CH11Code-2.pdf Membrane Structure

CHAPTER 11 (Membrane Structure) Membrane Assembly Begins in the Endoplasmic Reticulum 1. Phospholipid Synthesis Location: ○ Occurs in eukaryotic cells on the cytosolic surface of the endoplasmic reticulum (ER). Enzymes: ○ Enzymes involved are bound to...

CHAPTER 11 (Membrane Structure) Membrane Assembly Begins in the Endoplasmic Reticulum 1. Phospholipid Synthesis Location: ○ Occurs in eukaryotic cells on the cytosolic surface of the endoplasmic reticulum (ER). Enzymes: ○ Enzymes involved are bound to the ER membrane. Substrates: ○ New phospholipids are synthesized using free fatty acids as substrates. 2. Insertion into the Bilayer Monolayer Addition: ○ Newly synthesized phospholipids are exclusively deposited in the cytosolic half of the lipid bilayer. 3. Even Growth of Membranes Challenge of Asymmetry: ○ Addition of phospholipids in an unbalanced manner could lead to uneven membrane growth. Mechanism to Achieve Balance: ○ Flip-Flops: Spontaneous movement of lipids from one monolayer to another is rare. ○ Role of Scramblase: Scramblase is a type of transporter protein. Function: Randomly removes selected phospholipids from one half of the bilayer and inserts them into the other. Result: Ensures equal redistribution of newly made phospholipids between both monolayers. 4. Membrane Dynamics Fate of Newly Assembled Membranes: ○ Some of the newly formed membrane remains within the ER. ○ Others are transported to various cellular compartments, including: Golgi Apparatus Plasma Membrane Process Overview:  CHAPTER 11 (Membrane Structure) ○ The dynamic process of membrane budding from one organelle and fusing with another will be detailed in Chapter 15. Summary The assembly of phospholipids in the ER is crucial for maintaining the structural integrity and functionality of cell membranes. The action of scramblases plays a key role in achieving even membrane growth by redistributing newly synthesized phospholipids between the two halves of the bilayer. Certain Phospholipids Are Confined to One Side of the Membrane 1. Membrane Asymmetry Definition: Most cell membranes exhibit asymmetry, with different sets of phospholipids on each half of the bilayer. Origin of Asymmetry: ○ Membranes emerge from the ER with an even distribution of phospholipids. ○ Asymmetry is established in the Golgi apparatus. 2. Transport Proteins in the Golgi Flippases: ○ A family of phospholipid-handling transporters found in the Golgi membrane. ○ Function: Use ATP hydrolysis energy to transfer specific phospholipids. Move selected lipids from the noncytosolic side (facing exterior) to the cytosolic side. ○ Key Lipids Moved: Phosphatidylserine and Phosphatidylethanolamine are flipped to the cytosolic side. Phosphatidylcholine and Sphingomyelin remain concentrated in the noncytosolic layer. 3. Impact of Asymmetry Membrane Functionality: ○ Asymmetric distribution helps in bending and curving the membrane, which is vital for processes like vesicle budding. Orientation Preservation: ○ Membranes retain their orientation as they bud from one organelle and fuse with another or the plasma membrane.  CHAPTER 11 (Membrane Structure) 4. Cell Membrane Faces Distinct Faces: ○ Cytosolic Monolayer: Always faces the cytosol. ○ Noncytosolic Monolayer: Exposed to the exterior of the cell or the lumen of an organelle. Protein Orientation: ○ Proteins embedded in the membrane maintain their orientation, crucial for their functionality. 5. Role of Glycolipids Distribution: ○ Glycolipids are primarily found in the noncytosolic half of the bilayer. ○ Sugar groups face the cell exterior, forming a carbohydrate coat that protects the cell. Glycolipid Synthesis: ○ Occurs in the Golgi apparatus. ○ Enzymes that add sugars are oriented to only modify lipids in the noncytosolic half, trapping glycolipids there. Delivery to Plasma Membrane: ○ Once formed, glycolipids are delivered to the plasma membrane, displaying their sugars outward. 6. Phospholipid Distribution Asymmetric Distribution: ○ Phosphatidylcholine (red) and Sphingomyelin (brown) are concentrated in the noncytosolic monolayer. ○ Phosphatidylserine (light green) and Phosphatidylethanolamine (yellow) are mainly on the cytosolic side. ○ Phosphatidylinositols (dark green) play a role in cell signaling and are concentrated in the cytosolic half. 7. Special Functions of Lipids Inositol Phospholipids: ○ Minor components involved in relaying signals from the cell surface to the interior. ○ Concentrated in the cytosolic monolayer due to their signaling role. Conclusion The establishment and maintenance of phospholipid asymmetry are crucial for cell membrane function, influencing processes such as vesicle formation and protein  CHAPTER 11 (Membrane Structure) orientation. The Golgi apparatus plays a key role in this process through the action of flippases. Membrane Proteins Overview Cell Membrane Structure ○ Comprised of a lipid bilayer, which acts as a permeability barrier. ○ Hydrophilic molecules cannot easily pass through this barrier. Composition Mass Distribution in Plasma Membranes ○ Proteins: Approximately 50% of the mass. ○ Lipid: Remaining mass includes lipids and carbohydrates. Carbohydrates: Found on glycolipids and glycoproteins, present in relatively small amounts. Quantitative Comparison ○ Cell membranes contain about 50 times more lipid molecules than protein molecules due to the smaller size of lipids. Functions of Membrane Proteins 1. Transport ○ Proteins facilitate the movement of: Nutrients Metabolites Ions across the lipid bilayer. 2. Anchoring ○ Proteins anchor the membrane to macromolecules on either side. 3. Receptors ○ Detect chemical signals in the environment. ○ Relay signals into the cell interior. 4. Enzymatic Activity ○ Act as enzymes to catalyze specific reactions at the membrane. Specialization Each cell membrane type contains a unique set of proteins.  CHAPTER 11 (Membrane Structure) This diversity reflects the specialized functions of different membranes. Conclusion Understanding the structure and functions of membrane proteins is crucial for comprehending their role in cellular processes and interactions with the environment. Membrane Proteins Associate with the Lipid Bilayer in Different Ways Overview Membrane proteins exhibit diverse interactions with the lipid bilayer, despite its uniform structure. Types of Membrane Protein Interactions 1. Transmembrane Proteins ○ Definition: Proteins that extend through the lipid bilayer. Have portions of their mass on both sides of the membrane. ○ Characteristics: Amphipathic Nature: Contain both hydrophobic and hydrophilic regions. Hydrophobic Regions: Located in the interior of the bilayer. Nestled against the hydrophobic tails of lipid molecules. Hydrophilic Regions: Exposed to the aqueous environment on either side of the membrane. 2. Cytosolic Proteins ○ Location: Primarily found in the cytosol. ○ Association with Lipid Bilayer: Interact with the cytosolic half of the bilayer. Utilize an amphipathic α helix that is exposed on the protein's surface for association. 3. Lipid-Linked Proteins ○ Location: Reside entirely outside the lipid bilayer, either on one side or the other.  CHAPTER 11 (Membrane Structure) ○ Attachment Mechanism: Tethered to the membrane by one or more covalently attached lipid groups. 4. Peripheral Membrane Proteins ○ Binding Mechanism: Bound indirectly to one face of the membrane. Held in place by interactions with other membrane proteins. Classification of Membrane Proteins Integral Membrane Proteins: ○ Definition: Proteins that are directly attached to the lipid bilayer. This category includes: Transmembrane proteins Proteins associated with a lipid monolayer Lipid-linked proteins ○ Removal Method: These proteins can only be removed by disrupting the lipid bilayer using detergents. Peripheral Membrane Proteins: ○ Definition: Proteins not directly attached to the lipid bilayer. ○ Removal Method: Can be released from the membrane using gentler extraction procedures that disrupt protein–protein interactions while leaving the lipid bilayer intact. Conclusion Understanding the various types and classifications of membrane proteins is essential for grasping their roles and interactions within the cellular membrane. A Polypeptide Chain Usually Crosses the Lipid Bilayer as an α Helix Unique Orientation of Membrane Proteins Importance of Orientation: ○ Essential for the function of membrane proteins, particularly transmembrane receptor proteins. ○ Example: The signal-receiving part must be extracellular, while the signaling part is located in the cytosol.  CHAPTER 11 (Membrane Structure) Structure of Transmembrane Proteins Membrane-Spanning Segments: ○ Portions on either side of the lipid bilayer connected by specialized segments of the polypeptide chain. ○ These segments traverse the hydrophobic environment of the lipid bilayer. ○ Composed largely of amino acids with hydrophobic side chains, which prefer interactions with the lipid tails instead of water. Polypeptide Backbone Characteristics Hydrophilic Nature: ○ Peptide bonds between amino acids are typically polar, making the polypeptide backbone hydrophilic. ○ In the absence of water in the bilayer, backbone atoms form hydrogen bonds with each other. Formation of α Helices: ○ Hydrogen bonding is maximized in a regular α helix structure. ○ Most membrane-spanning segments of polypeptides traverse as α helices: Hydrophobic side chains are exposed on the outside, interacting with lipid tails. Hydrophilic backbone forms internal hydrogen bonds. Types of Transmembrane Proteins 1. Single-Pass Transmembrane Proteins: ○ Cross the membrane only once. ○ Often function as receptors for extracellular signals. 2. Multipass Transmembrane Proteins: ○ Consist of multiple α helices crossing the bilayer. ○ Form aqueous channels for small, water-soluble molecules. ○ Some regions may be amphipathic, with both hydrophobic and hydrophilic side chains arranged accordingly: Hydrophobic side chains face outward to contact lipid tails. Hydrophilic side chains line the pore. Function of Channels Selective Transport: ○ Channels allow for the selective transport of small, water-soluble molecules, including inorganic ions. Alternative Structures: β Sheets and β Barrels  CHAPTER 11 (Membrane Structure) β Barrel Structure: ○ Some transmembrane proteins cross the lipid bilayer as β sheets rolled into a cylinder (β barrel). ○ Amino Acid Arrangement: Hydrophilic side chains face the interior, lining the aqueous channel. Hydrophobic side chains are on the exterior, interacting with the lipid bilayer. ○ Example: Porin Proteins Form large, water-filled pores in mitochondrial and bacterial outer membranes. Allow passage of small nutrients, metabolites, and inorganic ions while blocking larger molecules. Conclusion Understanding the various structures and functions of membrane proteins, particularly how polypeptide chains traverse the lipid bilayer, is crucial for comprehending their roles in cellular processes. Membrane Proteins Can Be Solubilized in Detergents Importance of Understanding Protein Structure Significance: ○ Detailed knowledge of a protein's structure is essential for understanding its function. ○ Membrane proteins pose unique challenges due to their specific operational environment. Challenges with Membrane Proteins Environment: ○ Membrane proteins function in a partly aqueous and partly fatty environment. ○ Isolating these proteins while preserving their essential structure is difficult. Purification Process of Membrane Proteins 1. Initial Separation: ○ Before detailed examination, membrane proteins must be separated from other cellular proteins.  CHAPTER 11 (Membrane Structure) 2. Use of Detergents: ○ Purpose: Disrupts the lipid bilayer by breaking hydrophobic associations. ○ Types of Detergents: Small, amphipathic, lipid-like molecules. Differ from membrane phospholipids, which have two hydrophobic tails. Detergents possess a single (1) hydrophobic tail, giving them a conical shape. In water, they aggregate into micelles (irregular clusters), rather than forming bilayers like phospholipids. Mechanism of Action Interaction with Membranes: ○ When mixed in excess with membranes: The hydrophobic ends of detergent molecules interact with: Membrane-spanning hydrophobic regions of transmembrane proteins. Hydrophobic tails of phospholipid molecules. ○ This interaction disrupts the lipid bilayer, effectively separating membrane proteins from phospholipids. Formation of Protein-Detergent Complexes: ○ The hydrophilic ends of detergent molecules help draw the membrane proteins into the aqueous solution. ○ Resulting in the formation of protein–detergent complexes. ○ Simultaneously, detergents also solubilize phospholipids. Separation for Further Analysis Post-Solubilization: ○ Protein–detergent complexes can be separated from each other and from lipid–detergent complexes. ○ This separation allows for further analysis and study of the membrane proteins. Conclusion Understanding how detergents solubilize membrane proteins is crucial for studying their structure and function in detail, highlighting the unique challenges presented by their environment in the cell membrane.  CHAPTER 11 (Membrane Structure) We Know the Complete Structure of Relatively Few Membrane Proteins Limited Knowledge of Membrane Protein Structures Historical Context: ○ Most knowledge about membrane protein structures has come from indirect methods. ○ X-ray crystallography is the standard technique for determining protein structures directly but requires ordered crystalline arrays. Challenges with Membrane Proteins: ○ Membrane proteins must be purified in detergent micelles, which are often heterogeneous in size, complicating crystallization. ○ Compared to soluble proteins in the cytosol or extracellular fluids, membrane proteins are harder to crystallize. Advances in Structural Determination Recent Developments: ○ Advances in x-ray crystallography and new techniques like cryo-electron microscopy have improved the ability to determine membrane protein structures. ○ A growing number of membrane protein structures are now available at high resolution. Case Study: Bacteriorhodopsin Overview: ○ Bacteriorhodopsin is a small membrane protein abundant in the plasma membrane of Halobacterium salinarum, an archaean found in salt marshes. Function: ○ Acts as a membrane transport protein that pumps protons (H+) out of the cell. Structure: ○ Contains a single (1) chromophore called retinal, a light-absorbing nonprotein molecule, contributing to the protein's deep purple color. ○ Retinal is covalently attached to one of the transmembrane α helices. Mechanism of Action: ○ Upon absorbing a photon of light, retinal changes shape. ○ This conformational change induces a series of small structural changes in the surrounding α helices.  CHAPTER 11 (Membrane Structure) ○ The process results in the pumping of one proton from the retinal to the exterior of the organism (Figure 11–28). Biological Role Energy Generation: ○ In sunlight, thousands of bacteriorhodopsin molecules actively pump H+ out of the cell. ○ This action generates a concentration gradient of H+ across the plasma membrane. ○ The cell utilizes this proton gradient to store energy and convert it into ATP, a process discussed further in Chapter 14. Classification: ○ Bacteriorhodopsin is classified as a pump, which is a type of transmembrane protein that actively transports small organic molecules and inorganic ions across cell membranes. Conclusion Understanding the structures and functions of membrane proteins, such as bacteriorhodopsin, highlights the challenges faced in structural biology and the advances made in elucidating these complex molecules. The Plasma Membrane Is Reinforced by the Underlying Cell Cortex Structure of the Plasma Membrane Thin and Fragile Nature: ○ The plasma membrane is extremely thin, requiring nearly 10,000 membranes stacked to match the thickness of standard paper. Reinforcement Mechanisms: ○ Membranes are typically strengthened by a protein framework. ○ Proteins attach to the membrane through transmembrane proteins. Cell Wall vs. Cell Cortex Cell Wall (in Plants, Yeasts, and Bacteria): ○ Provides shape and mechanical properties. ○ Composed of a fibrous layer made up of proteins, sugars, and other macromolecules encasing the plasma membrane. Cell Cortex (in Animal Cells): ○ Stabilizes the plasma membrane through a meshwork of filamentous proteins.  CHAPTER 11 (Membrane Structure) ○ This network is attached to the underside of the membrane, providing structural support. Red Blood Cell Cortex Structure: ○ The cortex of human red blood cells has a simple, well-studied structure. ○ Red blood cells are small and possess a distinctive flattened shape (Figure 11–29A). Key Component: Spectrin ○ Spectrin is a dimeric protein, long and thin (about 100 nm in length). ○ Forms a lattice structure that supports the plasma membrane and maintains the biconcave shape of red blood cells. Connection to Membrane: ○ The spectrin network connects to the membrane via intracellular attachment proteins. ○ These proteins link spectrin to specific transmembrane proteins (Figure 11–29B, Movie 11.7). Impact of Genetic Alterations: ○ Genetic mutations leading to abnormal spectrin structure result in anemia. ○ Affected individuals have fewer red blood cells, which become spherical and fragile instead of flattened. Cortex in Other Animal Cells Composition: ○ Other animal cells contain proteins similar to spectrin and its associated attachment proteins. ○ These cortices are rich in actin and the motor protein myosin, making them more complex than that of red blood cells. Functions Beyond Mechanical Strength: ○ In addition to providing mechanical strength for blood vessel passage, other cell types utilize their cortex for: Selective uptake of materials from the environment. Changing shape and movement (discussed in Chapter 17). Restraining Protein Diffusion: ○ The cortex also plays a role in restraining the diffusion of proteins within the plasma membrane. Conclusion  CHAPTER 11 (Membrane Structure) The cell cortex is crucial for the structural integrity and functionality of animal cells, with specialized roles in various cell types beyond just mechanical support, highlighting its importance in cellular activities. A Cell Can Restrict the Movement of Its Membrane Proteins Lateral Diffusion of Membrane Proteins Fluid Nature of Membrane: ○ Membranes are described as two-dimensional fluids, allowing proteins and lipids to move freely within the plane of the bilayer. Experimental Evidence: ○ The movement was demonstrated by fusing a mouse cell with a human cell to form a hybrid cell. ○ Initially, mouse and human proteins remained confined to their respective halves of the hybrid cell. ○ Within approximately 30 minutes, the proteins became evenly mixed across the entire cell surface (Figure 11–30). Limitations of Free Movement Simplistic View of Membrane: ○ The idea of a membrane as a fluid sea where all proteins float freely is overly simplistic. Mechanisms for Protein Confinement: ○ Cells have developed methods to restrict the movement of specific proteins, creating functionally specialized regions known as membrane domains. Tethering Mechanisms External Tethering: ○ Plasma membrane proteins can be tethered to external structures, such as: Molecules in the extracellular matrix. Adjacent cells (discussed in Chapter 20). Internal Tethering: ○ Proteins may also be anchored to relatively immobile structures within the cell, notably the cell cortex (see Figure 11–29B). Barriers to Protein Movement Creation of Membrane Domains:  CHAPTER 11 (Membrane Structure) ○ Cells can create barriers to confine specific membrane components to distinct domains. Epithelial Cell Example: ○ In gut epithelial cells, it is crucial for transport proteins to be localized: Apical Surface: Transport proteins for nutrient uptake are confined here (facing the gut contents). Basal and Lateral Surfaces: Other transport proteins for solute export to tissues and bloodstream are restricted to these areas (see Figure 12–17). Role of Tight Junctions: ○ The asymmetric distribution of membrane proteins is maintained by tight junctions between adjacent epithelial cells (Figure 11–32). ○ Tight junctions are formed by specialized junctional proteins that create a continuous belt around the cell at contact points with neighbors. ○ This belt creates a seal between adjacent plasma membranes, preventing the diffusion of membrane proteins past the junction. Conclusion Cells utilize various mechanisms, including tethering and barrier formation, to restrict the movement of membrane proteins. This organization is essential for creating specialized functional domains on the cell surface, particularly in epithelial cells, where tight junctions play a crucial role in maintaining protein asymmetry. The Cell Surface Is Coated with Carbohydrate Overview of Cell Surface Carbohydrates Location of Carbohydrates: ○ Carbohydrates are covalently attached to some lipids and most proteins in the outer layer of the plasma membrane. Types of Carbohydrate-Modified Molecules: ○ Glycoproteins: Most membrane proteins have short sugar chains (oligosaccharides) linked to them. ○ Proteoglycans: Some membrane proteins contain one or more long (polysaccharide) chains. ○ Glycolipids: Lipids with carbohydrate components. Carbohydrate Layer (Glycocalyx): ○ All carbohydrate components (from glycoproteins, proteoglycans, and glycolipids) are located on the outside of the plasma membrane.  CHAPTER 11 (Membrane Structure) ○ This forms a protective sugar coating called the carbohydrate layer or glycocalyx (Figure 11–33). Functions of the Carbohydrate Layer Protection: ○ The carbohydrate layer helps protect the cell surface from mechanical damage. Hydration and Lubrication: ○ Oligosaccharides and polysaccharides attract water molecules, contributing to a slimy surface. ○ This property aids motile cells (like white blood cells) in squeezing through narrow spaces and prevents blood cells from sticking to each other or to blood vessel walls. Role in Cell–Cell Recognition and Adhesion Recognition and Binding: ○ Cell-surface carbohydrates are crucial for cell–cell recognition and adhesion. ○ Lectins: Specialized transmembrane proteins that bind to specific oligosaccharide side chains. Diversity of Oligosaccharides: ○ Oligosaccharide side chains, although typically fewer than 15 sugar units, are highly diverse. ○ Unlike proteins, sugars can be arranged in numerous configurations, often forming branched structures. ○ Even three different sugars can create hundreds of different trisaccharides through various covalent linkages. Specific Examples of Carbohydrate Functions Cell Type Recognition: ○ The carbohydrate layer acts like a distinctive uniform, unique to each cell type, facilitating recognition by other cells. Sperm-Egg Recognition: ○ Specific oligosaccharides play a role in the recognition of an egg by sperm (discussed in Chapter 19). Neutrophil Migration During Infection: ○ During bacterial infections, carbohydrates on neutrophils (white blood cells) are recognized by lectins on blood vessel lining cells. ○ This recognition prompts neutrophils to adhere to the blood vessel wall and migrate into infected tissue to combat invading bacteria (Figure 11–37).  CHAPTER 11 (Membrane Structure) Conclusion The carbohydrate layer on the cell surface is essential not only for protection and lubrication but also for mediating important interactions such as cell recognition and adhesion, playing a vital role in various physiological processes. –How We Know– MEASURING MEMBRANE FLOW Importance of Membrane Fluidity Definition and Role: ○ Fluidity is an essential feature of the lipid bilayer, crucial for maintaining cell membrane integrity and function. ○ Allows lateral movement of membrane-embedded proteins, facilitating various protein–protein interactions critical for cellular functions. Historical Context: ○ The significance of membrane fluidity was not recognized until the early 1970s. Methods for Measuring Membrane Fluidity Visual Measurement Techniques: ○ The most common approach involves labeling molecules that are native to the membrane. ○ Researchers then observe the movement and speed of these labeled molecules. Initial Findings: ○ Early experiments used labeled antibodies to tag membrane proteins, demonstrating their lateral movement within the membrane (referenced in Figure 11–30). ○ Observations suggested that membrane proteins diffuse freely in an "open sea" of lipids. Limitations of Initial Understanding Reevaluation of Diffusion: ○ While early findings indicated free diffusion, this perspective has since been recognized as overly simplistic.  CHAPTER 11 (Membrane Structure) ○ Further investigation revealed that protein movement is not entirely unrestricted, indicating the need for more sophisticated methods to study membrane fluidity. Advanced Measurement Techniques Need for Precision: ○ To obtain a more accurate understanding of membrane fluidity, researchers developed precise techniques for tracking protein movement. ○ These methods allow for detailed analysis of protein dynamics within the plasma membrane of living cells. Conclusion Understanding membrane fluidity is vital for comprehending cell membrane structure and function. The initial visual methods provided valuable insights, but further advancements are necessary to accurately depict the complexities of protein movement and interactions in the membrane. The FRAP attack Overview of FRAP Technique Definition: ○ Fluorescence Recovery After Photobleaching (FRAP) is a method used to measure the mobility and diffusion of membrane components (lipids or proteins). Steps in the FRAP Technique 1. Labeling Membrane Components: ○ Membrane proteins are labeled using: Fluorescent Antibodies: Cells are incubated with antibodies that have been tagged with a fluorescent marker. Covalent Attachment of Fluorescent Proteins: Techniques like those involving green fluorescent protein (GFP) can be used to attach a fluorescent marker to a membrane protein, utilizing DNA techniques (discussed in Chapter 10). 2. Photobleaching Process: ○ A small patch of the labeled membrane is irradiated with an intense pulse of light from a focused laser beam. ○ This irradiation irreversibly “bleaches” the fluorescent marker in the targeted area, typically about 1 μm². 3. Monitoring Recovery:  CHAPTER 11 (Membrane Structure) ○ After bleaching, the fluorescence in the area is monitored using a fluorescence microscope. ○ The recovery of fluorescence is tracked by measuring the time it takes for neighboring, unbleached fluorescent proteins to diffuse into the bleached region. Analysis of Results Fluorescence Recovery Measurement: ○ The rate of fluorescence recovery serves as a direct measure of the diffusion rate of protein molecules within the membrane. ○ These measurements provide insights into the fluidity and viscosity of the membrane. Findings: ○ Experiments indicate that cell membranes exhibit a viscosity comparable to that of olive oil, suggesting a significant degree of fluidity. Conclusion FRAP is a powerful technique that enables researchers to quantitatively assess the dynamics of membrane proteins, enhancing our understanding of cell membrane properties and behaviors. Through precise labeling and monitoring of diffusion rates, FRAP provides valuable insights into the fluidity of cellular membranes. One by one Limitations of FRAP Technique Population Measurement: ○ FRAP monitors the movement of large populations of proteins (hundreds or thousands) across a relatively large area of the membrane. ○ This approach does not allow for tracking the motion of individual molecules, complicating result analysis. Interpretation Challenges: ○ If labeled proteins do not migrate into the bleached zone during FRAP, it is unclear whether they are: Immobilized: Anchored in one position within the membrane. Restricted Movement: Limited to a small area due to barriers like cytoskeletal proteins, giving the appearance of being motionless. Development of Single-Particle Tracking (SPT) Microscopy  CHAPTER 11 (Membrane Structure) Objective: ○ To overcome the limitations of FRAP by enabling the observation of individual molecules or small clusters of molecules. Technique Description: ○ Labeling Method: Proteins are tagged with antibody-coated gold nanoparticles. ○ Appearance: The gold particles appear as tiny black dots under a light microscope. ○ Movement Tracking: The movement of these tagged protein molecules is monitored using video microscopy. Findings from SPT Studies Variety of Movement Patterns: ○ Membrane proteins exhibit diverse patterns of movement, which can include: Random Diffusion: Proteins moving freely in the membrane. Complete Immobility: Proteins remaining stationary. Dynamic Behavior: ○ Some proteins are capable of rapidly switching between different types of motion, highlighting the complexity of their movement within the membrane. Conclusion Single-particle tracking microscopy (SPT) provides a valuable method for studying the movement of individual membrane proteins, revealing intricate behaviors and dynamics that the FRAP technique cannot capture. By utilizing antibody-coated gold nanoparticles, researchers can gain insights into how membrane proteins interact with their environment, leading to a better understanding of cellular processes. Freed from cells Purpose of Isolation Researchers often aim to study specific membrane proteins in synthetic environments to: ○ Eliminate interference from other proteins. ○ Analyze the behavior and activity of the protein of interest without external influences. Isolation and Reconstitution Process  CHAPTER 11 (Membrane Structure) Isolation: Membrane proteins are extracted from cells. Purification: The target protein is purified from the extracted mixture. Reconstitution: The purified protein is incorporated into artificial phospholipid vesicles. ○ This setup allows the protein to maintain its proper structure and functionality. Advantages of Artificial Lipid Bilayers Enhanced Mobility: ○ Membrane proteins generally diffuse more freely and rapidly in artificial lipid bilayers compared to natural cell membranes. Crowding Effect: ○ Cell membranes are densely packed with various proteins and lipids, which can hinder protein movement. ○ The complexity and variety of lipids in cell membranes contribute to reduced mobility for most proteins. Tethering Influence: ○ Many membrane proteins in cells are anchored or tethered to: Extracellular Matrix: Interactions with the external environment. Cell Cortex: Attachment to the cytoskeletal structures just beneath the membrane. Impact on Research Studies using artificial lipid bilayers have significantly enhanced understanding of: ○ Membrane Protein Behavior: Insights into how proteins interact with lipids and their dynamics in a less complex environment. ○ Cell Membrane Architecture: Improved knowledge of how the structure and organization of cell membranes affect protein function. Conclusion Isolating and studying membrane proteins in synthetic lipid bilayers has revolutionized the field, providing clearer insights into protein mobility, functionality, and the overall architecture of cell membranes. This approach allows for more controlled experiments and a better understanding of the intrinsic properties of membrane proteins.  CHAPTER 11 (Membrane Structure) Essential Concepts Basic Structure of Cell Membranes Membrane Function: ○ Membranes create barriers that confine specific molecules to distinct compartments within cells. Lipid Bilayer: ○ Composed of a continuous double layer of lipid molecules. ○ Provides the fundamental structure and barrier function of all cell membranes. Properties of Membrane Lipids Amphipathic Nature: ○ Lipid molecules have both hydrophobic (water-repelling) and hydrophilic (water-attracting) regions. ○ This property leads to spontaneous assembly into bilayers when placed in water. ○ Bilayers form closed compartments that can reseal if torn. Major Classes of Membrane Lipids 1. Phospholipids 2. Sterols 3. Glycolipids Fluidity of the Lipid Bilayer Diffusion: ○ Individual lipid molecules can diffuse laterally within their own monolayer. ○ Lipid molecules do not spontaneously flip from one monolayer to the other. Composition Differences: ○ The two monolayers of a cell membrane exhibit different lipid compositions, reflecting their distinct functional roles. Temperature Adaptations  CHAPTER 11 (Membrane Structure) Cells adjust their lipid composition to maintain membrane fluidity at varying temperatures. Membrane Proteins Functionality: ○ Membrane proteins perform specialized functions, such as transporting small, water-soluble molecules across the lipid bilayer. Types of Membrane Proteins: ○ Transmembrane Proteins: Extend across the lipid bilayer. Typically structured as one or more α helices, or as a β sheet rolled into a barrel form. ○ Peripheral Membrane Proteins: Do not extend across the bilayer. Can be attached via: Noncovalent associations with other membrane proteins. Covalent attachment to lipids. Association of exposed amphipathic α helices with a single lipid monolayer. Support and Structure Cell Cortex: ○ Many cell membranes are reinforced by a framework of proteins, notably a meshwork of fibrous proteins forming the cell cortex underneath the plasma membrane. Confined Protein Movement Cells have mechanisms to restrict membrane proteins to specific domains. Proteins can be immobilized by attaching them to: ○ Intracellular macromolecules. ○ Extracellular structures. Carbohydrate Layer Surface Modifications:  CHAPTER 11 (Membrane Structure) ○ Many membrane proteins and some lipids have sugar chains attached, forming a carbohydrate layer. Functions of the Carbohydrate Layer: ○ Protects and lubricates the cell surface. ○ Plays a critical role in specific cell–cell recognition processes.