations of membrane environments. Lipid self-assembly often leads to these phases in biological contexts.
    • Other Lipid Phases: Hexagonal phases (H1, HII) are non-lamellar, tubular structures. Different orientations of the lipid tails with respect to each other lead to different structures.

    Membrane Protein Cryo-EM

    • Cryo-EM: This method utilizes cryo-electron microscopy to visualize biomolecules in solution, thereby allowing observation of macromolecular complexes and their dynamics. This method is crucial for obtaining and determining the structure of membrane proteins.
    • Cryo-Electron Microscopy (cryoEM) image classification: The method classifies and analyzes mixtures of macromolecular complexes which provides a way to characterize functional cycles of dynamic molecular machines.

    Model Membranes

    • Liposomes: Bilayer-encapsulated vesicles; important tools for studying membrane properties, vary in size (25 nm to 1 mm).
    • Model Membrane Types: Small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs), and giant unilamellar vesicles (GUVs). Bicelles are detergent-stabilized phospholipid bilayer discs.

    Black Lipid Membranes (BLMs)

    • BLMs: Lipid bilayers supported over teflon stretched until a single bilayer forms a separating membrane. This allows for electrophysiological measurement with electrodes.

    Patch Clamping

    • Patch Clamping: A technique used for studying the transmembrane currents of ion channels in cells and other processes in cells. Patch-clamping uses electrodes to measure the ion channels and other activity of the cell.

    Lipid Rafts

    • Lipid Rafts: Specialized regions of cell membranes enriched in sphingolipids and cholesterol; they may be important for regulating interactions between specific proteins and molecules. Can sequester proteins in a more specific way. Rafts appear as elevations, especially in high-resolution images captured with atomic force microscopy (AFM), due to the saturated and long sphingolipids.

    Membrane Interactions Summary

    • Interleaflet Coupling: Lipids' acyl chains interdigitate or interlock (especially the ethyl groups on each layer).
    • Asymmetric Distribution: Lipid arrangement (and ion distribution) varies between leaflets.
    • Negatively Charged Lipids: This feature of the membrane inner leaflets (e.g. phospholipids) influences protein-membrane interactions, particularly with proteins containing positively charged regions.
    • Lipid Self-Assembly: The self-assembly of lipids is crucial to membrane formation, including raft formation.
    • Hydrophobic Mismatch: Differences in lipid structure and packing lead to membrane deformation.
    • Specific Protein-Lipid Interactions: Proteins exhibit specific binding interactions with other membrane molecules like sphingolipids and cholesterol.

    Lecture Summary

    • Phospholipids form dynamic bilayers in polar solvents.
    • Temperature and composition of the membrane influences phases.
    • Cholesterol modulates fluidity and forms rafts.
    • Mesophases are another important structure for these systems (especially in lipid-water mixtures).
    • Detergents can be used to extract proteins.
    • Cryo-EM and X-ray crystallography are used for structural determination.

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    BS332 Lecture 2 2024 PDF

    Description

    Dive into the critical concepts of critical micelle concentration (CMC) and the role of detergents in protein crystallography. This quiz covers important aspects such as detergent functions, purification processes, and the significance of transferring to nanodiscs. Test your understanding of these essential biochemical techniques!

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