Biochemistry 162 Exam 2 Notes PDF

***[Biochemistry 162: Exam 2 -- Notes]*** ***[5.2 -- Myoglobin ]*** **[Globins are oxygen-binding proteins]** - Protein side chains lack affinity for O~2~ - Some transition metals bind O~2~ well but would generate **free radicals** if free in solution. - Organometallic compounds such...

***[Biochemistry 162: Exam 2 -- Notes]*** ***[5.2 -- Myoglobin ]*** **[Globins are oxygen-binding proteins]** - Protein side chains lack affinity for O~2~ - Some transition metals bind O~2~ well but would generate **free radicals** if free in solution. - Organometallic compounds such as heme are more suitable, but Fe^2+^ in free heme could be oxidized to Fe^3+^. - Solution - Capture the oxygen molecule with heme that is protein bound. - Myoglobin is the main oxygen storage protein. - Hemoglobin is a circulating oxygen-binding protein. **[Could myoglobin transport O~2~?]** - pO~2~ in lungs is about 13 kPa: It sure binds oxygen well - pO~2~ in tissues is about 4 kPa: It will **not** release it. - Would lowering the affinity (P~50~) of myoglobin to oxygen help? **[For effective transport affinity must vary with pO~2~]** - Transport protein must: - Bind oxygen tightly at high concentration (lungs) - Bind oxygen weakly at low concentration (tissues) - Can only be achieved if the affinity changes with oxygen concentration. - Enter: **Hemoglobin.** - A diagram of a state Description automatically generated ***[5.3 -- Cooperativity]*** **[How can affinity to oxygen change? ]** - Must be a protein with multiple **binding sites.** - Binding sites must be able to **interact with each other.** - This phenomenon is called **cooperativity** - Positive cooperativity - First binding event increases affinity at remaining sites - **Recognized by sigmoidal binding curves** - Negative cooperativity - First binding event reduces affinity at remaining sites. - Alcohol dehydrogenase, GAP dehydrogenase - Sites "fire" alternately; unclear what the advantage is - **Binding proteins** and **enzymes** can be cooperative **[Model of Positive Cooperativity]** - Empty protein has 2 binding sites - Binding of ligand at one site causes other site to become less wiggly - Second site is better able to bind the second ligand.  **[Cooperativity: Quantitative Description]** - Cooperative proteins have multiple ligand-binding sites - [*P* + *nL*  ↔ *PL*~*n*~]{.math.inline} - So K~a~ becomes: - K~a~ = [\$\\frac{\\lbrack PL\_{n}\\rbrack}{\\left\\lbrack P \\right\\rbrack\\left\\lbrack L \\right\\rbrack\^{n}}\$]{.math.inline} - And θ becomes: - θ = = [\$\\frac{\\left\\lbrack L \\right\\rbrack\^{n}}{\\left\\lbrack L \\right\\rbrack\^{n} + K\_{d}}\$]{.math.inline} - Taking the log of both sides gives the **Hill Equation:** - Log[\$(\\frac{\\theta}{1\\ --\\ \\theta\\ })\$]{.math.inline} = nlog\[L\] -- logK~d~ - n = the Hill Coefficient (the degree of cooperativity) - n = 1 No cooperativity - n \> 1 Positive cooperativity - n \< 1 Negative cooperativity **[The Hill Plot of Cooperativity: ]** - At low oxygen, Hemoglobin behaves as a monomer that binds weakly. - At medium oxygen, Hemoglobin transitions from low to high affinity. - At high oxygen, Hemoglobin behaves as a monomer that binds tightly. - n ≤ the number of binding sites A diagram of a graph Description automatically generated **[Two Model of Cooperativity: Concerted vs. Sequential:]**  **[Cooperativity is a special case of allosteric regulation: ]** - Allosteric protein - Binding of a ligand to one site affects the binding properties of a different site, on the same protein. - Can be positive or negative - Homotropic - Normal ligand of the protein is the allosteric regulator - Heterotropic - Different ligand affects binding of the normal ligand. - **Cooperativity = positive homotropic regulation.** ***[5.4 -- Hemoglobin ]*** **[Hemoglobin binds oxygen cooperatively]** - Hemoglobin (Hb) is a tetramer of 2 subunits (α2β2) - Each subunit is similar to myoglobin **[Sequence Similarity between Hemoglobin and Myoglobin]** A diagram of a dna structure Description automatically generated with medium confidence **[R and T States of Hemoglobin]** - T = **Tense** state - More interactions, more rigid - **Lower affinity** for O~2~ - R = **Relaxed** state - Fewer Interactions, more flexible - **Higher affinity** for O~2~ - O~2~ binding triggers a T R conformational change - Conformational change from the T state to the R state involves **breaking ion pairs** between the α1--β2 interface. - This constitutes the "communication" between subunits.  **[Subunit Interactions in Hemoglobin]** A diagram of a structure Description automatically generated **[T -- State Interactions]**  **[T -- State is stabilized by salt-bridges]** A diagram of a line with numbers and letters Description automatically generated with medium confidence **[Conformational change is triggered by oxygen binding ]**  **[Spectroscopic Detection of Oxygen Binding to Myoglobin ]** - The heme group is a strong **chromophore** that absorbs both in ultraviolet and visible range. - Ferrous form **(Fe^2+^)** without oxygen has an intense Soret band at 429 nm. - Oxygen binding alters the electronic properties of the heme, and shifts the position of the Soret band to 414 nm. - **Binding of oxygen can be monitored by UV-Vis spectrophotometry.** - **Deoxyhemoglobin (in venous blood) appears purplish in color and oxyhemoglobin (in arterial blood) is red.** ***[5.5 -- Effect of pH and CO~2~]*** **[pH and CO~2~ affect O~2~ binding to Hb ]** A diagram of an anhydrate cell Description automatically generated **[Acid promotes oxygen release: Bohr Effect]** - Actively metabolizing tissues generate H^+^, lowering the pH of the blood near the tissues relative to the lungs. - Hemoglobin Affinity for oxygen depends on the pH - H^+^ binds to hemoglobin and stabilizes the T state - Protonates His146 which then forms a salt bridge with Asp94 - Leads to the release of O~2~ (in the tissues) - Each subunit does this -- so there are four "Bohr protons" - T**he pH difference between lungs and metabolic tissues increases efficiency of the O~2~ transport** - This is known as the **Bohr effect.** **[pH Effect on O~2~ Binding to Hemoglobin ]** - Curve shifts to the RIGHT when binding becomes weaker (more T-state) - Curve shifts to the LEFT when binding becomes tighter (more R-state)  **[Lower pH increases O~2~ release ]** A diagram of a function Description automatically generated **[Hemoglobin and CO~2~ Export]** - CO~2~ is produced by metabolism in tissues and must be exported. - 15--20% of CO~2~ is exported in the form of a carbamate on the amino terminal residues of each of the polypeptide subunits.  - **Notice:** - **The formation of a carbamate yields a proton which can contribute to the Bohr Effect.** - **The carbamate forms additional salt bridges stabilizing the T-state** - The rest of the CO~2~ is exported as dissolved bicarbonate. - Formed by carbonic anhydrase, and also producing a proton **[CO~2~ drives O~2~ release even further]** A diagram of a diagram of different types of saturation Description automatically generated with medium confidence ***[5.6 -- 2,3,-BPG and Hemoglobin ]*** - 2,3-Bisphosphoglycerate Regulates O~2~ Binding - Negative heterotropic regulator of Hemoglobin function. - Present at mM concentrations in erythrocytes - Produced from an intermediate in glycolysis - Small negatively charged molecule, binds to the positively charged central cavity of Hemoglobin. - Stabilizes the T-state -  **[2,3-BPG Binds to the Central Cavity of Hemoglobin]** A diagram of a b-state Description automatically generated  The positively charged molecules are lining the binding pocket because BPG is a high negatively charged molecule **[What is the purpose of BPG and having a binding site? ]** - 2,3-BPG allows for O~2~ release in the tissues and adaptation to changes in altitude. - At sea level, pO~2~ is higher so Hemoglobin carries more O~2~ - At high altitude, pO~2~ is lower, so Hemoglobin carries less O~2~ - To compensate, cells produce more BPG at high altitudes to drive more Hemoglobin into the T-state - Although T-state binds O~2~ poorly, it releases O~2~ very well. - This adaptation allows time for more red blood cells to be made. A diagram of a sea level Description automatically generated ***[5.7 -- CO Poisoning and Sickle-Cell Anemia]*** **[Binding of Carbon Monoxide]** - CO has similar size and shape of O~2~; fits in same binding site - **CO binds** to heme over 20,000 times **better** than O~2~ because the carbon in CO has a filled lone electron paiur that can be donated to vacant d-orbitals on the Fe^2+^. - Protein pocket decreases affinity for CO, but it still binds about 250 times better to hemoglobin than oxygen. - CO is highly toxic as it competes with oxygen. It blocks the function of **myoglobin, hemoglobin, and mitochondrial cytochromes** that are involved in oxidative phosphorylation.  **[Heme binding to protein affects CO vs. O~2~ binding ]** - CO binding to Hemoglobin has 2 effects: - Competes with O~2~ for Fe^2+^, so fewer binding sites available for O~2­~ - This is not a problem if you have enough Hemoglobin or red blood cells - CO and O~2~ lock Hemoglobin into the R-state (high affinity for O~2~) - CO binds Hemoglobin tightly, other subunits remain in R-state despite low pH, CO~2~, and BPG: O~2~ not released. - Tissues starve while Hemoglobin holds O~2~ A diagram of a molecule Description automatically generated **[Sickle-cell anemia is due to a mutation in hemoglobin]** - Glu6 Val in the β chain of Hemoglobin - The new Valine side chain can bind to a different hemoglobin molecule to form a fiber - Fibers cause red blood cells to sickle; can't flow - Pain, anemia, infections. Untreated homozygous patients usually die young. Bone marrow transplant cures. - Heterozygous individuals exhibit a resistance to malaria. - Made worse by exertion (low O~2~ drives Hemoglobin into T-state, worsens fiber formation, leads to athlete sudden deaths when undiagnosed. **[Formation of Hemoglobin Strands in Sickle-Cell Anemia and a Cure]** - Possible cure for Sickle-cell in clinical trials - 2019 clinical trial of CRISPR gene therapy - Victoria Gray's bone marrow was removed, gene-edited in the lab to cause stem cells to produce fetal Hemoglobin, which binds O~2~ tightly and overcomes symptoms of Sickle Cell disease. - It works -- and it is a once-for-life treatment - Also works for other inherited Hemoglobin diseases, like β-thalassemia.  8. ***[-- Antibodies]*** **[Two Types of Immune Systems]** - Cellular immune system - Targets **own cells** that have been infected - Also clears up virus particles and infecting bacteria - **Key players: Macrophages, Killer T cells (T~c~), and inflammatory T cells (TH~1~).** - Humoral "fluid" immune system - Targets **extracellular** pathogens - Can also recognize foreign proteins - Make soluble **antibodies** - Keeps "memory" of past infections - **Key players: B-lymphocytes and Helper T-cells (TH~2~)** **[Cellular Immune System ]** - Antibodies bind to fragments displayed on the surface of invading cells. - Phagocytes: specialized cells that eat invaders - Macrophages: Large phagocytes that ingest bacteria that are tagged by antibodies **[Humoral Immune System]** - Vertebrates also fight infections with soluble antibodies that specifically bind **antigens**. - **Antigens** are substances that stimulate production of **antibodies** - Typically, macromolecular in nature - Recognized as foreign by the immune system - Coat proteins of bacteria and viruses - Surface carbohydrates of cells or viruses - **Antibodies** are proteins that are produced by B cells and specifically bind to antigens - Binding will mark the antigen for destruction or interfere with its function - A given antibody will bind to a small region (epitope) of the antigen - One antigen can have several epitopes **[Antibodies: Immunoglobulin G]** - Composed of 2 heavy chains and 2 light chains - Composed of constant domains and variable domains - Light chains: one constant and one variable domain - Heavy chains: three constant and one variable domain - Variable domains of each chain make up antigen-binding site (2 per antibody) - Variable domains contain regions that are hypervariable (specifically the antigen-binding site) - Confers high antigen specificity A diagram of a structure Description automatically generated **[Antigens bind via Induced fit]** - Antigen binding causes significant structural changes to the antibody A close-up of several molecules Description automatically generated **[Antibody specificity is an important analytical reagent]**  **[Antibody detection can be colormetric or luminescent]** ***[Question Time: ]*** ***[Chapter 5 -- ]*** - Binding Proteins - Saturation Curve - Cooperativity - Hill Plot - Hb + Mb: Regulation - SCD/Malaria ***[Chapter 6 -- ]*** ***[6.1 -- Enzyme Fundamentals]*** **[Chapter 6 Enzymes]** - Key topics about enzyme function: - Physiological significance of enzymes - Origin of catalytic power of enzymes - Chemical mechanisms of catalysis - Mechanisms of chymotrypsin and lysozyme - Description of enzyme kinetics and inhibition **[What are enzymes?]** - **Enzymes are catalysts** - **Increase reaction rates without being used up** - Most enzymes are globular portiens - However, some RNA (ribozymes; e.g., ribosomal RNA) alos catalyze reactions - Study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s. - Study of enzymes has dominated biochemistry in the past and contributes to do so - Eduard Buchner: Zymase = yeast juice that ferments sugar to make ethanol (1900) **[Why biocatalysis over inorganic catalysts? ]** - Greater reaction specificity: avoids side products - Milder reaction conditions: conducive to conditions in cells - Higher reaction rates: in a biologically useful timeframe - Capacity for regulation: control of biological pathways - Metabolites have many potential pathways of decomposition - Enzymes make the desired one most favorable  **[Enzymatic Substrate Selectivity ]** A diagram of chemical formulas Description automatically generated with medium confidence **[Reaction Conditions Compatible with Life]** - 37ºC - pH = 7 - Mild oxidizing/reducing conditions - Currently limited to reactions required by natural selection; can be engineered. **[Seven Classes of Enzymes: Defined by the Reactions Catalyzed]**  ***[6.2 -- Enzyme Catalysis ]*** **[Enzyme-Substrate Complex]** - Enzymes act by binding substrates - The noncovalent enzyme substrate complex is known as the **Michaelis complex** - Description of chemical interactions - Development of kinetic equations E + S ES EP E + P A black text on a white background Description automatically generated  **[Enzymatic Catalysis]** - Enzymes do not affect equilibrium (∆G) - Reactions that face significant activation barriers (∆G^+-^) that must be surmounted during the reaction are SLOW. - Enzymes increase reaction rates (k) by decreasing ∆G^+-^ A diagram of a function Description automatically generated  **[Enzymes Decrease G^+-^ ]** A diagram of a function Description automatically generated **[Rate Enhancement by Enzymes]**  **[How to Lower ∆G^≠^]** - Enzymes use binding energy to pay entropy cost - Uncatalzyed biomolecular reactions - **Two free** reactants **single** restricted transition state conversion is **entropically unfavorable** - Uncatalyzed unimolecular reactions - Flexible reactant rigid transition state conversion is **entropically unfavorable** for flexible reactants - Catalyzed reactions - Enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complex - **Entropy cost is paid during binding** - **Rigid reactant complex transition state conversion is entropically okay.** **[Support for the Proximity Model]** - Propinquity: Things that are in close proximity tend to react more with each other - A model reaction of anhydride formation shows how rate increases - D orientation - Requires random collision - Put reactive groups nearby effective concentration increases - Orients reactive groups effective concentration very high A diagram of chemical formulas Description automatically generated with medium confidence **[How to Lower ∆G^≠^]** - Enzymes bind transition states best - The idea was proposed by Linus Pauling in 1946 - Enzyme active sites are complimentary to the transition state of the reaction - Enzymes bind transition states better than substrates - Stronger/additional interactions with the transition state as compared to the ground state lower the activation barrier - Largely ∆H^+-^ effect ***[6.3 -- Stickase Example ]*** **[Illustration of TS Stabilization Idea: Imaginary Stickase]**  **[Stick Binding Protein (SBP)]** - Binding proteins are good transporters but poor enzymes A white background with black text Description automatically generated  **[Stickase ]** - Magnets (M) help stick to adopt the bended shape (transition state) A diagram of a transition process Description automatically generated  ***[6.4 -- Types of Catalysis]*** **[Catalytic Mechanisms ]** - **Acid -- base catalysis**: give and take protons - **Covalent catalysis**: change reaction paths - **Metal ion catalysis**: use redox cofactors, pK~a~ shifters - **Electrostatic catalysis**: preferential interactions with TS **[General Acid--Base Catalysis]** A diagram of a reaction Description automatically generated **[Amino Acids in General Acid--Base Catalysis]**  **[Covalent Catalysis ]** - A transient covalent bond between the enzyme and the substrate - Changes the reaction Pathway - Uncatalyzed: A black text with letters and arrows Description automatically generated with medium confidence - Catalyzed  - Requires a nucleophile on the enzyme (X) - Can be a reactive **serine, thiolate, amine, or carboxylate** A table with text and images Description automatically generated with medium confidence **[Metal Ion Catalysis ]** - Involves a metal ion bound to the enzyme - Interacts with substrate to facilitate binding - Stabilizes negative charges - Participates in oxidation reactions - We will largely ignore these mechanisms. ***[6.5 -- Chymotrypsin ]*** **[Chymotrypsin is a zymogen]**  **[Active Site of Chymotrypsin with Substrate ]** ***[6.6 Chymotrypsin has Burst Kinetics ]***  A diagram of a ph and a cat Description automatically generated ***[6.7 -- Chymotrypsin Catalytic Cycle ]*** **[Serine Proteases: Chymotrypsin]**  **[Chymotrypsin Mechanism -- Step 1: Substrate Binding ]** A diagram of a molecule Description automatically generated **[Chymotrypsin Mechanism -- Step 2: Nucleophilic Attack ]**  **[Chymotrypsin Mechanism -- Step 3: Substrate Cleavage ]** A diagram of a molecule Description automatically generated **[Chymotrypsin Mechanism -- Step 4: Water Comes In ]**  **[Chymotrypsin Mechanism -- Step 5: Water Attacks]** A diagram of a molecule Description automatically generated **[Chymotrypsin Mechanism -- Step 6: Break-off from the Enzyme]**  **[Chymotrypsin Mechanism -- Step 7: Product Dissociates]** A diagram of a molecule Description automatically generated **[Aspartyl Proteases ]**  ***[6.8 -- Lysozyme ]*** **[Peptidoglycan and Lysozyme ]** - Peptidoglycan is a polysaccharide found in many bacterial cell walls - Cleavage of the cell wall leads to the lysis of bacteria - Lysozyme is an antibacterial enzyme A diagram of a chemical reaction Description automatically generated **[General Acid-Base + Covalent Catalysis: Cleavage of Peptidoglycan by Lysozyme ]** X-ray structures of lysozyme with bound substrate analogs show that the C-1 carbon is located between Glu 35 and Asp 52 residues.  ***[6.9 -- Lysozyme Mechanism and Catalytic Cycle ]*** **[Cleavage of Peptidoglycan by Lysozyme: Two Successive S~N~2 Steps Model ]** - Asp 52 acts as a nucleophile to attacks the anomeric carbon in the first S~N~2 step - Glu 35 acts as a general acid and protonates the leaving group in the transition state - Water hydrolyzes the covalent glycosyl-enzyme intermediate - Glu 35 acts as a general base to deprotonate water in the second S~N~2 step. A diagram of a chemical reaction Description automatically generated  A diagram of a chemical structure Description automatically generated ***[6.10 -- Fundamentals of Enzyme Kinetics ]*** **[What is enzyme kinetics? ]** - Kinetics is the study of the rate at which compounds react - Rate of enzymatic reaction is affected by: - Enzyme (wild-type vs. mutant) - Substrate (preferred vs. alternative) - Effectors (activators & inhibitors) - Temperature (too low, optimal, too high) - pH (too low, optimal, too high) - Age of the enzyme (they have a shelf-life) **[The goal of enzyme kinetics ]** - Compare catalytic ability of two enzymes - Wild-type vs. mutant - Species 1 vs. Species 2 - Compare an enzyme's preference for alternative substrates - Substrate 1 vs. Substrate 2 - Determine the enzyme mechanism - Sequential -- all substrates bind before chemistry - Ordered: A binds first, then B - Random: A and B bind in any order - Ping--Pong -- two chemical steps, covalent enzyme-substrate intermediate - Understand how inhibitors work (Drug development) - Reversible: Competitive, Uncompetitive, Noncompetitive (Mixed) - Irreversible: forms a covalent adduct that permanently disables enzyme - Potency: IC~50~ or K~i~ - Understand regulation of metabolic enzymes ***[6.11 -- Measuring Enzyme Activity ]*** **[Quantifying enzyme catalysis ]** - Use specific activity (µmol/min/µg) - Compares gross activity under one set of conditions (T, pH, \[S\]); fast, simple. - Can be used for impure or pure proteins (screening in cell lysates) - Normally only good for comparisons within the same lab - Use Michaelis-Menten constants k~cat~ and K~m~ - Inherent chemical properties of an enzyme-substrate pair - Still depend on conditions, but fewer (T, pH) - Can always calculate K~m~ : does not depend on \[E\] - Purified proteins required for k~cat~ : must know \[E\] - Should be repeatable across many labs - Measures the **rate** of reaction as a function of **substrate concentration.** **[How to do Kinetic Measurements]** - Experiment: 1. Mix enzyme + substrate(s) 2. Record rate of substrate disappearance/product formation as a function of time (the velocity of reaction) 3. Plot initial velocity vs. substrate concentration 4. Change substrate concentration and repeat  **[How to collect kinetic data: approach]** - Modality - Colorimetric: UV-Vis-IR-NMR Spectroscopy/Fluorimetry - Radiometric: Radiolabeled substrate/product physically separated and counted - Direct: Substrate or product is detected via spectroscopy or radioactivity - Indirect: Coupled Enzyme Assay; primary enzyme produces a product that is a substrate for a second enzyme -- product of second enzyme is detectable - Continuous: undisturbed reaction monitored by spectroscopy (Beer's Law) - Discontinuous: remove sample form reaction periodically and quench to stop reaction and measure product (Standard Curve, radioactive cpm) - Which modalities can be used for different kinds of enzymes? ***[6.12 -- Michaelis -- Menten Equation ]*** - \[E\] + \[S\] \[ES\] \[EP\] \[E\] + \[P\] - Assume all P is equivalent (EP + P) - Assume no P present at start of reaction (initial rate) - Assume \[ES\] is constant throughout the measurement (steady-state) - Assume \[S\] \>\> \[E\] (saturation conditions) **[Deriving Michaelis-Menten 1]** - Simplified reaction scheme A close-up of a number Description automatically generated - Steady state assumption  - Rate of ES formation = rate of ES destruction A black text with black letters Description automatically generated - Collection of rate constants gives "Michaelis" constant - K~m~­ approaches K~d~ when k~2~ \ - Complex lipid-based structures that form pliable sheets - Composed of a variety of lipids and proteins - Some membrane lipids and proteins are glycosylated - All cells have a cell membrane, which separates the cell from its surrounding - Eukaryotic cells have various internal membranes that divide the internal space into compartments **[Electron Micrograph of Biological Membranes]** A close-up of a cell Description automatically generated **[Functions of Membranes]** - Define the boundaries of the cell - Allow import and export - Selective import of nutrients (e.g. lactose) - Selective export of waste and toxins (e.g., antibiotics) - Retain metabolites and ions within the cell - Sense external signs and transmit information into the cell - Provide compartmentalization within the cell - Separate energy-producing reactions from energy-consuming ones - Keep proteolytic enzymes away from important cellular proteins - Produce and transmit nerve signals - Store energy as a proton gradient - Support synthesis of ATP **[Common Features of Membranes ]** - Sheet-like flexible structure, 30 -- 100 Å (3 -- 10 nm) thick - Main structure is composed of two leaflets of lipids (**bilayer**) - With the exception of archaebacteria: monolayer of bifunctional lipids - Form spontaneously in aqueous solutions and are stabilized by noncovalent forces, especially hydrophobic effect - Protein molecules span the lipid bilayer - Asymmetric - Some lipids are found preferably "inside" - Some lipids are found preferably "outside" - Carbohydrate moieties are always outside the cell - Electrically polarized (inside negative \~ --60 mV) - Fluid structures: two-dimensional solution of oriented lipids **[Fluid Mosaic Model of Membranes]** - Proposed in 1972 (Singer and Nicholson, UCSD) - Lipids form a viscous, two dimensional solvent into which proteins are inserted and integrated more or less deeply - Integral proteins are firmly associated with the membrane, often spanning the bilayer - Peripheral proteins are weakly associated and can be removed easily - Some are noncovalently attached - Some are linked to membrane lipids  ***[11.3 -- Membrane Composition ]*** **[The Composition of Membranes]** - Lipid composition of membranes is different in: **Different organisms tissues, and organelles** - Ratio of lipid to protein varies - Type of phospholipid varies - Abundance and type of sterols varies - Prokaryotes lack sterols - Cholesterol predominant in the plasma membrane, virtually absent in mitochondria - Galactolipids abundant in plant chloroplasts but almost absent in animals **[Membrane composition is highly variable in different organisms]** A table with numbers and text Description automatically generated  **[Membrane bilayers are asymmetric ]** - Two leaflets have different lipid compositions - Outer leaflet is often more positively charged - Phosphatidylserine outside has a special meaning: - Platelets: Activates blood clotting - Other cells: Marks the cell for destruction **[Membrane composition asymmetry ]**  ***[11.4 -- Membrane Proteins]*** **[Functions of Proteins in Membranes]** - Receptors: detecting signals from outside - Light (opsin) - Hormones (insulin receptor) - Neurotransmitters (acetylcholine receptor) - Pheromones (taste and smell receptors) - Channels, gates, pumps - Nutrients (maltoporin) - Ions (K--channel) - Neurotransmitters (serotonin reuptake protein) - Enzymes - Lipid biosynthesis (some acyltransferases) - ATP synthesis (F~0~F~1~ATPase/ATP synthase) **[Three Types of Membrane Proteins]** A diagram of a cell membrane Description automatically generated **[Peripheral Membrane Proteins]** - Associate with the polar head groups of membranes - Relatively loosely associated with membrane - Through ionic interactions with the lipids or aqueous domains of integral membrane proteins - Removed by disrupting ionic interactions either with high salt or change in pH - Purified peripheral membrane proteins are no longer associated with any lipids **[Integral Membrane Proteins]** - Span the entire membrane - Have asymmetry like the membrane - Different domains in different compartments - Tightly associated with membrane - Hydrophobic stretches in the protein interact with the hydrophobic regions of the membrane - Removed by detergents that disrupt the membrane - Purified integral membrane proteins still have phospholipids associated with them **[Integral Membrane Proteins: α-helices]**  **[Integral Membrane Proteins: β-sheets]** A diagram of a dna structure Description automatically generated  **[Amino acids in membrane proteins cluster in distinct regions]** - Transmembrane segments are predominantly hydrophobic - Tyr and Trp cluster at nonpolar interface - Charged amino acids are only found in aqueous domains A diagram of a structure Description automatically generated **[Lipid-linked Membrane Proteins ]**  **[Lipid Anchors]** - Some membrane proteins are lipoproteins - They contain a covalently linked lipid molecule - Long chain fatty acids - Isoprenoids - Sterols - Glycosylated phosphatidylinositol (PGI) - The lipid part can become part of the membrane - The protein is now anchored to the membrane - Reversible process - Allows targeting of proteins - Some, such as GPI anchors are found only on the outer face of plasma membrane **[Cell membranes are asymmetric]** - Every component of the membrane exhibits asymmetry - Lipids - Outer and inner leaflets have different lipid compositions - Proteins - Individual peripheral membrane proteins are only associated with one side of the membrane - Integral membrane proteins have different domains on different sides of the membrane - Specific lipid modification of proteins targets the protein to a specific leaflet - Carbohydrates - Only on the outside of cells ***[11.5 -- Membrane Phases]*** **[Physical Properties of Membranes]** - Can exist in various **phases** and undergo phase transitions - **Dynamic** and **flexible** structures - **Not permeable** to large polar solutes and ions - **Permeable** to small polar solutes and nonpolar compounds (H~2~O, CO~2~) - Permeability can be **temporarily increased** by chemical treatment or electrical shock - When we want to add foreign DNA into the cell **[Membrane Phases]** - Depending on their composition and the temperature lipid bilayer can be in gel or fluid phase - Gel Phase: Individual molecules do not move around - Fluid Phase: individual molecules can move around - Heating causes phase transition from the gel to fluid - Under physiological conditions, membranes are more fluid-like than gel-like - Must be fluid for proper function A diagram of different types of molecules Description automatically generated **[Organisms can adjust the membrane composition]** - Membrane fluidity is determined mainly by the fatty acid composition - More fluid membranes require **shorter** and more **unsaturated** fatty acids - Remember differences in T~M~ of fatty acids - At higher temperatures cells need more saturated fatty acids - To maintain integrity - At lower temperatures cells need more unsaturated fatty acids - To maintain fluidity  **[Sterols increase membrane rigidity and permeability]** - Cell membranes of many eukaryotes contain **sterols** - Cholesterol in animals - Phytosterols in plants - Ergosterol in fungi - Sterols break up phospholipids that are too tightly arranged - Sterols create islands of structure for phospholipids that are too mobile A diagram of a molecule Description automatically generated with medium confidence ***[11.6 Membrane Lipids in Motion]*** **[Membrane Dynamics: Lateral Diffusion is Fast]** - Individual lipids undergo fast lateral diffusion within the leaflet  **[Membrane Dynamics: Transverse Diffusion is Slow]** - Spontaneous flips from one leaflet to another are rare because the charged head group must transverse the hydrophobic tail region of the membrane A diagram of a transbolation Description automatically generated with medium confidence **[How do we know about lateral? ]** - Fluorescence Recovery After Photobleaching (FRAP) - Monitors lateral lipid diffusion in the outer leaflet - Rates of lateral diffusion are fast (up to 1mm/sec) - A lipid can circumnavigate an E.coli cell in one second **[FRAP]**  A diagram of lines and text Description automatically generated with medium confidence  **[How do we know about transverse?]** - Membranes are asymmetric - Outer leaflet contains PC and SM - Inner leaflet contains PE, PS, and PI - All lipids are made inside cells, so something must manage the asymmetry. Transporters? - Kennedy and Rothman (1977) - Double-label experiment to show how fast lipids flip - Evidence for "flippases", "floppases", and "scramblases" A diagram of a number of different types of substances Description automatically generated with medium confidence  **[Kennedy-Rothman Experiment]** - Separately label inner leaflet and outer-leaflet lipids to see how they move between leaflets - Newly synthesized PE goes into the inner leaflet - ^3^H- glycerol was supplied and then chased with unlabeled glycerol to label some of the new PE - Outer leaflet PE was chemically labeled with TNBS either immediately or after a 3-minute pause. A diagram of a cell line Description automatically generated with medium confidence  A diagram of a graph Description automatically generated with medium confidence This suggests that phospholipids in cells are moved between leaflets using enzyme transporters ***[11.7 -- Membrane Rafts, Curvature, and Fusion]*** **[Membrane Rafts]** - Lipid distribution in a single leaflet is not random or uniform - Lipid rafts - Contain clusters of glycosphingolipids with longer-than-usual tails - Are more ordered - Contain specific doubly or triply acylated proteins - Allow segregation of proteins in the membrane  **[Caveolin forces membrane curvature]** A diagram of a structure Description automatically generated **[Other Modes of Membrane Curvature]**  **[Membrane Fusion]** - Membranes can fuse with each other without losing continuity - Fusion can be spontaneous or protein-mediated - Examples of protein-mediated fusion are: - Entry of influenza virus into the host cell - Release of neurotransmitters at nerve synapses **[Examples of Membrane Fusion]** Diagram of a diagram of a human body Description automatically generated with medium confidence **[Neurotransmitter Release]**  **[Neurotransmitter Release: Step 1]** Diagram of a diagram showing the formation of a plasma membrane Description automatically generated **[Neurotransmitter Release: Step 2]**  **[Neurotransmitter Release: Step 3]** A diagram of zipping causes and tension on a leaflet Description automatically generated **[Neurotransmitter Release: Step 4]**  **[Neurotransmitter Release: Step 5]** A diagram of fusion pore Description automatically generated **[Neurotransmitter Release: Step 6]**  ***[11.8 -- Transport Across Membrane]*** **[Transport Across Membranes]** - Cell membranes are permeable to small nonpolar molecules that passively diffuse through the membrane - Passive diffusion of polar molecules involves desolvation and thus has a high activation barrier - Transport across the membrane can be facilitated by proteins that provide an alternative diffusion path - Such proteins are called **transporters** or permeases **[Types of Transport]** Diagram of a cell membrane Description automatically generated **[Polar solutes need alternative paths to cross cell membranes]**  **[Three Classes of Transport Systems]** A diagram of a structure Description automatically generated  **[Glucose Transporter in the Membrane]** A diagram of a molecule Description automatically generated **[Model for Glucose Transport]**  **[Kinetics of Glucose Transport]** A diagram of a normal and normal glucose Description automatically generated **[There are multiple glucose transporters]** - A Na^+^ glucose symporter and a glucose uniporter operate on opposite sides of epithelial cells - Cells can also have asymmetry, with distinct proteins confined to one side  **[Bicarbonate transporter is an antiporter]** - Maintains the electrochemical potential across the membrane Diagram of carbon dioxide anhydrms Description automatically generated **[Two Types of Active Transport]**  **[ABC transporters use ATP hydrolysis to drive transport of substrates]** - ABC = **ATP-binding Cassette** (cassette refers to a common motif seen in DNA Sequences of Transport proteins **[Ion channels maintain gradients for active transport]** - Potassium Ion Channel: - Large K^+^ ion goes thru - Smaller Na^+^ ion can't - How? A diagram of a protein structure Description automatically generated with medium confidence