092409-BCH311

092409-BCH311 - CH3COOH --> H^+ = CH3COO^pH = pKa +...

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Unformatted text preview: CH3COOH --> H^+ = CH3COO^pH = pKa + log [A-]/[Ha] log [0.9*0.45]/(0.1*0.1) pKa + 1.6 4.76 + 1.6 6.36 Na^+ Protein Three-Dimensional Structure • Biologically active proteins are polymers of amino acids linked by covalent peptide bonds • While many different conformations (3D structures) are possible, usually only one structure has biological activity - this is called the native conformation • Four levels of protein structure are commonly defined: Secondary (2°) Structure • Secondary structure is the hydrogen-bonded arrangement of the backbone of the protein • The two main forms of secondary structure are α-helices and β-sheets • Within each amino acid residue there are two bonds with reasonably free rotation: (1) the bond between the Cα and the amino N of that residue, and (2) the bond between the Cα and the carbonyl C of that residue • The combination of the planar peptide bond and the two freely rotating bonds has important implications for the 3D conformation of the protein • A peptide-chain backbone can be visualized as a set of playing cards, each card representing a planar peptide bond • The cards are linked at opposite corners by swivels, representing the bonds about which there is considerable freedom of rotation Secondary (2°) Structure • The angles φ (phi) and υ (psi), also called Ramachandran angles, are used to designate rotations around the Cα-N and Cα-C bonds, respectively • The conformation of a protein backbone can be described by specifying the values of φ and υ for each residue (-180° to 180°) • Two kinds of secondary structures that occur frequently in proteins are the repeating α-helix and β-sheet hydrogen bonded structures • The φ and υ angles repeat themselves in contiguous (connected/in contact) amino acids in regular secondary structures the side group usually go out of the plane in the secondary structure Fig. 4-1, p. 89 The α-helix • The driving force for the formation of the α-helix is the optimal arrangment of amino acids to achieve maximal hydrogen bonding within the protein backbone • The α-helix is stabilized by H-bonds parallel to the helix axis within the polypeptide backbone beta-helix is stabilized by H-bonds perpendicular • Counting from the N-terminal end, the C-O (hydrogen bond acceptor) group of each amino acid is H-bonded to the N-H (hydrogen bond donor) group of an amino acid four residues away (1 H-bonded to 5, 2 to 6, 3 to 7 etc) • The helical conformation allows a linear arrangement of the atoms involved in the H-bonds, which gives the bonds maximum strength and thus makes the helical conformation very stable • There are 3.6 residues for each turn of the helix, and the pitch of the helix is 5.4 Å (or angstrom = 10-10 m), helix is right-handed • Proteins have varying amounts of α-helical structures, ranging from a few percent to nearly 100% • Note that this arrangement facilitates protrusion of the R side chains on the external surface of the α-helix The α-helix • From left to right, ball and stick model showing terminology; ball and stick model with planar groups shaded; computer generated space filling model Fig. 4-2a, p. 91 The α-helix - Hemoglobin • Model of the hemoglobin protein, showing the helical regions Fig. 4-2b, p. 91 β-Hemoglobin and Myohemerythrin • Three-dimensional structure of two proteins with substantial amounts of α-helix in their structures • The helices are represented by the regularly coiled sections of the ribbon diagram. Myohemerythrin is an oxygen-carrying protein in invertebrates Fig. 4-3, p. 92 The α-helix - Disruption • Several factors can disrupt the α-helix - proline creates a bend in the backbone because of its cyclic structure • Proline cannot fit into the α-helix because (1) rotation around the C-N bond is severely restricted, and (2) prolines α-amino group cannot participate in intrachain hydrogen bonding • Electrostatic repulsion between positively-charged R groups of lysine or arginine, or negatively-charged R groups of glutamate or aspartate residues, can also prevent the formation of α-helices • Steric repulsion (or crowding) because of bulky R groups can also prevent the formation of α-helices The β-sheet • The arrangement of atoms in the β-sheet conformation differs markedly from that in the α-helix • The peptide backbone in the β-sheet is almost completely extended • Hydrogen bonds can be formed between different parts of a single chain that is doubled back on itself (intrachain bonds) or between different chains (interchain bonds) • If the peptide chains run in the same direction a parallel β-sheet is formed • When alternating chains run in opposite directions, an antiparallel β-sheet is formed • The hydrogen bonding between peptide chains in the β-sheet gives rise to a repeated zig-zag structure • The hydrogen bonds run perpendicular to the direction of the protein chain, not parallel as is the case for the α-helix • Similar to that of α-helix, the amino acid side chains protrude outward The parallel β-sheet • The peptide chains run in the same direction, from N- to C-terminal Fig. 4-4a, p. 92 The antiparallel β-sheet • The peptide chains run in the opposite directions forming an antiparallel configuration Fig. 4-4b, p. 92 Protein Ribbon Diagrams • Leishmania major coproporphyrinogen oxidase an essential enzyme in the heme biosynthetic pathway • α-helices are represented as cylindrical spiral ribbons; β-sheets as thick arrows; non-repetitive loops as round ropes Protein Ribbon Diagrams • Leishmania major mitochondrial ribonuclease Supersecondary structures • The α-helix and β-sheet, as well as other secondary structures, are combined in many ways as the polypeptide chain folds back on itself in a protein • The combination of α-helices and β-sheets produces various kinds of supersecondary structures in proteins • The most common feature of this sort is the βαβ unit, in which two parallel strands of β-sheet are connected by a stretch of α-helix • Another example is a αα unit, also known as a helix-turn-helix • The αα unit consists of two antiparallel α-helices • In this arrangement, energetically favorable contacts exist between the amino acid side chains in the two stretches of helix Supersecondary structures • Arrows indicate the direction of the polypeptide chains. Top, a βαβ unit. Bottom, an αα unit, also known as a helix-turn-helix Fig. 4-4a, p. 92 The Collagen Triple Helix • Collagen, the major component of bone and connective tissue, is the most abundant protein in vertebrates • It is organized into water-insoluble fibers of great strength • A collagen fiber consists of three polypeptide chains wrapped around each other in a ropelike twist, or triple helix • Each of the three chains has a repeating sequence of X-P-G or X-hP-G (hP is hydroxyproline and X is any amino acid) • Proline and hydroxyproline can constitute up to 30% of the residues in collagen • The triple helix of collagen is arranged so that every third residue on each chain is inside the helix - only glycine is small enough to fit into the available space • The three individual collagen chains are themselves helices that differ from the α-helix • The three strands are also held together by hydrogen bonds involving hydroxyproline and hydroxylysine The Collagen Triple Helix • Collagen is both intra-molecularly and intermolecularly linked by covalent bonds formed by reactions of lysine and histidine residues • The amount of crosslinking within collagen fibers increases with age • Collagen in which the proline is not hydroxylated to hydroxyproline is less stable than normal collagen • Symptoms of scurvy, such as bleeding gums and skin discoloration, are the results of fragile collagen • The enzyme that hydroxylates proline (proline hydroxylase) and thus maintains the normal state of collagen requires ascorbic acid (vitamin C) to remain active Fig. 4-11, p. 97 Two Types of Protein Conformation: Fibrous and Globular • Often it is difficult to draw a clear distinction between secondary and tertiary protein structure • The nature of the side chains in a protein (part of the tertiary structure) can influence the folding of the backbone (the secondary structure) • Silk fibers consist largely of the protein fibroin and, like collagen, has a fibrous structure yet, unlike collagen, consists largely of β-sheets • Fibers of wool consist largely of the protein keratin (the major protein of hair, horns, animal hooves, reptile shells etc), which is largely α-helical • Collagen, silk and wool are all fibrous proteins • In other proteins, the backbone folds back on itself to produce a more or less spherical shape - these are called globular proteins • Globular proteins, unlike fibrous proteins, are water-soluble and have compact structures; their tertiary and quaternary structures can be quite complex Fig. 4-12, p. 98 Summary 4-3, p. 98 secondary structure doesn't include the side chain, yet the tertiray does Tertiary (3°) Structure of Proteins • The three-dimensional arrangement of all the atoms in the molecule - this now includes the R side chains • The conformations of the side chains and the positions of any prosthetic groups (any non-amino acid group attached to the protein) are part of the tertiary structure, as is the arrangement of the α-helices and β-sheets with respect to each other • In a fibrous protein, the overall shape of which is a long rod, the secondary structure also provides much of the information about the tertiary structure • The helical backbone does not fold back upon itself, and the only aspect of the tertiary structure that is not specified by the secondary structure is the arrangement of the atoms of the side chains • For a globular protein, considerably more information is needed - how do the αhelices and β-sheets fold back on each other, and how does that influence the interactions between side chain R groups? Forces Involved in Tertiary (3°) Structures • • The primary structure of proteins - the order of amino acids - depends on the formation of covalent peptide bonds Higher-order levels of structure, such as the conformation of the backbone (2° structure) and the positions of all the atoms in the protein (3° structure) usually depends on noncovalent interactions If a protein consists of several subunits, the interaction of the subunits (4° structure) also depends on noncovalent interactions - contributing to the most stable structure for a given protein Several types of noncovalent interactions can occur; a. backbone hydrogen bonding (2° structure), b. hydrogen bonding between the side chains of amino acids c. hydrophobic interactions between nonpolar amino acids d. electrostatic interactions between oppositely charged R side groups e. electrostatic interactions between metal ions and charged R side groups In addition to these noncovalent interactions, covalent interactions in the form of disulfide bridges/bonds can occur between cysteine side chains • • • Tertiary Structure of Proteins • The three-dimensional conformation of a protein is the result of the interplay of all these stabilizing forces (both covalent and noncovalent) Fig. 4-13, p. 100 Myoglobin • Myoglobin is a classic example of a globular protein, and an excellent case study for the 3D arrangement of a protein • Myoglobin is a relatively small (M.W. = 16.7 kDa) oxygen-binding protein of muscle cells • It functions both to store oxygen and to facilitate oxygen diffusion in rapidly contracting muscle tissue, and is a sensitive marker of muscle injury, e.g. heart attack • Myoglobin was the first protein to be completely crystallized (using X-ray crystallography) - revealing its 3° structure • The complete myoglobin molecule consists of a single polypeptide chain of 153 amino acid residues and includes a prosthetic group, the heme group, • The myoglobin molecule (including the heme group) has a compact structure, with the interior atoms very close to each other The structure of myoglobin • The peptide backbone and the heme group are shown overlain on the space-filling model • The α-helical segments are designated by the letters A through H. The NH3+ and COO- indicate the N- and C-terminal ends, respectively Fig. 4-15, p. 102 Myoglobin - Heme prosthetic group • Myoglobin has eight α-helical regions and no β-sheets - approximately 75% of the residues in myoglobin are found in these α-helices, which are designated A though H • Polar amino acids are on the exterior of the molecule while the interior contains almost exclusively nonpolar amino acids • Two histidine residues are found in the interior; they are involved in interactions with the heme group and bound oxygen • The prosthetic group of myoglobin is called heme, which consists of a metal ion Fe(II) and protoporphyrin IX • The heme group is planar and fits into a hydrophobic pocket in the protein • The heme group is held in position by hydrophobic attractions between the porphyrin ring and the nonpolar side chains of the protein Myoglobin - Heme prosthetic group • The porphyrin part consists of four five-membered rings based on the pyrrole structure; these four rings are linked by bridging methine (-CH=) groups to form a square planar structure Fig. 4-16a, p. 103 Myoglobin - Heme prosthetic group • The Fe(II) metal ion has six coordination sites, and it forms six metal-ion complexation bonds • Four of the six sites are occupied by the N atoms of the four pyrrole-type rings Fig. 4-16b, p. 103 Myoglobin - Heme prosthetic group • The fifth coordination site of Fe(II) is occupied by one of the N atoms of the imidazole side chain of histidine residue F8 (eight residue in helical segment F), • The oxygen is bound at the sixth coordination site of the Fe(II) • The other histidine residue E7 lies on the same side of the heme group as the bound oxygen, and controls the access of oxygen to the hydrophobic pocket Fig. 4-17, p. 104 Myoglobin - CO and O2 binding • More than one molecule can bind to heme: the affinity of free heme for carbon monoxide (CO) is 25,000 times greater than its affinity for O2 • However, when CO is forced to bind at an angle in myoglobin because of the steric block by His E7, its advantage over oxygen drops by several orders of magnitude • This protects against the possibility that traces of CO produced during metabolism would occupy all the oxygen-binding sites of the heme • Nevertheless, CO is a potent poison in larger quantities because of its effects on O2 binding to hemoglobin • It is also important to note that although our metabolism requires that hemoglobin and myoglobin bind oxygen, it would be equally disastrous if the heme never let oxygen go Myoglobin - CO and O2 binding • Oxygen and carbon monoxide binding to the heme group of myoglobin - the presence of the E7 histidine forces a 120° angle to the O2 or CO Fig. 4-18, p. 105 Summary 4-4, p. 106 Quaternary (4°) Structure of Proteins • Quaternary structure is the final level of protein structure and pertains to proteins that consist of more than one polypeptide chain - each chain is called a subunit, the number of which can range from two to more than a dozen • The chains may be identical (homo-) or different (hetero-) • Commonly occurring examples are dimers (2), trimers (3), and tetramers (4), of polypeptide chains • These polypeptide chains interact with one another noncovalently via electrostatic attractions, hydrogen bonds, and hydrophobic interactions • As a result of these noncovalent interactions, subtle changes in structure at one site on a protein molecule may cause drastic changes in properties at a distant site - proteins that exhibit this property are called allosteric • A classic illustration of the quaternary structure and its effect on protein properties is a comparison of hemoglobin, an allosteric protein, with myoglobin, which consists of a single polypeptide chain Hemoglobin • Hemoglobin is a tetramer, consisting of four polypeptide chains, two α-chains and two β-chains - in oligomeric proteins, the types of polypeptide chains are designated with Greek letters • The two α-chains of hemoglobin are identical, as are the two β-chains • The overall structure of hemoglobin is designated as α2 β2 - a heterotetramer • Both the α-chains and the β-chains of hemoglobin are similar to myoglobin - the α-chain is 141 residues long, the β-chain is 146 residues long (myoglobin is 153 residues long) • Many of the amino acids of the α-chain, the β-chain, and myoglobin are homologous - that is, the same amino acid residues are in the same positions • The heme group is also the same in myoglobin and hemoglobin • Myoglobin binds one O2 molecule while hemoglobin binds four • Both hemoglobin and myoglobin bind O2 reversibly, but the binding of O2 to hemoglobin exhibits positive cooperativity, whereas O2 binding to myoglobin does not Hemoglobin • The overall structure of hemoglobin is that of a heterotetramer and is designated as α2β2 • Both the α-chains and the β-chains of hemoglobin are similar to myoglobin - and all four polypeptide chains can bind O2 through the heme group Fig. 4-21, p. 107 Hemoglobin - Positive Cooperativity • O2 binding by hemoglobin exhibits positive cooperativity - this means that the binding of one O2 molecule stimulates the binding of another • Above is a graph of the O2-binding properties of myoglobin and hemoglobin - the O2-binding curve of myoglobin is hyperbolic while that of hemoglobin is sigmoidal • The shape of the hemoglobin binding curve indicates that the binding of the first O2 molecule facilitates the binding of the second and so on - cooperative binding • Important to note that in spite of cooperative binding, myoglobin has a higher O2 binding capacity than hemoglobin Fig. 4-22, p. 107 How does hemoglobin work? • The two different types of behavior exhibited by myoglobin and hemoglobin are related to the functions of these proteins • Myoglobin has the primary function of O2 storage in the muscles - it must bind strongly to O2 at low pressures - at 1 torr partial pressure of O2 myoglobin is 50% saturated • The primary function of hemoglobin is O2 transport - it must be able to both bind strongly to O2 and release oxygen easily, depending on the conditions • In the alveoli of the lungs the O2 pressure is 100 torr - at this pressure hemoglobin is 100% saturated with O2 • In the capillaries of active muscles the O2 pressure is 20 torr - at this pressure hemoglobin is <50% saturated with O2 - in other words hemoglobin gives up O2 easily in capillaries, where the need for O2 is great • Structural changes during binding of small molecules are characteristic of allosteric proteins such as hemoglobin • Hemoglobin has different quaternary structures in the bound (oxygenated) and unbound (deoxygenated) forms Hemoglobin • Hemoglobin has different quaternary structures in the bound (oxygenated) and unbound (deoxygenated) forms • In deoxygenated hemoglobin (a) the two βsubunits are much farther apart, compared with oxygenated hemoglobin (b) - thus, there is much less room at the center of oxyhemoglobin • This change is so marked that the two forms of hemoglobin have different crystal structures Fig. 4-23, p. 109 Conformational changes that accompany hemoglobin function • Other ligands are involved in cooperative effects when O2 binds to hemoglobin, e.g. both H+ and CO2, which themselves bind to hemoglobin, and affect the affinity of hemoglobin for O2 by altering the proteins 3D structure in subtle but important ways • The effect of H+ is called the Bohr effect, after its discoverer, Christian Bohr • In contrast, the O2-binding ability of myoglobin is not affected by the presence of H+ or CO2 • An increase in the concentration of H+ (i.e. a lowering of the pH) reduces the affinity of hemoglobin for O2: Increasing the [H+] causes the protonation of several amino acids, including H146 of the β-chains • The protonated H146 residue is attracted to and stabilized by a salt bridge (ionic bond) to D94 - this favors the deoxygenated form of hemoglobin • Actively metabolizing tissue, which requires O2, releases H+, thus acidifying the local environment • Hemoglobin has a lower affinity for O2 under these conditions and O2 is released where needed Hemoglobin - The Bohr Effect • In actively metabolizing tissue, hemoglobin releases O2-and binds both CO2 and H+ • In the alveoli of the lungs, hemoglobin releases CO2 and H+ and binds to O2 Fig. 4-24, p. 110 Hemoglobin - The Bohr Effect • The O2 saturation curves for both myoglobin and hemoglobin • The affinity of myoglobin for O2 is unaffected by pH • However, at lower pH, the affinity of hemoglobin for O2 is significantly reduced Fig. 4-25, p. 110 Hemoglobin - CO2 Binding • Large amounts of CO2 are produced during metabolism • The CO2 in turn forms carbonic acid, H2CO3 - pKa = 6.35 • The normal pH of blood is 7.4 - as a result, about 90% of dissolved CO2 will be present as the bicarbonate ion, HCO3-, releasing H+ • The presence of larger amounts of H+ as a result of CO2 production favors the quaternary structure of deoxygenated hemoglobin - hence the affinity of hemoglobin for O2 is lowered • The HCO3- is transported to the lungs, where it combines with the H+ released when hemoglobin is oxygenated, producing H2CO3 • In turn H2CO3 liberates CO2, which is then exhaled • In the presence of large amounts of H+ and CO2, as in respiring tissue, hemoglobin releases O2 • The presence of large amounts of O2 in the lungs reverses the process, causing hemoglobin to bind O2 - oxygenated hemoglobin can then transport O2 to the tissues Hemoglobin - 2,3-bisphosphoglycerate • Hemoglobin in blood is also bound by another ligand 2,3-bisphosphoglycerate (BPG), with drastic effects on its O2-binding capacity • BPG binds to hemoglobin is the central pocket between all four subunits - only one molecule of BPG binds per hemoglobin heterotetramer • The binding of BPG to hemoglobin is electrostatic; between the negative charges of the BPG and positive charges on several amino acid side chains • In the presence of BPG the partial pressure at which 50% of hemoglobin is bound to O2 is 26 torr - if BPG were not present the O2-binding capacity would be much higher, and little O2 would be released in the capillaries • Stripped hemoglobin, which can be isolated from blood and from which the endogenous BPG has been removed, displays this behavior Hemoglobin - 2,3-bisphosphoglycerate (You do not need to remember the structure of BPG) Fig. 4-26, p. 110 Hemoglobin - 2,3-bisphosphoglycerate Hemoglobin - BPG binding • A comparison of the O2 binding properties of hemoglobin in the absence and presence of BPG • The presence of BPG markedly decreases the affinity of hemoglobin for O2 Fig. 4-28, p. 111 Fetal hemoglobin (Hb F) • BPG also plays an integral role in supplying the growing fetus with O2 • The fetus obtains O2 from the mothers bloodstream via the placenta • Fetal hemoglobin (Hb F) has a higher affinity for O2 than does maternal/adult hemoglobin (Hb A), for two major reasons: • The first is the presence of two different polypeptide chains - the subunit structure of Hb F is α2γ2, where the β chains of adult hemoglobin (Hb A) have been replaced by the γ chains, which are similar but not identical in structure • The second feature is that Hb F binds less strongly to BPG than does Hb A - in the β chain of adult hemoglobin, H143 makes a salt bridge to BPG - in Hb F the γ chain has a H143 S143 substitution • This change of a positively charged amino acid for a neutral one diminishes the number of contacts between the Hb F and BPG, effectively reducing the allosteric effect enough to give Hb F a higher binding curve than Hb A Hb F versus Hb A • A comparison of the O2 binding capacity of Hb F and Hb A • Hb F binds less strongly to BPG and, consequently, has a greater binding affinity for O2 than does Hb A (maternal hemoglobin) Fig. 4-29, p. 111 Sickle Cell Hemoglobin (Hb S) • Another type of hemoglobin that has been extensively studied is sickle-cell anemia hemoglobin (Hb S) • In Hb S, the β-chains have a single amino acid substitution of a glutamic acid to valine (E V) • This substitution of a nonpolar amino acid for a polar one causes the characteristic effects of this disease • The nonpolar amino acid is on the surface of the β-subunit and leads to aggregation of the molecules through nonpolar interactions Hb S - Sickle cell anemia Summary 4-5, p. 112 ...
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This note was uploaded on 12/05/2009 for the course BCH 311 taught by Professor Howlett during the Fall '08 term at Rhode Island.

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