Protein Structure and Function.pdf

Protein Structure and Function Presented by Dr. Kherie Rowe Professor of Biochemistry [email protected] Reading: Lippincott Reviews in Biochemistry, 8th Ed., Chapters 1, 2 and 4....

Protein Structure and Function Presented by Dr. Kherie Rowe Professor of Biochemistry [email protected] Reading: Lippincott Reviews in Biochemistry, 8th Ed., Chapters 1, 2 and 4. 1 1 Learning Objectives 1. Explain what determines the primary structure of a protein. 2. Explain the nature of the amino acids including the following concepts: chirality, stereoisomers, and functional groups. 3. Given the 1 letter or 3 letter code for an amino acid, provide the full name for that amino acid and vice versa. 4. Given the name or code for an amino acid, identify the functional group that is on its side chain. 5. Given a pKa, determine at what pH the amino acid or chemical will buffer best. 6. Given a primary structure of a peptide, determine the net charge at physiological pH. 7. Compare and contrast the kinds of bonds involved in stabilizing or determining primary structure, secondary structure and tertiary structure. 8. Explain the physiological role that the following proteins have in the body: hemoglobin, myoglobin, collagen, superoxide dismutase. 2 2 Proteins Proteins, the workhorses of the Functions cell, are the most abundant and Source of Energy functionally versatile of the cellular macromolecules. Source of amino acids to rebuild Are the product of information new proteins carried on the genome Enzymes Biopolymers of a-amino acids Movement joined together by peptide bonds Communication (and disulfide bridges). Transport Only 20 amino acids are used – but some are modified post- Structural integrity translationally etc. 3 3  Amino Acids are the Building Blocks of Proteins 4 Chirality: all amino acids have D and L-chirality except glycine. 4 Amino Acid Categories Amino Acids can be categorized on the basis of their side chain properties: o Size o Hydrophobicity o Polarity o Charge o Aromaticity Some AAs have specific effects on secondary structure: Pro, Gly 5 5 Amino Acid Three- And One- Letter Symbols 6 6 Functional Groups The functional groups of proteins are usually found in their amino acid side chains. Hydroxyl – Ser, Thr, Tyr All of these can be phosphorylated on their side chains. Methyl/aliphatic – Ala, Val, Ile, Leu, Met Met can donate its methyl group to other molecules that need a carbon atom to make bigger molecules. Methyls and larger aliphatic groups are hydrophobic and contribute strongly to folding. Carboxy – Asp, Glu Amino – Lys Sulfhydryl – Cys Cys can form disulfide bonds, which can help stabilize tertiary or the quaternary structure of proteins, or serve as redox reaction. 7 7 Characteristics of the Peptide Bond 1. No bond rotation Peptides are linear chains of amino acids. The peptide, amide bond has double-bond character, and does not rotate: This is a key aspect of higher order structure. There are two angles of rotation associated with each amino acid: N-Ca and Ca-CO. These are restricted by the R groups. 8 8 Amino Acids: The Building Blocks of Proteins Amino acid chains – typically drawn in the direction of amino-group to carboxy-group Backbone atoms include nitrogen, alpha-carbon, and carboxylate group, with the associated hydrogen. Side chains, also called R-groups, sprout from the alpha-carbon. These side-chain functional groups include alcohols, thiols, thioethers, carboxylic acids, carboxamides, and a variety of basic groups. When combined in various sequences, this array of functional groups accounts for the broad spectrum of protein function. For instance, the chemical reactivity associated with these groups is essential to the function of enzymes, the proteins that catalyze specific chemical reactions in biological systems. 9 9 Characteristics of the Peptide Bond 2. Generally, a trans-bond Because of the steric interference of the R-groups when in the cis-position 10 10 Characteristics of the Peptide Bond 3. Uncharged but polar The -C=O and the -NH group of the peptide bond do not ionize but do participate in hydrogen bonding. 11 11 Each Peptide Bond Can Form Two Hydrogen Bonds 12 12  Proline is the Exception! 13 Hydrogen bond: a weak bond between two molecules resulting from an electrostatic attraction between a proton in one molecule and an electronegative atom in the other. 13 Four Levels of Protein Structure 1° Primary structure 2° Secondary structure 3° Tertiary structure 4° Quaternary structure 14 14 I. Primary Structure The order in which amino acids are joined together Can include the location of any disulfide bonds Polypeptide backbone Stabilized by covalent bonds (not by non-covalent bonds) 15 15 II. Secondary Structure Three Kinds: A. Alpha helix B. Beta sheets C. Beta turns/ random coil Defines the steric relationship between amino acids that are close to each other in the primary amino acid sequence. Brought about by linking the carbonyl and amide groups of the peptide bonds by means of hydrogen bonds (H-bonds). 16 16 A. α-Helix Forms spontaneously A rigid, rod-like coiled structure R groups pointed outwards The lowest energy and most stable conformation for a polypeptide chain Hydrogen bonds are +/- parallel to the axis of the helix Stability arises from the formation of the maximum possible number of H- bonds 17 17 Hydrogen-Bonding In α-Helix Each peptide bond forms 2 H-bonds: one to the peptide bond of the 4th residue above and one to the peptide bond of the 4th residue below 3.6 amino acid residues per 360° turn (5.4 Å); 13 atoms/turn; 1.5 Å rise/residue R groups extend outward Right-handed 18 18 Destabilization of the α-Helix Proline is an α-helix breaker. It interrupts the helical structure because there is no -H on the amide N and because the ring exerts geometric constraint. Bulky hydrophobic residues disrupt because of steric problems. Charged residues disrupt because of charge repulsion/attraction. 19 19  Composed of 2 or more peptide chains or segments b-Pleated Sheet of polypeptide chains that are arranged either parallel or anti-parallel to each other 20 20 The a-carbon and its R group side chain alternate slightly above and slightly below the plane of the main chain of the polypeptide (ruffled or pleated appearance). May have 2 - 15 strands. 21 21 C. β-Turn (β-bends, Reverse Turn) Most common form of turns They cause a change in direction of the polypeptide chain Formed by a hydrogen bond from one main chain carbonyl oxygen to the main chain N-H group 3 residues along the chain Pro-Gly sequence is common in beta bends Glycine – has the smallest R group Proline – causes a kink in the polypeptide chain 22 22  Secondary Structure Examples Rhodopsin: Membrane Protein, alpha- Beta-barrel Porin: A transmembrane IgG: Globular, all beta sheet Myoglobin: Globular, Alpha-helical helical protein 23 Two alpha-helical proteins, myoglobin and rhodopsin. Rhodopsin is a transmembrane protein, whereas hemoglobin is completely soluble. Examples of beta-sheet proteins – Beta barrels. This is an example of a porin – a transmembrane protein with a large pore. Other beta-barrels exist in soluble proteins. The IgG fold is a fold (or domain) seen in many proteins but was first seen in crystal structures of immunoglobulins. Note that IgG is held together with disulfide bonds into its quaternary structure. Each domain in an antibody molecule has a similar structure of two beta sheets packed tightly against each other in a compressed antiparallel beta barrel. This conserved structure is termed the immunoglobulin fold Note: Beta pleated sheet structures can be extensive and do not have to interact with nearby residues. This differentiates them from alpha-helices where hydrogen bonding is obligatorily to residues near in the sequence. 23 An Enzyme: Cu, Zn-Superoxide Dismutase Superoxide dismutase catalyzes the dismutation of the superoxide (O2.-) radical into either molecular oxygen, O2 or to hydrogen peroxide, H2O2. Superoxide dismutase has a dimeric structure, with a monomer molecular mass of 16,000 Da. Cu and Zn are cofactors. Each subunit consists of eight antiparallel β-sheets called a β-barrel structure. 24 Mutations in the first SOD enzyme (SOD1) can cause familial amyotrophic lateral sclerosis (ALS, a form of motor neuron disease). 24  Tertiary Structure – Types of Bonds 1. Hydrogen bonds 2. Hydrophobic interactions 3. Van der Waals interactions 4. Ionic bonds (rare) 5. Disulfide bridges 25 Hydrogen bonds occur in inorganic molecules, such as water, and organic molecules, such as DNA and proteins. Van der Waals attractions can occur between any two or more molecules and are dependent on slight fluctuations of the electron densities. Hydrophobic interactions and Van der Waal's forces are caused by different interactions. Hydrophobic interactions occur when nonpolar (hydrophobic) amino acids associate with each other and cluster together to hide from water, usually on the inside of a protein. The Raven textbook describes Van der Waal's forces as "Weak attractions between atoms due to oppositely polarized electron clouds." Prof. Mehrtens simplifies the definition by describing Van der Waal's forces as occurring when molecules are so close that the proton of one atom attracts another molecule's electrons. This occurs at the "Van der Waal's radius," which is a specific distance. They are weak and transiet, but many of them together can be stabilizing. Hydrophobic interactions are also weak, but stronger than Van der Waal's forces. 25 Disulfide Bonds A covalent linkage between 2 cysteine residues to form a cystine residue 26 26 Amino Acid Sequence Specifies 3D Structure The 20 amino acids can be linked in combinations specific for a given protein. Long chains of amino acids fold in a pattern dependent on the exact order of amino acids. A genetic (point) mutation leading to one incorrect amino acid substitution in a protein comprising thousands of amino acids can result in that protein having a different shape and little or even no biological activity or new, unexpected properties. 27 27 Defective Enzymes – Hunter Disease A mutation in the gene for iduronate- 2-sulfatase changes a single amino acid Arg468Gln. The mutated enzyme is inactive and heparan - and dermatan sulfates cannot be broken down. Causes severe mucopolysaccharidosis – previously fatal, but a recombinant enzyme “Elaprase” is now available. 28 28 Modular Protein Domains A compact, independently folding unit in a protein that often performs a specific function. They only occur in large proteins (> 200 residues) 29 29  Quaternary Structure: Hemoglobin vs Myoglobin Myoglobin: Monomeric protein Hemoglobin: A tetrameric protein This is a single poly-peptide protein. This is made from 4 poly-peptide chains; 2a, 2b. It has a single domain (globin fold) Each chain has a single domain (globin fold). It has primary, secondary and tertiary structure. It has primary, secondary and tertiary structure. It has quaternary structure It does not have quaternary structure. because 4 polypeptides are assembled into a functional whole protein. Figures from http://slideplayer.com/slide/4462886/ 30 30 Sickle Cell Hemoglobin A single point mutation in the gene coding for b-globin (GAG to GTG) brings about a single amino acid change in the protein – glutamate 6, an acidic and hydrophilic amino acid, is replaced by valine, a hydrophobic one. 31 31 Another Example of Quaternary Structure Fatty acid synthase complex – an obligatory homodimer Heptahelical receptor - associated G-protein – an obligatory heterotrimer 32 32 Fibrous Proteins Long cylindrical (rod- or rope-like) shape is common. Low solubility in water With a structural rather than a dynamic role in the cell or organism Often contain larger amounts of regular secondary structure than globular proteins Examples are collagen, elastin, keratin In their purest form, fibrous proteins forgo true tertiary structure for very strong secondary and quaternary structures 33 33 Collagen Most abundant protein in the human body Comprises about 25% of total mammalian protein Present in all tissues and organs Provides the framework that gives the tissues their form and structural strength Types and organization are dictated by the structural role played in a particular organ Composed of 3 left-handed polypeptide helices twisted around each other to form a right-handed super-helix – Triple Helix Stabilized by H-bonds between individual polypeptide chains Helices have approximately 3 residues per turn Amino acid sequence primarily consists of large numbers of repeating triplets with the sequence of Gly-X-Y X is frequently proline and Y is often hydroxyproline or hydroxylysine Repeating structure is absolute requirement for the formation of the Triple Helix 34 34 Collagen 35 35 Why Gly-Pro-HyP/HLy? Glycine – the only residue with an R-group small enough to fit within the central core of the triple helix and small enough to allow close proximity of chains to each other Proline and Hydroxyproline confer rigidity because their ring is conformationally inflexible. Hydroxyproline is involved in H-bond formation that helps to stabilize the triple helix. Hydroxylysine is site of attachment of carbohydrate moieties, most commonly galactose and glucose. 36 36 Post-Translational Modification – Examples There are many possible modifications (hundreds) Phosphorylation is one example that often serves a regulatory role. Other modifications can serve other roles. 37 37 Acid Base Chemistry: Henderson-Hasselbalch Equation HA ↔ A + H+ [ +][ ] K = = equilibrium constant for the dissociation of a weak acid. [ ] Higher Ka the greater the tendency to dissociate a proton pH = ― log[H+] [ ] pH = pK + log [ ] pKa is the pH where the acid and conjugate base forms of a compound are equal in concentration 38 38  The Henderson–Hasselbalch equation describes the relation of pH to the acid (AH) and conjugate base forms (A-) of a titratable group, in biological systems. (pKa, the negative log of the acid dissociation [𝐴 ] 𝑝𝐻 = 𝑝𝐾 + 𝑙𝑜𝑔 constant, is used) [𝐴𝐻] The equation can be used to do the following: pKa is the pH where the acid and o Estimate the pH of a buffer solution conjugate base forms of a o Find the equilibrium pH in acid-base compound are equal in reactions concentration. o Calculate the isoelectric point of proteins 39 39  Henderson-Hasselbalch Equation Acidic residues: pKa [𝐴 ] Asp 3.86 𝑝𝐻 = 𝑝𝐾 + 𝑙𝑜𝑔 Glu 4.25 [𝐴𝐻] The amino and carboxylic acid pKa values on each amino acids are only Basic Residues: relevant for the two residues at the ends. The rest are in amide bonds and not tritratable. For most large proteins, the end aminoLys acids are a10.5 minor contribution to the over-all charge properties. Arg 12.5 -COOH -COO- Fraction AH pKa of a terminal Amino group is about 9 to 10 for individual amino acids. pKa of a terminal Carboxylate group is about 2.0, for individual amino acids. More important are the side chain pKa values – they His 6.0 determine the overall charge and solubility of a protein. Cysteine Cys of pH. The graph shows the fraction of AH still present as a function 8.3It is a titration curve, as you add increasing amounts of base. 40 pH: 40 41 41 42 42 Buffering Capacity The pKa is the best buffering point of a pH buffer. The buffering capacity of a solution is the change in pH for the amount of acid (or base) added. The less change in pH, the better the buffering. Imagine a titration experiment where you have a solution of a buffering compound (e.g., Histidine or phosphate), and you then slowly add acid (or base). Basically, at the pKa, the titratable compound soaks up the added acid or base, and the pH changes little. Once the pH moves away from the pKa, the compound is fully titrated to its acid or base form, and then any further added acid or base, will give a large change in H+ concentration; it no longer has buffering capacity. 43 43 Isoelectric Point The isoelectric point, pI, is the pH value where the molecule is net neutral (net charge is zero). The pI value is less important for individual amino acids than the protein as a whole. The pH that proteins are net neutral at, often makes them less soluble, and they may precipitate out of solution. Most proteins (but by no means all), are more acidic, meaning that they have a pI of less than 7; typical values are 5-6. However, the pI values can range up to basic values near the pKa of lysine. This just depends on the number of acidic and basic residues in the protein. 44 44 Net Protein Charge Example: The following polypeptide would have the following charges, at pH 7.4, on the side chains of the amino acids in the polypeptide: +1 0 -1 0 +1 +1 0 -1 -1 0 0 -1 -1 +NH 3-Val-Glu-Pro-Arg-Lys-Ile-Asp-Glu-Gln-Thr-Glu-COO - Note that the N-terminus is always a +1 at pH 7.4 and the C-terminus is always a -1 at pH 7.4, unless they are modified. The net charge on this peptide is -2 = 5(-1 charges) + 3(+1 charges). 45 45 Key Points You have to know the Henderson-Hasselbalch equation. Know what a pKa is and how it relates to buffering capacity and a titration curve Know what the charge would be on the important side chains at normal and acidic and basic pH values Know what buffering capacity means and where a compound buffers best Know what an Isoelectric Point (pI) is 46 46 Summary Amino acids constitute proteins according to the genetic code. Amino acids contain an α-carbon that is covalently linked to an amino group, a carboxylic acid group, and a distinctive R group, or side chain group. Only the L-amino acid stereoisomers are used by the ribosome. Proteins are composed of 20 amino acids, designated by a 3-letter code or a 1-letter code. The primary structure of a protein is its sequence of amino acids. The secondary structure of a protein typically defined through hydrogen bond interactions and can take 3 forms: alpha helix, a beta-pleated sheet, and a random coil. The tertiary structure of a protein is held together by several kinds of interactions: Hydrophobic interactions, van der Waals interactions, ionic bonds, disulfide bridges. The Henderson–Hasselbalch equation can be used to determine the charge on side chains. The pKa is the pH at which a functional group will buffer the best. The overall charge of a polypeptide/protein is the sum total of all the + and – charges. 47 47