Chemical Constitution & Functional Groups & Interactions PDF

CHEMICAL CONSTITUTION & FUNCTIONAL GROUPS & INTERACTIONS INCLUDES SOME REVISIONS FROM IB AND CHEMISTRY 4 All bonds (interactions) on the molecular and atomic level are electric in nature. These forces arise due to the existence of charge and...

CHEMICAL CONSTITUTION & FUNCTIONAL GROUPS & INTERACTIONS INCLUDES SOME REVISIONS FROM IB AND CHEMISTRY 4 All bonds (interactions) on the molecular and atomic level are electric in nature. These forces arise due to the existence of charge and follow Coulomb´s law: 5 The ability of an atom to attract the electron pair in a covalent bond to itself Non-polar bond similar atoms have the same electronegativity; they will both pull on the electrons to the same extent; the electrons will be equally shared. BENEFIT Polar bond different atoms have different electronegativities; BENEFIT one will pull the electron pair closer to its end; it will be slightly more negative than average, −; or more positive, +; the other will be slightly less negative,BENEFIT a dipole is formed and the bond is said to be polar; greater electronegativity difference =BENEFIT greater polarity. 6 A scale to measure electronegativity. INCREASE Values increase across periods; Values decrease down groups; Fluorine has the highest value. INCREASE H BENEFIT 2.1 Li Be B C N O F BENEFIT 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Na Mg Al Si P S Cl BENEFIT 0.9 1.2 1.5 1.8 2.1 2.5 3.0 K Br BENEFIT 0.8 2.8 7 Intramolecular forces – Forces that hold atoms together within a molecules. Intermolecular forces – Forces that exist between molecules, weaker than intramolecular forces. 8  Intermolecular Intramolecular 1. Hydrogen Bonds 1. Covalent 2. Salt-bridges 2. Ionic 3. Permanent Dipole - Permanent Dipole 3. Metallic 4. Permanent Dipole - Induced Dipole Relative strength: BENEFIT Dispersion Forces (< 1 kcal/mol) < Dipole-Dipole 5. Instantaneous Dipole-Induced Dipole Interactions (2-5 kcal/mol) < Hydrogen Bonds (12-16 kcal/mol) Etc. 6. PI Interactions The nomenclature can be different in different scientific areas 9 1. Covalent Bond - bond formed between atoms that have similar electronegativities, the affinity or desire for electrons. Because both atoms have similar affinity for electrons and neither tends to donate them, they share electrons in order to achieve octet configuration and become more stable. Bonds force depends, as seen, on proximity: Triple > double > single. Nonpolar Covalent Bond - formed between same atoms or atoms with very similar electronegativities (equal sharing). Polar Covalent Bond - formed when atoms of slightly different electronegativities share electrons (not equal sharing). 10 2. Ionic Bond – bond formed by the complete transfer of valence electron(s) between atoms. It is a type of chemical bond that generates two oppositely charged ions. 3. Metallic Bond - occurs between atoms of metals, in which the valence electrons are free to move through the lattice. This bond is formed via the attraction of the mobile electrons—referred to as sea of electrons - and the fixed positively charged metal ions. 11 12  Intermolecular Intramolecular 1. Hydrogen Bonds 1. Covalent 2. Salt-bridges 2. Ionic 3. Permanent Dipole - Permanent Dipole 3. Metallic 4. Permanent Dipole - Induced Dipole Relative strength: BENEFIT Dispersion Forces (< 1 kcal/mol) < Dipole-Dipole 5. Instantaneous Dipole-Induced Dipole Interactions (2-5 kcal/mol) < Hydrogen Bonds (12-16 kcal/mol) Etc. 6. PI Interactions The nomenclature can be different in different scientific areas 13 1. Hydrogen Bonds – A bond shared by two electronegative atoms. It is the strongest intermolecular force. H-bond is a special form of electrostatic dipole-dipole force, its not really a true chemical bond. The partially positive end of hydrogen is attracted to the partially negative end of the oxygen (O), nitrogen (N), or fluorine (F) of another molecule. Strength of H-bond: F>O>>N. Hydrogen bond should not be confused with a covalent bond to hydrogen. 14 H-bonds with donor-acceptor distances of: 2.2-2.5 Å - “strong, mostly covalent”; energies 40-14 kcal/mol 2.5-3.2 Å - “moderate, mostly electrostatic”; energies 15-4 kcal/mol 3.2-4.0 Å - “weak, electrostatic”; energies pH mantains proton 36 GLY-ASP - Calculate pI 1- Protonate all ionizable groups 2- Find maximum charge 3- Find pKa before/after zero net species 37 K-E-H - Calculate pI 1- Protonate all ionizable groups; 2- Find maximum charge; 3- Find pKa before/after zero net Species. 38 LYS-GLU-HIS - Calculate pI 1- Protonate all ionizable groups; 2- Find maximum charge; 3- Find pKa before/after zero net Species. 39 LYS-GLU-HIS - Calculate pI 1- Protonate all ionizable groups 2- Find maximum charge 3- Find pKa before/after zero net species 40 LYS-GLU-HIS - Calculate pI 1- Protonate all ionizable groups 2- Find maximum charge 3- Find pKa before/after zero net species 41 LYS-GLU-HIS - Calculate pI 1- Protonate all ionizable groups 2- Find maximum charge 3- Find pKa before/after zero net species 42 CHARGE Peptide: CYS-HIS-ALA-ARG-GLY-GLU - Calculate pI and charge at pH = ሼ1,4,7,10,13ሽ 43 CYS-HIS-ALA-ARG-GLY-GLU - Calculate pI and charge at pH = ሼ1,4,7,10,13ሽ 44 CYS-HIS-ALA-ARG-GLY-GLU - Calculate pI and charge at pH = ሼ1,4,7,10,13ሽ 45 CYS-HIS-ALA-ARG-GLY-GLU - Calculate pI and charge at pH = ሼ1,4,7,10,13ሽ 46 PRIMARY STRUCTURE Linear; Ordered; 1 Dimensional (1D). At one end is an amino acid with a free amino group the (the N-terminus) and at the other is an amino acid with a free carboxyl group the (the C-terminus). A perfectly linear amino acid polymer is neither functional nor energetically favorable → folding! PEPTIDE BOND 0.133 nm – shorter than a typical single bond but longer than a double bond. 40% double bond character. Backbone represented on the left.  PEPTIDE BOND An amide type of covalent chemical bond. Links two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino "acid" and N2 (nitrogen number two) of another along a peptide or protein chain. The dehydration condensation of two amino acids to form a peptide bond (red) with expulsion of water (blue). Number of amino acid residues is calculated by dividing molecular weight by 110 as the average molecular weight of proteins amino acids is near 128 (kDa) and: 128-18 of dehydration upon peptide bond formation accounts for 110 (kDa). Accounts for the double bond within the peptide bond shifting between carbon-oxygen to carbon-nitrogen (Hybrid of the 2 resonance structures with electrons delocalized over the carbonyl O, the carbonyl C and the amide N. Constrains the peptide bond, so that it CANNOT rotate, which gives the polypeptide sequences a backbone with little room for conformational change. 5 PEPTIDE BOND RESONANCE Planar Geometry. The six atoms of the peptide bond group are planar (C, C=O, N-H, C). Rotation in the polymer occurs art C Inherent dipole (N partially positive; O partially negative). Peptide bond is polar! 6 PEPTIDE BOND CONFIGURATION The double bond structure means that can there are two degrees of freedom per residue for the peptide chain and so they can be in: Trans – the oxygen and hydrogen atoms face two different directions ( angle is 180º); Cis – the oxygen and hydrogen atoms face the same directions  angle is 0º). Peptide bonds in protein structures are mainly found in trans conformation with a torsion angle x close to 180º. Only a very low proportion is observed in cis conformation with x angle around 0º due to steric interference between side chains. 7 PSI and PHI ANGLES Bonds between the amino group and the - carbon and between the -carbon atom and carbonyl group are pure single bonds. The rotation around N-C bond of the peptide groups is called φ (phi) and the one around Ca-C is called ψ (psi). 8 PSI and PHI ANGLES Each of these angles is defined by the relative position of four atoms of backbone. Clockwise angles are positive and counterclockwise angles are negative with each having a 180º sweep. Each rotation angles range from -180º to 180º. 9 PSI and PHI ANGLES Adjacent rigid peptides units may rotate about these bonds taking on various orientations. These rotations are restricted by steric interference limiting possible angles: Between main-chain and side-chain atoms of adjacent residues; Between carbonyl oxygens on adjacent residues. Only 10% of the {φ, ψ} combinations are generally observed for proteins 10 RAMACHANDRAN PLOT Plot of φ vs ψ Space filing model of peptides/proteins that Space filing model of peptides that shows shows values of psi and phi angles that are values of psi and phi angles that are sterically sterically permitted in a polypeptide chain. permitted in a polypeptide chain. Two degrees of freedom: φ (phi) angle = rotation about N – C  (psi) angle = rotation about C – C The computed angles which are sterically allowed fall on certain regions of plot. White = sterically disallowed conformations (atoms come closer than sum of van der Waals radii); Green = sterically allowed conformations. 11 Acid-base behavior depends of: N- and C-terminal; Different ionizable R groups within protein. The polypeptide chain can form H-Bonds: Hydrogen-bond donor: N-H group Hydrogen-bond acceptor: C=O group Size of protein is highly variable but usually lower than 50 amino acids is considered a peptide. Number of amino acid residues is calculated by dividing molecular weight by 110 as the average molecular weight of proteins amino acids is near 128: 128-18 of dehydration upon peptide bond formation accounts for 110. 12 What is the approximate molecular weight of a protein with 682 amino-acids residues in a single polypeptide chain? Assuming an average of 110 by residue (corrected for loss of water in formation of peptide bond), a protein containing 682 residues has an Mr of approximately 682 × 110 = 75000 14 PROTEIN FOLDING Occurs in the cytosol. Involves localized spatial interaction among primary elements (i.e. amino-acids). Gives conformations that reduce energy (thermodynamically favourable). Yields secondary structure. SECONDARY STRUCTURE Non-linear; Three-dimensional (3D). Repeating values of φ and ψ along the chain result in regular structure. The ability to do this is dependent on steric considerations...i.e. secondary structure is dependent to some degree on primary structure (sequence). Formed and stabilized by hydrogen bonding, electrostatic and van der Waals interactions. SECONDARY STRUCTURE Hydrogen bonds are key to determine protein structure as they are directional: the participating dipoles must be aligned properly. Hydrogen bonds are dipole-dipole interactions formed between heteroatoms in which one heteroatom (e.g. nitrogen) contains a bond to hydrogen and the other (e.g. oxygen) contains an available lone pair of electrons. SECONDARY STRUCTURE Non-polar amino acid stabilize the folded state of a protein through van der Waals interactions. These forces are non-directional. Although individual interactions are weak, the more surface area of contact, the stronger the interactions. SECONDARY STRUCTURE Other factors that affect secondary structure Prosthetic groups: Coenzymes Cations Intramolecular/Intermolecular bonds: disulfides dityrosine aldol cross-linking RAMACHANDRAN Some parameters describe helices: PLOT 1. Axis: Line of symmetry about which the helix is wound. 2. Diameter: Largest lateral dimension of the helix. 3. Number of turns; number of residues per helical turn, n. 4. Pitch – length/distance between any two points on the helix that are exactly one turn apart (measured in nm). By convention, a positive value of n denotes a right-handed helix. 22 -HELIX Most abundant secondary structure. 3D arrangement of amino acids with the polypeptide chain in a corkscrew shape. Looks like a coiled “telephone cord”. -HELIX The dipoles of hydrogen bonding backbone atoms are in near perfect alignment. The radius of the helix allows for favourable van der Waals interactions across the helical axis. Side chains are well staggered minimizing steric interference. -HELIX Repeating values of : φ, ~ -57° and ψ ~ -47° give a right-handed helical fold (the alpha-helix). e.g. cytochrome c, an alpha helical protein. -HELIX 3.7 amino acids per turn. Average length: 10 amino acids or 3 turns. Varies from 5 to 40 amino acids. Held by internally H-bonds between the H of –N-H group and the –O of C=O of every fourth amino acid along the chain.  -HELIX RAMACHANDRAN PLOT Amino acids with a side chain whose movement is largely restricted in an alpha helix (branched at beta carbon like threonine or valine) are disfavoured, i.e. occur less often in alpha helices than in other secondary structure elements. Glycine, with its many possible main chain conformations, is also rarely found in helices. Proline is considered a helix breaker because its main chain nitrogen is not available for hydrogen bonding. Prolines are often found near the beginning or end of an alpha helix. Proline in an -helix induces alteration - proline kink. 27 3.10 HELIX i Less common (10-15%). Stabilized by hydrogen bonds of the type (i, i+3); Ni+3 → Oi. Smaller radius, compared to the α-helix. 3 residues/turn. Rise 0.20 nm/residue. Helix pitch 0.60 nm. φ = -49°, ψ = -26°. π-HELIX Right-handed (although left-handed is also possible); 4.1 residues/turn. Rise ~0.115 nm/residue; Helix pitch ~0.41 nm. H-bonds: Ni+5 → Oi. φ = -57°, ψ = -80° (approx.) Most π-helices are only 7 residues in length and do not adopt regularly repeating (φ, ψ) dihedral angles throughout the entire structure like that of α-helices or β-sheets. π-HELIX Created by the insertion of a single additional amino acid into a pre-existing α-helix. α-helices and π-helices can be inter-converted by the insertion and deletion of a single amino acid. Given both the relatively high rate of occurrence of π-helices and their noted association with functional sites (i.e. active sites) of proteins, this ability to interconvert between α-helices and π-helices has been an important mechanism of altering and diversifying protein functionality over the course of evolution. BETA-SHEET Polypeptide chains are arranged side by side. Similarly, repetitive values in the region of: φ = -110 to –140 and ψ = +110 to +135 give beta sheets. Plastocyanin is composed mostly of beta. BETA-SHEET Hydrogen bonds form between chains in which the N−H groups in the backbone of one strand establish hydrogen bonds with the C=O groups in the backbone of the adjacent strands. R groups of extend above and below the sheet. Rise per residue: 3.47 Angstroms for antiparallel strand; 3.25 Angstroms for parallel strands. BETA-SHEET Try, Trp and Phe are typically found in beta- sheets as these structural arrangements has enough pace to accommodate their large side-chains. -branched amino-acids (Thr, Val, Ile) are favored at the middle of -sheets Pro is found in the edge of -sheets to avoid “edge-to-edge” association between proteins, which could lead to aggregation and amyloid formation. BETA-SHEET - PARALLEL N-termini of successive strands are oriented in the same direction. Residue i may form hydrogen bonds to residues j − 1 and j + 1; this is known as a wide pair of hydrogen bonds. By contrast, residue j may hydrogen-bond to different residues altogether, or to none at all. Lower stability. BETA-SHEET - ANTIPARALLEL Successive β-strands alternate directions so that the N-terminus of one strand is adjacent to the C-terminus of the next. Strongest inter-strand stability because it allows the inter-strand hydrogen bonds between carbonyls and amines to be planar, which is their preferred orientation. -TURN Allows the peptide chain to reverse direction. Carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away. Proline (and in less extend glycine) are prevalent in beta turns. TURNS & RANDOM COILS Loops & Turns ( turns) 1/3 globular protein; Mostly at surface of protein; C=O H-bonded to the NH three residues away ; Proline and glycine. Random coil Can't assign 2° structure, adopts multiple conformations depending on conditions but not random - energy minima; Flexible linkers, hinges. MIXED Such β sheets can be purely antiparallel, purely parallel, or mixed. Valine, threonine and Isoleucine tend to be present in β-sheets. Proline tends to disrupt β strands. Proteins can have a mix of various secondary structure motifs. TERCIARY STRUCTURE Determined by a variety of interactions (bond formation) among R groups and between R groups and the polypeptide backbone. The weak interactions include: 1. Hydrogen bonds among polar side chains; 2. Ionic bonds between charged R groups (basic and acidic amino acids); TERCIARY STRUCTURE 3. Hydrophobic interactions among hydrophobic ( nonpolar) R groups. Strong covalent bonds include disulfide bridges, that form between the sulfhydryl groups (SH) of cysteine monomers, stabilize the structure. TERCIARY STRUCTURE Specific overall shape of a protein Cross links between R groups of amino-acids in chain: Disulfide –S–S– The amino acid cysteine forms a bond with another cysteine through its R group. Ionic –COO– -------- H3N+ – Charged R groups bond together. TERCIARY STRUCTURE H-bonds –C=O ------ HO – Polar R groups of amino-acids form bonds with other polar R groups. Hydrophobic –CH3 H3C– These amino-acids orient themselves towards the middle of the chain avoiding water. Hydrophilic –CH3 OH– Amino-acids orient themselves towards the water. QUATERNARY STRUCTURE Non-linear; 3 Dimensional (3D). Results from the aggregation (combination) of two or more polypeptide subunits held together by non-covalent interaction like H- bonds, ionic or hydrophobic interactions. Favorable, functional structures occur frequently and have been categorized. OLIGOMERS Proteins made of: One subunit = monomer; Two subunits = dimer (e.g. G-protein coupled receptor dimers – GPCRs- on the left); Three subunits = trimer; Four subunits = tetramer;  OLIGOMERS Each polypeptide chain in such a protein is called a subunit. Oligomeric proteins can be made of multiple polypeptides that are: identical → homooligomers (homo = same), or different → heterooligomers (hetero = different). The simplest: a homodimer. https://www.medical-supply.ie/product/idimerize-inducible-homodimer-system/ Protein size has limits due to: the genetic coding capacity of nucleic acids It is simply more efficient to make many copies of a small polypeptide than one copy of a very large protein. In fact, most proteins with a molecular weight greater than 100,000 have multiple subunits, identical or different. the accuracy of the protein biosynthetic process The error frequency is low (about 1 mistake per 10,000 amino acid residues added), but even this low rate results in a high probability of a damaged protein if the protein is very large. Simply put, the potential for incorporating a “wrong” amino acid in a protein is greater for a large protein than for a small one. QUATERNARY STRUCTURE Protein are classified in two types based on higher order structure: Fibrous proteins: Polypeptide chains arranged in long stands or sheets. Globular proteins: Polypeptide chains folded into a spherical or globular shape. FIBROUS PROTEINS E.g.: a keratin, collagen and silk fibroin. Structural function to provide strength and flexibility. The fundamental structural unit is simple repeat of an element of secondary structure. Mostly water insoluble. Collagen is the main fibrous component of skin, bone, tendon, cartilage, and teeth. It contains three helical polypeptide chains, each nearly 1000 residues long. Glycine appears at every third residue in the amino acid sequence, and the sequence glycine-proline-hydroxyproline recurs frequently. Hydroxyproline is a major component of the protein collagen, comprising roughly 13.5% of mammalian collagen. Proline and hydroxyproline confer rigidity on the collagen molecule by permitting the sharp twisting of the collagen helix. 51 Keratin structural molecules are normally long and thin, insoluble in water, very high tensile strength, and arranged to form fibers. Composed of long rods, twisted together, laid down in criss-cross matrix form. 52 Fibrinogen, the protein of the blood plasma, is converted into the insoluble protein fibrin during the clotting process. Once fibrin is activated, it assembles to form tough fibrils. The chains at the centre reach over and bind into pockets on the large globular heads of neighbouring fibrin molecules. Also, fibrin associates head-to-head, which is made stronger in a clot by crosslinking, making the interaction permanent. Finally, the longer chains coloured orange here can extend over to neighbouring fibrils to form even larger structures, binding in a small pocket coloured green here. 53 Myosin contains many amino acids with positively and negatively charged side chains; they form 18 and 16 %. Myosin catalyses the hydrolytic cleavage of ATP (adenosine triphosphate). Myosin combines easily with another muscle protein called actin, the molecular weight of which is about 50,000; it forms 12 to 15 percent of the muscle proteins. In muscle, actin and myosin filaments are oriented parallel to each other and to the long axis of the muscle. The actin filaments are linked to each other lengthwise by fine threads called S filaments. During contraction, the S filaments shorten, so that the actin filaments slide toward each other, past the myosin filaments, thus causing a shortening of the muscle. 54 Comparison of compact globular structure of GLOBULAR BSA with its extended forms ( helices and sheets) PROTEINS E.g.: Enzymes and regulatory proteins Different segments of polypeptide chains fold back on each other resulting in a compact form. Either dissolve or form colloidal suspensions in water. Generally more sensitive to temperature & pH change than fibrous protein counterparts. Denaturation is a process in which a protein loses its native shape due to the disruption of weak chemical bonds and interactions, thereby becoming biologically inactive. Denatured protein becomes insoluble in the solvent in which it was originally soluble. Denaturation is usually irreversible. In the case of proteins : A loss of three-dimensional structure, sufficient to cause loss of function. Change in physical, chemical and biological properties of protein molecules. Loss of secondary structure: lose all regular -helices and -sheets and adopt random coil. Loss of tertiary structure: disruption of covalent interactions (disulfide bridges), non-covalent dipole-dipole (polar aa) and induced dipole (non-polar aa). Loss of quaternary structure: subunits disrupted. Changing pH denatures proteins because it changes the charges on many of the side chains. This disrupts electrostatic attractions and hydrogen bonds. Certain reagents such as urea and guanidine hydrochloride denature proteins by forming hydrogen bonds to the protein groups that are stronger than the hydrogen bonds formed between the groups. Detergents such as sodium dodecyl sulphate denature proteins by associating with the non-polar groups of protein, thus interfering with the normal hydrophobic interactions. Organic solvents such as acetone alcohols denature proteins by disrupting hydrophobic interactions. Proteins can also be denatured by heat. Heat increase molecular motion, which can disrupt the attractive forces. None of these agents breaks peptide bonds, are not hydrolyzed, so the primary structure of a protein remains intact when it is denatured. The denatured state does not necessarily equate with complete unfolding of the protein and randomization of conformation. Under most conditions, denatured proteins exist in a set of partially folded states that are poorly understood. Most proteins can be denatured by heat, which affects the weak interactions in a protein (primarily hydrogen bonds) in a complex manner. If the temperature is increased slowly, a protein’s conformation generally remains intact until an abrupt loss of structure (and function) occurs over a narrow temperature range. In cooking, this stress that causes denaturation is typically heat. As it heats, its proteins coagulate. Agitation also denatures protein. We see this clearly in the whipping of egg whites. In nature, you can see the denaturation of protein in the waves at the beach. The constant churning creates foam from various proteins in the sea water. HYDROSTATIC PRESSURE (5,000 – 10,000 atm) Pressure destabilization of hydrophobic aggregates by using an information theory model of hydrophobic interactions. Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure with water molecules penetrating their core. UV RADIATION UV radiation supplies kinetic energy to protein molecules, causing their atoms to vibrate more rapidly and disrupting relatively weak hydrogen bonding and dispersion forces. ACIDS AND BASES Acids and bases disrupt salt bridges held together by ionic charges. Double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added. This reaction occurs in the digestive system, when the acidic gastric juices cause the curdling (coagulating) of milk. Examples: Acetic acid; Trichloroacetic acid 12% in water; Sulfosalicylic acid; Sodium bicarbonate. ORGANIC SOLVENTS (ETHER, ALCOHOL) Alcohol Disrupts Hydrogen Bonding: New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains. E.g. In the prion protein, TYR128 is hydrogen bonded to ASP178, which cause one part of the chain to be bonding with a part some distance away. After denaturation, there is substantial structural changes. SALTS OF HEAVY METALS (PB, HG) Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights. Since salts are ionic, they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt. This reaction is used for its disinfectant properties in external applications. E.g. AgNO3 is used to prevent gonorrhea infections in the eyes of newborn infants. Silver nitrate is also used in the treatment of nose and throat infections, as well as to cauterize wounds. “Heavy metals may also disrupt disulfide bonds because of their high affinity and attraction for sulfur and will also lead to the denaturation of proteins.” REDUCING AGENTS DISRUPT DISULFIDE BONDS: Disulfide bonds are formed by oxidation of the sulfhydryl groups on cysteine. If oxidizing agents cause the formation of a disulfide bond, then reducing agents, of course, act on any disulfide bonds to split it apart. Reducing agents add hydrogen atoms to make the thiol group, -SH. Agents that break disulphide bonds by reduction include: 2-Mercaptoethanol; Dithiothreitol; TCEP (tris(2-carboxyethyl)phosphine). DETERGENTS Detergents are amphiphilic molecules (both hydrophobic and hydrophilic parts). Disrupt hydrophobic interactions: hydrophobic parts of the detergent associate; with the hydrophobic parts of the protein (coating with detergent molecules); hydrophilic ends of the detergent molecules interact favorably with water (nonpolar parts of the protein become coated with polar groups that allow their association with water); hydrophobic parts of the protein no longer need to associate with each other Dissociation of the non-polar R groups can lead to unfolding of the protein chain (same effect as in nonpolar solvents). The restoration of native structure of a protein by returning the protein solution to native conditions is known as ‘renaturation’ of the protein. Most of the times, proteins can be renatured simply by reversing the process used to denature them: Gradually decrease temperature of solution: If temperature is reduced too fast, the protein may be trapped in a local minimum rather than reaching native state, with insufficient energy to escape the local minimum. Remove denaturant (organic solvent): Reconstituting the protein in aqueous solution of appropriate pH and osmolarity is generally sufficient to renature protein. Re-adjust pH to native conditions: For most aqueous proteins, this is in the range of 6.5–7.5. However, if the protein in question is from a compartment with a different pH (such as the thylakoid lumen or lysosome), then renaturation would occur at that pH. It should be noted that the principles of protein folding, and renaturation apply only to aqueous proteins, and not membrane proteins. Why do egg whites go from clear to opaque when you fry an egg? Egg whites contain large amounts of proteins called albumins, and the albumins normally have a specific 3D shape, thanks to bonds formed between different amino acids in the protein. Heating causes these bonds to break and exposes hydrophobic (water-hating) amino acids usually kept on the inside of the protein. The hydrophobic amino acids, trying to get away from the water surrounding them in the egg white, will stick to one another, forming a protein network that gives the egg white structure while turning it white and opaque. From protein denaturation, another delicious breakfast. 69 70 71 72 73 Group of specialized proteins that contain heme group as a tightly bound prosthetic group (non-protein compound permanently associated with a protein). The heme group role dependents on the environment created by the 3D structure: Enzyme catalase - active site; Cytochrome - electron carrier; Myoglobin and hemoglobin - oxygen carrier. Hemoglobin Biological role: transport oxygen (4 binding sites); Binds oxygen in the lungs and transports it to the peripheral tissues; Circulates in erythrocytes; Affinity for oxygen is modulated to bind tightly in the lungs and release easily in tissues, allosteric properties of hemoglobin. Myoglobin Biological role: stores oxygen (1 binding site); Increases O2 solubility in tissues (muscles), binds to it and keeps it until needed. 75 Structure subunit similar: Eight -helices; Contain heme group; Mb monomeric proteins; Hb heterotetramer (22) – composed of 4 polypeptides held together by non-covalent interaction; Each subunit is like myoglobin and contains a heme group. There are two identical  dimers. 76 Structure subunit similar: Compact structure; 80% is an -helix terminated by Proline or -bends/turns; Interior is composed of non-polar amino-acids that are packed together stabilized by hydrophobic interactions; Charged amino-acids are located at the surface. 77 Heme group: Organic component – protoporphyrin (4 pyrrole rings linked by methane bridges); Inorganic component - iron atom (Fe2+); FERROUS state, oxidation state of +2. Iron is at the center of protoporphyrin has 6 coordination bonds: 4 nitrogen atoms, two O2. At one side of protoporphyrin plane, iron is bound to a histidine reside, proximal histidine, that helps to stabilize the O2 binding non-covalently to the heme iron. This region is stabilized by another histidine called the distal histidine of the protein (H-bond to oxygen). 78 In deoxyhemoglobin, the iron atom remains unbound to oxygen. In this state, Fe atom is too large to fit into the center of the protoporphyrin ring and so the iron remains below the protoporphyrin. The binding of the oxygen atom to the iron pulls away electrons of the iron, making it smaller. This allows it to fit into the protoporphyrin plane. Since the iron atom is attached to the proximal histidine, it causes it to move, shifting the entire polypeptide chain. The shift causes a conformational change in the surface-to-surface interactions between the adjacent subunits. 79 Quaternary structure of deoxyhemoglobin is in T-state (taut or tense state), constrained by a network of ionic bonds and H-bonds; low affinity for O2. Oxyhemoglobin contains a more relaxes conformation and exists in the R-state (relaxed state); higher affinity for O2 that leads to rupture of some ionic and H-bonds and more freedom of movement. 80 Upon oxygenation, one a dimer rotates 15º with respect to the second ad dimers, leading to the cooperativity behavior of hemoglobin: the binding of an oxygen to the heme group increases the tendency of the other heme group to bind to another oxygen molecule. Conclusion: O2 binds to deoxyhemoglobin inducing a conformational change from the T-state to R- state, which is the cause of the cooperative behavior of hemoglobin. 81 Myoglobin (storage) Simple chemical curve; As the oxygen concentration increases, the curve rises sharply and quickly levels off; At pO2 of 2 mmHg, half of myoglobin molecules are saturated with O2. 50% This means that: Mb binds to O2 molecules quickly; Mb has a string affinity for O2; Mb does not release O2 until the pO2 drops to a low quantity. Physiological consequences – stores oxygen. THIS HAPPENS AS MB IS A SINGLE CHAIN. 82 Hemoglobin (carrier) Sigmoidal curve (S-sharped); Hb binds to oxygen with lower affinity than Mb; Only at concentration of 26 mmHg, is half of hemoglobin saturated with O2. 50% This means that hemoglobin: Binds oxygen less strongly than Mb; Has a lower affinity for oxygen than Mb; Releases O2 much more readily than Mb. THIS HAPPENS AS Hb BINDS O2 COOPERATIVELY. Binding of a O2 at one site of one chain makes the other unoccupied sites more likely to bind O2 (the same to unload). 83 Physiological consequences – oxygen carrier. Biological facts: In our Lungs, the partial pressure of oxygen is around 100 mmHg. In Resting tissue, the partial pressure of oxygen is around 40 mmHg. In Exercising tissue, the partial pressure of oxygen is around 20 mmHg. 84 Myoglobin as O2 carrier: Lungs Resting Tissue: 98% - 97% = 1% oxygen unload Lungs Exercising Tissue: 98%-91% = 7% oxygen unload Hemoglobin as O2 carrier: Lungs Resting Tissue: 98% - 77% = 21% oxygen unload Lungs Exercising Tissue: 98%-32% = 66% oxygen unload 85  According to this model, Hb exists either in the T or R-states. binding of oxygen simply shifts the equilibrium between these two states. As such, the affinity of other heme only increase when hemoglobin assumes the R-state, which contrast to what is seen experimentally. This model stats that even if oxygen binds, the hemoglobin does not change to the R- state! Wrong. Does not consider also that the remaining Hb also change. 86 The binding of oxygen stimulates a conformational change in the polypeptide to which it binds that in turn leads to a conformational change in the nearby polypeptide chains. As subunits change in conformation, their affinity for oxygen increases. Notice that according to this model there are intermediate states between R- and T-states and that R-state is only achieved when all four sites are occupied. Again, contrast to what is seen experimentally. Conclus 87 The sigmoidal curve for hemoglobin is formed from the combination of the T-state curve and R-state curve. Conclusion: Both fail, and cooperativity nature of oxygen binding must be defined using both models. 88 CO binds tightly to one or more heme iron forming carbon monoxyhemoglobin (HbCO) and hemoglobine shifts to R-form. O2 affinity increases, shifting O2 binding curve to hyperbolic. Consequently Hb cannot deliver O2 to tissue; looses its high- quality characteristics as a transporter. 89 The O2 curve of hemoglobin in red blood Cells (RBCs) differ considerably from pure hemoglobin. In RBCs, hemoglobin can unload 98-32% = 66 % of oxygen in going from lungs to exercising tissue. Pure hemoglobin unloads 98-90% = 8% oxygen when moving over the same partial pressure difference. The T-state of hemoglobin in its pure form is highly unstable as it contains a pocket of positive charge at the center of tetramer This instability pushes equilibrium towards the R-state, Physiologically this means that pure hemoglobin would bind O2 too strongly and would not release it to tissue. When O2 begins to bind to the heme groups, in the lungs, the center pocket collapses and the 2,3-BPG is kicked out, leading to the relaxed state. 90 2,3 – BPG is naturally occurring molecule founds in cells, it is formed as intermediate during glycolysis Acts as an allosteric effector to hemoglobin causing a low O2 affinity. 2,3-BPG, a highly anionic molecule, can fit perfectly into the positively charged pocket of the T-state. By binding, it stabilizes the T-state and pushes the equilibrium towards the T-state, which makes it less attached to O2. 91 Some molecules can bind proteins and modulate activity by allosteric interaction. Allosteric modulators or allosteric effectors bind reversibly to site separate from functional binding or active site. Modulation of activity occurs through change in protein conformation. 2,3 biphosphoglycerate (BPG), CO2 and protons are allosteric effectors of Hb binding to O2. 2,3 - BPG 92 As the fetus is developing, it expresses a slightly different hemoglobin, in which β chain is replaced with a  chain (22). One major difference between  and  chains is that His143 is replaces by a neutral serine residue. Due to this substitution, the center pocket has a smaller positive charge and 2, 3-BPG do not bind as well as to adult hemoglobin. The reduced affinity for 2,3-BPG results in fetal hemoglobin having a higher affinity for oxygen, binding oxygen when the mother’s hemoglobin is releasing oxygen. Fetal hemoglobin, with its higher affinity for O2, can successfully deliver O2 do developing fetus. 93 2,3-BPG is not the only allosteric effector that improves hemoglobin efficiency. H+ and carbon dioxide, produced by actively respiring tissues, are also allosteric effectors, enhancing oxygen release by hemoglobin. At lower pH (higher amount of H+) or at higher concentrations of CO2, salt bridges (ionic bonds) form that stabilize the T state. Shift curve to the right, thereby decreasing affinity of hemoglobin to O2, allowing it to unload more O2 to tissues 94 98 - 32 = 66% is released to the tissues in the absence of CO2 and pH=7.4. 98 – 21 = 77% is released to the tissues in the absence of CO2 and at a lower pH=7.2. 98 - 10 = 88% is released to the tissues in the presence of CO2 and pH=7.2. The pH effect The amino group of the terminal residues at the -subunits and the histidine reside 146 and 122 are responsible for this effect. They have a pKa around 7 and will become protonated at a lower pH. 95 When protonated, His146 on the  chain is positively charged and participates in a salt-bridge (ionic bond) with Asp94, stabilizing the T-state. The amino-termini of the hemoglobin chains lie at the interface between  and  chains. CO2 can interact with the positively charged amino termini to formed negatively charged carbamate groups that form ionic interactions that also stabilize the T-state. The T-state is favoured decreasing oxygen affinity. 96 Non-polar CO2 moves into the RBC where it is converted by carbonic anhydrase into hydrogen ions and bicarbonate ions. Higher concentrations of CO2 more hydrogen ions will be produced, decreasing haemoglobin affinity as a results of salt-bridge formation. 97 At higher altitudes, the atmospheric pressure is lower than at sea level. This means that less oxygen will enter our blood plasma through the lungs and ultimately the tissue. In order to increase the amount of 02 delivered to out tissues, our body responds in a variety of ways. Na immediate response involves a higher rate of breathing (hyperventilation) and a quicker heart rate, These responses however are relatively inefficient, short-lasting and can be dangerous. A much more efficient and safer way to increase the amount of oxygen delivered to our tissue is to change the amount of 2,3-BPG in the blood, increase the number of hemoglobin molecules and ultimately increase the RBC count in the blood. This method is not only safer but also long-lasting. 98 The increasing of 2,3-BPG will shift the curve to the right, which will decrease the affinity of hemoglobin for oxygen. This means that hemoglobin will be able to unload and deliver more oxygen to the tissues. 99 100 101 102 103 104 105