Copy of Lehninger Chapter 5.pdf

10/2/2023 5 Protein Function 1 1 Proteins Function by Interacting Dynamically with Other Molecules • two types of interactions: – protein acting as a reaction catalyst, or enzyme, alters the chemical configuration or composition of a bound molecule – neither the chemical configuration nor the co...

10/2/2023 5 Protein Function 1 1 Proteins Function by Interacting Dynamically with Other Molecules • two types of interactions: – protein acting as a reaction catalyst, or enzyme, alters the chemical configuration or composition of a bound molecule – neither the chemical configuration nor the composition of the bound molecule is changed 2 2 Principle 1 (1 of 4) The functions of many proteins involve the reversible binding of other molecules. A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. The transient nature of protein-ligand interactions is critical to life, allowing an organism to respond rapidly and reversibly to changing environmental and metabolic circumstances. 3 3 1 10/2/2023 Principle 2 (1 of 4) A ligand binds a protein at a binding site that is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. The interaction is specific: the protein can discriminate among the thousands of different molecules in its environment and selectively bind only one or a few types. A given protein may have separate binding sites for several different ligands. These specific molecular interactions are crucial in maintaining the high degree of order in a living system. 4 4 Principle 3 (1 of 2) Proteins are flexible. Changes in conformation may be subtle, reflecting molecular vibrations and small movements of amino acid residues throughout the protein. Changes in conformation may also be more dramatic, with major segments of the protein structure moving as much as several nanometers. Specific conformational changes are frequently essential to a protein’s function. 5 5 Principle 4 (1 of 2) The binding of a protein and a ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. The structural adaptation that occurs between protein and ligand is called induced fit. 6 6 2 10/2/2023 Principle 5 (1 of 4) In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits. 7 7 Principle 6 (1 of 3) Interactions between ligands and proteins may be regulated. 8 8 5.1 Reversible Binding of a Protein to a Ligand: OxygenBinding Proteins 9 9 3 10/2/2023 Principle 1 (2 of 4) The functions of many proteins involve the reversible binding of other molecules. A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. The transient nature of protein-ligand interactions is critical to life, allowing an organism to respond rapidly and reversibly to changing environmental and metabolic circumstances. 10 10 Heme Prosthetic Group • heme = proteinbound prosthetic group – present in myoglobin and hemoglobin – consists of a complex organic ring structure, protoporphyrin, with a bound Fe2+ atom 11 11 Oxygen Can Bind to a Heme Prosthetic Group • oxygen: – poorly soluble in aqueous solutions – diffusion through tissues is ineffective over large distances – transition metals have strong tendency to bind (iron, copper) 12 12 4 10/2/2023 Coordination Bonds of Iron • six coordination bonds: – four to nitrogen atoms in the flat porphyrin ring – two perpendicular to the porphyrin 13 13 Principle 2 (2 of 4) A ligand binds a protein at a binding site that is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. The interaction is specific: the protein can discriminate among the thousands of different molecules in its environment and selectively bind only one or a few types. A given protein may have separate binding sites for several different ligands. These specific molecular interactions are crucial in maintaining the high degree of order in a living system. 14 14 Perpendicular Coordination Bonds • two perpendicular coordination bonds: – one is occupied by a sidechain nitrogen of a highly conserved proximal His residue – one is the binding site for molecular oxygen (O2) • Fe2+ binds O2 reversibly • Fe3+ does not bind O2 15 15 5 10/2/2023 Globins Are a Family of OxygenBinding Proteins • globins = widespread protein family – highly conserved tertiary structure: eight α-helical segments connected by bends (globin fold) – most function in O2 transport or storage 16 16 Types of Globins • four types in humans and other mammals: – myoglobin = monomeric, facilitates O2 diffusion in muscle tissue – hemoglobin = tetrameric, responsible for O2 transport in the bloodstream – neuroglobin = monomeric, expressed largely in neurons to protect the brain from low O2 or restricted blood supply – cytoglobin = monomeric, regulates levels of nitric oxide, a localized signal for muscle relaxation 17 17 Myoglobin Has a Single Binding Site for Oxygen • myoglobin: – 153 residues + one molecule of heme – bends named after the α-helical segments they connect • His93 = ninety-third residue from the amino terminal end • His F8 = eighth residue in α helix F 18 18 6 10/2/2023 Protein-Ligand Interactions Can Be Described Quantitatively • a simple equilibrium expression describes the reversible binding of a protein (P) to a ligand (L): (5-1) 19 19 when given Association Constant • association constant (Ka) = provides a measure of rd - Ra= ta the affinity of the ligand L for the protein – higher Ka = higher affinity – equivalent to the ratio of the rates of the forward (association) and the reverse (dissociation) reactions that form the PL complex (5-2) 20 20 [L] Remains Constant (5-2) (5-3) • when [L] >>> [ligandbinding sites], the binding of the ligand by the protein does not appreciably change [L] 21 21 7 10/2/2023 Binding Equilibrium (5-4) (5-5) x = y/(y + z) describes a hyperbola 22 22 Graphical Representations of Ligand Binding (5-5) • [L] at which ½ of the available ligand-binding sites are occupied (Y = 0.5) corresponds to 1/Ka 23 23 Dissociation Constant • dissociation constant (Kd) = reciprocal of Ka – equilibrium constant for the release of ligand – lower Kd = higher affinity (5-6) (5-7) • when [L] = Kd, ½ of the ligandbinding sites are occupied (5-8) 24 24 8 · d = the dissociation (mstant represents the ligand concentration at which half of the avaliable receptor binding SitS are occupied It iS a measure of affinity between the ligand and the recepter ... . higher - weak binding , I affinity 10/2/2023 Representative Kd Values 25 25 Binding of O2 to Myoglobin (5-9) • Kd equals the [O2] at which ½ of the available ligandbinding sites are occupied, or [O2]0.5: (5-10) 26 26 Partial Pressure of O2 (5-11) 27 27 9 10/2/2023 Principle 3 (2 of 2) Proteins are flexible. Changes in conformation may be subtle, reflecting molecular vibrations and small movements of amino acid residues throughout the protein. Changes in conformation may also be more dramatic, with major segments of the protein structure moving as much as several nanometers. Specific conformational changes are frequently essential to a protein’s function. 28 28 Protein Structure Affects How Ligands Bind • carbon monoxide (CO) binds free heme more than 20,000 times better than does O2 – differences in the orbital structures affect binding geometries 29 29 Myoglobin’s Distal His Increases Heme’s Affinity for O2 • hydrogen bond between the imidazole side chain of His E7 and bound O2 electrostatically stabilizes the Fe-O2 polar complex • 20,000-fold stronger binding affinity of free heme for CO compared with O2 declines to ~40-fold Rotates to open and close the pocket 30 30 10 10/2/2023 Hemoglobin Transports Oxygen in Blood • erythrocytes (red blood cells) transport O2 – formed from hemocytoblasts (precursor stem cells) – main function is to carry hemoglobin • arterial blood = ~96% saturated with O2 • peripheral blood = ~64% saturated with O2 31 31 Principle 5 (2 of 4) In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits. 32 32 all missed In person R Hemoglobin Subunits Are Structurally Similar to Myoglobin "Hgb" ~ • hemoglobin: - 4 submits – tetrameric protein with 4 heme groups – adult hemoglobin has two globin types: two α chains (141 residues each) and two β chains · (146 residues each)alpolicies monomer ↳ tetramer 33 33 11 10/2/2023 Structural Conservation of Globins -> hum & Mys of put entip eachother Low sequence similarity 31 structure myoglobin High structural similarity the in us humo . figure shows the evaitich infivence on the 34 globin Fold 34 The Quaternary Structure of Hemoglobin • strong interactions between unlike subunits – hydrophobic effect – hydrogen bonds the submits – ion pairs (salt bridges) interact ↳ charged aminoacias • α1β1 (and α2β2) interface involves >30 residues Show • α1β2 (and α2β1) interface involves 19 residues 35 35 Principle 4 (2 of 2) The binding of a protein and a ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. The structural adaptation that occurs between protein and ligand is called induced fit. 36 36 12 - 10/2/2023 My0- de storage Hemo- pickup * It can & drop off bind On in either state Hemoglobin Undergoes a Structural Change on Binding Oxygen - Greater number of ion pairs hemoglobin: – R state = O2 has a " higher affinity for hemoglobin – T state = more stable when O2 is absent, predominant conformation of deoxyhemoglobin V= caries Why by and • two conformations of · ↓R hemoglobin drop has to bind 02 off ↳T (iniuys) · T state = higher on pairs 37 37 Ion Pairs Stabilize the T State • T state is stabilized by a greater number of ion pairs, -> when O2 is binded to any 4 of the monomers many of which lie at the α1β2 (and α2β1) interface salt Bridge 38 38 Conformational Change in Hemoglobin • O2 binding to hemoglobin in the T state triggers a conformational change to the R state - > so that all 4 subunits can now – αβ subunit pairs slide past each other and rotate – the pocket between the β subunits narrow – some ion pairs that stabilize the T state break and ↳ to allow the some new ones form bind 02 submits to slick 39 39 13 10/2/2023 The T → R Transition ↳ becomes move Anotice gets the pocket smallet planar 40 40 Changes in Conformation Near Heme "pucker" "Planar" - stabolizes the Onbinding 41 41 Principle 5 (3 of 4) In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits. 42 42 14 10/2/2023 Hemoglobin Binds Oxygen Cooperatively • Hemoglobin must bind oxygen efficiently in the lungs (pO2 = 13.3 kPa) and release efficiently in tissues (pO2 = 4 kPa) ○ Myoglobin would be unsuited; would not release in tissues due to high affinity • Hemoglobin solves problem by undergoing transition from low-affinity state (T) to high-affinity state (R) Hemo - works be · bi transitich from + > R ... need to monomeric globins stive their raes which relates to their , SAMCAWT 43 · 43 Hemoglobin Binds Oxygen Cooperatively • hemoglobin ↓Pickitup has a hybrid sigmoid binding curve for oxygen * need ↳ the R a sigmoid only way multisubunit conformational ↳ no pichip to small /"S shaped" curve have get this is to protein that indergoes to change differences . in Allows /"cooperative" a a "sensitivity" concentration . 44 44 Principle 6 (2 of 3) Interactions between ligands and proteins may be regulated. 45 45 15 10/2/2023 causes Allosteric Proteins · hemoglobin - O is an modulater 02 affecting 02 -amore I other activating humotropic • allosteric protein (e.g., hemoglobin) = binding of a ligand to one site affects the binding properties of another site on the same protein – modulators = ligands that bind to an allosteric protein to induce a conformational change – homotropic = normal ligand and modulator are identical – heterotropic = modulator is a molecule other than the normal ligand rotipottncantareE · I -> type of allosteric > types of protein modulater (CO2) I M thems -> home On 46 · -> 46 = Hetero = CO2 activating protein modulater = Inhibiter Carbon Monoxide Can Bind to Hemoglobin • CO has ~250-fold greater affinity for hemoglobin than does O2 so to ball- ↳ still nave sigmoid HgD shape 47 47 Cooperative Ligand Binding Can Be Described Quantitatively • for a protein with n binding sites, the equilibrium becomes: P + nL ⇌ PLn (5-12) Hemo : tems+ 42 = PLu 48 48 16 10/2/2023 The Ka and Y for Cooperative Ligand Binding • the expression for the association constant becomes: (5-13) ↳ association • the expression for Y is: (5-14) 49 49 The Hill Equation (5-14) • rearranging, then taking the log of both sides, yields: - (5-15) dent hud to know now its derived the Hill equation: (5-16) 50 50 Hill Plots and Hill Coefficients What a hill plot is & What It Hells you using the hill equation -> • Hill plot = plot of log [Y/(1 − Y)] versus log [L] • Hill coefficient = nH = slope of a Hill plot – If nH = 1, ligand binding is not cooperative binding is independent – nH > 1 indicates positive cooperativity helps Activating– nH < 1 indicates negative cooperativity -> Inhibiter binding I binding stops any situation where binding does not activate -> -> ↳ * UH bi at It Will = never= would the same require time of # on binding binding sites all proteins af all sites , I does not binding binding impact another chother another ... to be binded interest * 51 51 17