Chapter 4: The Three-Dimensional Structure of Proteins PDF

Chapter 4 The Three-Dimensional Structure of Proteins © 2018 Cengage Learning. All Rights Reserved. Chapter Outline Part 1 (4-1) Protein structure and function (4-2) Primary structure of proteins (4-3) Secondary s...

Chapter 4 The Three-Dimensional Structure of Proteins © 2018 Cengage Learning. All Rights Reserved. Chapter Outline Part 1 (4-1) Protein structure and function (4-2) Primary structure of proteins (4-3) Secondary structure of proteins Part 2 (4-4) Tertiary structure of proteins (4-5) Quaternary structure of proteins © 2018 Cengage Learning. All Rights Reserved. 4-1 Protein Structure Biologically active proteins are polymers that consist of amino acids linked by covalent peptide bonds Many conformations are possible for proteins due to flexibility of amino acids Native conformations: 3-D shapes of proteins with biological activity Many proteins have large segments of random structure as they have no obvious regular repeating structure © 2018 Cengage Learning. All Rights Reserved. What are the levels of protein structure? Primary structure (1°): Order in which amino acids are covalently linked together Read from the N-terminal end to the C-terminal end Secondary structure (2°): Ordered 3-D arrangement in space of the backbone atoms in a polypeptide chain Tertiary structure (3°): 3-D arrangement of all atoms in a protein, including those inside chains and in prosthetic groups Quaternary structure (4°): Arrangement of subunits with respect to one another Subunits: Individual parts of a large molecule © 2018 Cengage Learning. All Rights Reserved. 4-2 Primary Structure of Proteins Amino acid sequence helps determine the 3-D conformation of a protein, which determines its properties Changes in one amino acid residue in a sequence can alter biological functions Example - Hemoglobin associated with sickle-cell anemia (Glu6 is replaced by Val) Determination of this sequence is a routine operation in classical biochemistry. It consists of several steps. © 2018 Cengage Learning. All Rights Reserved. 4-3 Secondary Structure of Proteins Is the hydrogen-bonded arrangement of the backbone of the protein. Peptide chains are linked at opposite corners by swivels Each amino acid residue has two bonds with free rotation that are designated by the Ramachandran angles, phi (Φ) and psi (Ψ). Fig. 4.1 Bond between the α-carbon and amino nitrogen of that residue (Φ). Bond between the α-carbon and carboxyl carbon of that residue (Ψ). Types - α-helix and β-pleated sheet arrangements © 2018 Cengage Learning. All Rights Reserved. Figure 4.1 - Peptide Conformation Is Described by Phi and Psi Angles The peptide bonds exist in a plane, and the bond between the carbonyl group and the amide group does not rotate. Two bonds coming from the alpha carbon can rotate, and secondary structure can be described by the two angles, phi and psi, shown. When the two planes are parallel, both angles are assigned the value of 180 degrees. © 2018 Cengage Learning. All Rights Reserved. The α-Helix Stabilized by hydrogen bonds parallel to the helix axis within the backbone of a single polypeptide chain The helical conformation allows for a linear arrangement of the atoms involved in H-bonds Which provides maximum bond strength and makes the helical conformation very stable. Features (Fig. 4.2) Coil of the helix is clockwise or right-handed There are 3.6 residues for each turn of the helix Linear distance between corresponding points on successive turns (pitch) is 5.4 Å, & 0.54 nm in SI units © 2018 Cengage Learning. All Rights Reserved. Figure 4.2 - Four Different Graphic Representations of the α-Helix (A)From left to right, a stick representation with the H bonds as dotted lines; a space-filling model; a hybrid of a ribbon diagram and a stick figure that shows the path of the peptide backbone (B)A three-dimensional structure representation of the alpha helices of a subunit of hemoglobin © 2018 Cengage Learning. All Rights Reserved. Factors That Disrupt the α-Helix Bending of the polypeptide backbone because of proline Restricts rotation due to its cyclic structure Results in absence of the N—H for hydrogen bonding in the α-amino group Strong electrostatic repulsion caused by the proximity of several side chains of like charges Examples - Lys and Arg or Glu and Asp Steric repulsion, or crowding, caused by the proximity of bulky side chains Example - Val, Ile, and Thr © 2018 Cengage Learning. All Rights Reserved. α-Helix (continued 1) Each peptide bond is s-trans and planar C—O of each peptide bond is hydrogen bonded to the N—H of the fourth amino acid residue The α-helix is stabilized by hydrogen bonds parallel to the helical axis within the backbone of a single chain. All the side chains lie outside the helix. © 2018 Cengage Learning. All Rights Reserved. The β-Pleated Sheet Hydrogen bonds between peptide chains can be interchain or intrachain Polypeptide chains lie adjacent to one another If chains run in the same direction, the sheet will be parallel If chains run in the opposite direction, the sheet will be antiparallel (Fig. 4.4). The H-bonding gives rise to a zigzag structure and are perpendicular to the direction of the protein chain (Fig.4.5). R groups alternate above and below the plane © 2018 Cengage Learning. All Rights Reserved. Figure 4.4 - Hydrogen Bonding in β-Pleated Sheets (A) Parallel β-pleated sheets (B) Antiparallel β- pleated sheets © 2018 Cengage Learning. All Rights Reserved. Figure 4.5 - Three-Dimensional Form of the Antiparallel β- Pleated Sheet Arrangement The antiparallel orientation allows for maximum hydrogen bonding because the oxygen, nitrogen, and hydrogen are in a straight line. © 2018 Cengage Learning. All Rights Reserved. Irregularities in Regular Structures Found in shorter stretches than with the α-helix Sometimes break up the regular nature of the α-helix The most common is the 310 helix which has 3- residues per turn and 10 atoms in the ring formed by making the H-bond. Other common helices are 27 and 4.416 following the same nomenclature. A β-bulge is a common nonrepetitive irregularity found in antiparallel β-sheets. It occurs between two normal β-structure hydrogen bonds and involves two residues on one strand and one on the other. See Figure 4.6 © 2018 Cengage Learning. All Rights Reserved. Reverse Turns (RT) Parts of proteins where the polypeptide chain folds back on itself. RT marks a transition between one secondary structure and another. For steric reasons, glycine is frequently encountered in RT at which the peptide chain changes direction. The single hydrogen of the side chain prevents crowding (Fig. 4.7A and B). Proline is also encountered in reverse turns because of its cyclic structure (Fig. 4.7C). A reverse turn is region of the polypeptide having a hydrogen bond from one main chain carbonyl oxygen to the main chain N-H group 3 residues along the chain (i.e., Oi to N i+3). © 2018 Cengage Learning. All Rights Reserved. Figure 4.7 - Structures of Reverse Turns Arrows indicate the directions of the polypeptide chains. © 2018 Cengage Learning. All Rights Reserved. Supersecondary Structures and Domains Supersecondary structures - Result from the combination of α- and β-strands βαβ unit - Two parallel strands of β-sheet are connected by a stretch of α-helix αα unit - Contains two antiparallel α-helices Also called helix-turn-helix β-meander - Antiparallel sheet is formed by a series of tight reverse turns connecting stretches of the polypeptide chain Greek key - Formed when a polypeptide chain doubles back on itself See Fig. 4.8 © 2018 Cengage Learning. All Rights Reserved. Figure 4.8 - Schematic Diagrams of Supersecondary Structures Arrows indicate the directions of the polypeptide chains. (A)A (βαβ) unit. (B)An (αα) unit. (C)A (β-meander). (D)The (Greek key). (E)The Greek key motif in protein structure resembles the geometric patterns on this ancient Greek vase, giving rise to the name. © 2018 Cengage Learning. All Rights Reserved. Supersecondary Structures and Domains (continued) Motif: a repetitive supersecondary structure. β-barrel - Created when β-sheets are extensive enough to fold back on themselves. Fig. 4.9. Motifs are important, since they: 1. Provide information about the folding of proteins 2. Do not help predict the biological function of the protein A domain is defined as a polypeptide chain or a part of a polypeptide chain that can fold independently into a stable tertiary structure. Domains are also units of function; proteins may comprise a single domain or as many as several dozen domains. There is no fundamental structural distinction between a domain and a subunit. Many proteins that have the same type of function have similar protein sequences; consequently, domains with similar conformations are associated with the given function. Example: 3-different types of domains by which proteins bind to DNA. © 2018 Cengage Learning. All Rights Reserved. 4-4 Tertiary Structure of Proteins 3-D arrangement of all atoms in a molecule Includes the conformations of side chains and the positions of any prosthetic groups In a fibrous protein the shape is a long rod. Backbone of the protein does not fold back on itself Arrangement of the atoms of the side chains is not specified by the secondary structure For a globular protein Tertiary structure helps determine the way in which helical and pleated-sheet sections fold back on each other The interactions between side chains play an essential role in the folding of proteins. © 2018 Cengage Learning. All Rights Reserved. Forces Involved in Tertiary Structures Tertiary structures depend on noncovalent interactions Backbone hydrogen bonding between polar side chains of amino acids Hydrophobic interaction between nonpolar side chains Electrostatic attraction between side chains of opposite charge (Fig. 4.13) Complexing several side chains to a single metal ion Disulfide bonds between side chains of cysteines They restrict folding patterns available to polypeptide chains They are absent in myoglobin and hemoglobin but present in chymotrypsin and trypsin. © 2018 Cengage Learning. All Rights Reserved. Figure 4.13 - Forces That Stabilize the Tertiary Structure of Proteins Note that the helical structure and sheet structure are two kinds of backbone hydrogen bonding. Although backbone hydrogen bonding is part of secondary structure, the conformation of the backbone constrains the possible arrangement of the side chains. © 2018 Cengage Learning. All Rights Reserved. Myoglobin (Mb)-An Example of Protein Structure. Consists of a single polypeptide chain of 153 amino acid residues and includes a prosthetic group, the heme group, in a hydrophobic pocket. Heme: Iron-containing cyclic compound. Mb Has a compact structure. Mb has an eight α-helical regions,(A-H) which are stabilized by hydrogen bonding in the polypeptide backbone. Mb has no β-pleated sheet regions. Exterior contains most of the polar side chains Interior contains nonpolar side chains and two polar histidine residues that are involved in interactions with the heme group and bound O © 2018 Cengage Learning. All Rights Reserved. Figure 4.15 - Structure of Myoglobin The peptide backbone and the heme group are shown overlain on the space-filling model. The helical segments are designated by the letters A through H. The terms NH3+ and COO– indicate the N-terminal and C-terminal ends, respectively. © 2018 Cengage Learning. All Rights Reserved. Heme Group (Fig. 4.16) Its presence affects conformation of the polypeptide. Consists of: A metal ion, Fe(II), and an organic part, protoporphyrin IX Has six coordination sites and forms six metal–ion complexation bonds Four sites are occupied by N atoms of the four pyrrole-type rings of the porphyrin Fifth coordination site is occupied by one of the N atoms of the imidazole side chain of histidine residue F8 O2 is bound at the sixth coordination site Consists of four five-membered rings based on the pyrrole structure that are linked by methine (—CH═) groups © 2018 Cengage Learning. All Rights Reserved. Figure 4.17 - The Oxygen-Binding Site of Myoglobin The porphyrin ring occupies four of the six coordination sites of the Fe(II). Histidine F8 (His F8) occupies the fifth coordination site of the iron (see text). Oxygen is bound at the sixth coordination site of the iron, and histidine E7 lies close to the oxygen. © 2018 Cengage Learning. All Rights Reserved. Oxygen: Imperfect Binding to the Heme Group More than one molecule can bind to heme Affinity of free heme for carbon monoxide (CO) is 25,000 times greater than its affinity for O2 When CO is forced to bind at an angle, then its advantage over O2 drops by 2- orders of magnitude. CO is a potent poison in large quantities. © 2018 Cengage Learning. All Rights Reserved. 4-5 Quaternary Structure of Proteins Pertains to proteins that consist of more than one polypeptide chain Each chain is called a subunit Oligomers: Molecules that are made up of several smaller subunits Include dimers, trimers, and tetramers Chains interact with one another noncovalently via electrostatic attractions, hydrogen bonds, and hydrophobic interactions Allosteric: Property of multisubunit proteins such that a conformational change in one subunit induces a drastic change in another subunit Examples of a protein with quaternary structure. Insulin, Hemoglobin and© 2018Immunoglobulin Cengage Learning. All Rights Reserved. Hemoglobin (Hb) Hb is a tetramer, consisting of 4-polypeptide chains, Two α-chains 141 residues long Two β-chains 146 residues long Overall structure - α2β2 Most amino acids of the α-chain, β-chain, and myoglobin (153 residues long) are homologous © 2018 Cengage Learning. All Rights Reserved. Figure 4.21 - The Structure of Hemoglobin Hemoglobin (α2β2) is a tetramer consisting of four polypeptide chains (two α- chains and two β-chains). © 2018 Cengage Learning. All Rights Reserved. Oxygen Binding of Hemoglobin (Hb) One molecule of myoglobin binds one O2 Hemoglobin can bind up to four molecules of O2 Binding of O2 to hemoglobin exhibits positive cooperativity When one O2 molecule is bound, it becomes easier for the next O2 to bind. © 2018 Cengage Learning. All Rights Reserved. Figure 4.22 - A Comparison of the oxygen-binding behaviour of myoglobin and hemoglobin The oxygen-binding curve of myoglobin is hyperbolic, whereas that of hemoglobin is sigmoidal. Myoglobin is 50% saturated with oxygen at 1 torr partial pressure; hemoglobin does not reach 50% saturation until the partial pressure of oxygen reaches 26 torr. © 2018 Cengage Learning. All Rights Reserved. How does hemoglobin work? The two different types of behavior exhibited by Mb & Hb are related to the functions of these proteins. Hemoglobin Function - Oxygen transport Requirement - Bind strongly to O2 and release O2 easily Saturation - 50% at 26 torr partial pressure of O2 In the capillaries of active muscles pressure of oxygen is 20 torr. In other words, Hb gives oxygen easily in capillaries, where the need for oxygen is great. Myoglobin Function - Oxygen storage Requirement - Bind strongly to O2 at very low pressures Saturation - 50% at 1 torr partial pressure of O2 © 2018 Cengage Learning. All Rights Reserved. Figure 4.23 - The Structures of (A) Deoxyhemoglobin and (B) Oxyhemoglobin Structure of oxygenated hemoglobin is different from that of deoxygenated hemoglobin There is much less room at the center of oxyhemoglobin, due to the moving a way of the cationic (+) groups. © 2018 Cengage Learning. All Rights Reserved. Conformational Changes That Accompany Hemoglobin Function Other ligands are involved in cooperative effects when O2 binds to Hb. Both H+ and CO2 which bind to Hb can affect protein affinity for O2 by altering 3-D structure in subtle but important ways. The effect of H+ is called the Bohr effect (Fig. 4.24) Increase in [H+] reduces O2 affinity of Hb and causes protonation of key amino acids like His146 of the β-chains. Now His146 is stabilized by, a salt bridge to Asp94, which favors the deoxygenated form of Hb. In the active muscles an↑ [H+] lowers Hb affinity for O2 which releases O2 (Fig.4.25). © 2018 Cengage Learning. All Rights Reserved. Figure 4.24 - The General Features of the Bohr Effect In actively metabolizing tissue, hemoglobin releases oxygen and binds both CO2 and H+. In the lungs, hemoglobin releases both CO2 and H+ and binds oxygen. © 2018 Cengage Learning. All Rights Reserved. Conformational Changes That Accompany Hemoglobin Function (continued 1) Hb’s acid–base properties are affected by O2–binding properties: The oxygenated form of Hb is a stronger acid since it has a lower pKa than its deoxygenated form Deoxygenated hemoglobin has higher affinity for H+ than the oxygenated form. Table 4.1 - A Summary of the Bohr Effect © 2018 Cengage Learning. All Rights Reserved. Conformational Changes That Accompany Hemoglobin Function (continued 2) Hb in blood is also bound to another ligand, 2,3- bisphosphoglycerate (BPG) (Fig. 4.26). Binding is electrostatic; specific interactions take place between the –ve charges on BPG and the +ve charges on the protein (Fig.4.27) In the presence of BPG (P50= 26 torr.), and oxygen will be released. If BPG is not present (P50=1 torr), and little O2 would be released in the capillaries. Fig. 4.28 The presence of BPG stabilizes the deoxy-Hb. © 2018 Cengage Learning. All Rights Reserved. Figure 4.26 - The Structure of BPG BPG (2,3- bisphosphogly cerate) is an important allosteric effector of hemoglobin. BPG plays a role in the supply of O2 to the growing fetus. © 2018 Cengage Learning. All Rights Reserved. Figure 4.27 - Binding of BPG to Deoxyhemoglobin Note the electrostatic interactions between the BPG and the protein. © 2018 Cengage Learning. All Rights Reserved. Figure 4.28 A comparison of the oxygen-binding properties of Hb in the presence and absence of BPG. © 2018 Cengage Learning. All Rights Reserved. Figure 4.29 A comparison of the oxygen-binding capacity of fetal and maternal Hb. HbF (α2γ2) binds less strongly to BPG and as a result: HbF has a higher affinity for oxygen than HbA, allowing for efficient transfer of oxygen from the mother to the fetus. © 2018 Cengage Learning. All Rights Reserved.