Protein Structure and Function PDF

CHAPTER 3 Protein Structure and Function Outline 3.1 Proteins and amino acids 3.2 Proteins: primary structure 3.3 Proteins: secondary, tertiary, and quaternary structures 3.4 Enzymes 3.5 Factors affecting enzyme activity 3.1 Proteins and amino acids Derived from the Greek word...

CHAPTER 3 Protein Structure and Function Outline 3.1 Proteins and amino acids 3.2 Proteins: primary structure 3.3 Proteins: secondary, tertiary, and quaternary structures 3.4 Enzymes 3.5 Factors affecting enzyme activity 3.1 Proteins and amino acids Derived from the Greek word proteios meaning “first” to indicate the central roles that proteins play in living organisms Proteins are the indispensable agents of biological function, and amino acids are the building blocks of proteins. The stunning diversity of the thousands of proteins found in nature arises from the intrinsic properties of only 20 commonly occurring amino acids. These features include: (1) the capacity to polymerize (2) novel acid–base properties (3) varied structure and chemical functionality in the amino acid side chains, and (4) Chirality (or handedness, means that an object or molecule cannot be superimposed on its mirror image by any translations or rotations) Functions of proteins (1) Enzymes are biological catalysts. Majority of the enzymes that have been studied are proteins. Reactions that would take days or weeks or require extremely high temperatures without enzymes are completed in an instant. For example, the digestive enzymes pepsin, trypsin, and chymotrypsin break down proteins in our diet so that subunits can be absorbed for use by our cells. Without enzymes, the body cannot absorb nutrients. Functions of proteins (2) Defense proteins include antibodies (also called immunoglobulins) which are specific protein molecules produced by specialized cells of the immune system in response to foreign antigens. These foreign invaders include bacteria and viruses that infect the body. Each antibody has regions that precisely fit and bind to a single antigen. It helps to end the infection by binding to the antigen and helping to destroy it or remove it from the body. Functions of proteins (3) Transport proteins carry materials from one place to another in the body. The protein transferrin transports iron from the liver to the bone marrow, where it is used to synthesize the heme group for hemoglobin. Transferrin is synthesized and Transferrin-Iron secreted into serum mostly by the liver. Synthesis of transferrin is regulated by iron. Functions of proteins Iron alone is extremely reactive. If iron is not bound by specific serum carriers and/or storage proteins within the body, it can viciously interact with vascular, cellular, and subcellular structures. Therefore, after absorption, it is bound to the plasma protein transferrin (TF) for safe transport. Iron uptake from transferrin involves the binding of transferrin to the transferrin receptor, internalization of transferrin within an endocytic vesicle by receptor-mediated endocytosis and the release of iron from the protein by a decrease in endosomal pH (4.0-6.5). A reduction in pH induces the release of iron from transferrin in a process that involves a conformational change in the protein from a closed to an open form due to pH change. Functions of proteins (3) Transport proteins carry materials from one place to another in the body. The proteins hemoglobin and myoglobin are responsible for transport and storage of oxygen in higher organisms, respectively. Functions of proteins (4) Regulatory proteins control many aspects of cell function, including metabolism and reproduction. We can function only within a limited set of conditions. For life to exist, body temperature, the pH of the blood, and blood glucose levels must be carefully regulated. Functions of proteins Many of the hormones that regulate body function, such as insulin and glucagon, are proteins. https://www.healthgrades.com/right-care/endocrinology-and-metabolism/glucagon http://www.danzettwoch.com/howthatworks/images/zettwoch_adrenaline.jpg http://www.danzettwoch.com/howthatworks/images/zettwoch_adrenaline.jpg Functions of proteins https://healthnews.com/beauty/skin-care/keratin-how-to-know-if-my-levels-are-low/ (5) Structural proteins provide mechanical support to large animals and provide them with their outer coverings. Our hair and fingernails are largely composed of the protein keratin. Other proteins provide mechanical strength for our bones, tendons, and skin. Without such support, large, multicellular organisms like ourselves could not exist. https://a-z-animals.com/reference/keratin/ Epidermolysis bullosa and the “butterfly babies” People with EB have genetic mutations resulting to abnormal structural proteins in the skin. Epidermolysis bullosa and the “butterfly babies” Recessive Dystrophic EB is the most severe, chronic type of EB. Blistering begins at birth or shortly afterwards. Much of the skin is covered in blisters and there is extensive internal blistering. Children can develop deformities caused by the recurrent scarring of the fingers and toes (pseudosyndactyly) and the hands and arms become fixed in stiff positions (contractures). It is painfully difficult for a child with recessive Dystrophic EB to ingest food due to the internal blistering that occurs in the mouth, esophagus, and gastrointestinal tract. https://www.stanfordchildrens.org/en/service/dermatology/epidermolysis-bullosa/faq Functions of proteins (6) Movement proteins are necessary for all forms of movement. Our muscles, including that most important muscle, the heart, contract and expand through the interaction of actin and myosin proteins. Sperm can swim because they have long flagella made up of proteins. Functions of proteins Functions of proteins (7) Nutrient proteins serve as sources of amino acids for embryos or infants. Egg albumen and casein in milk are examples of nutrient storage proteins. Amino acids As the name implies, these compounds contain both an amine and an acid Hundreds are formed both naturally and synthetically; Only 20 are common in nature; All 20 are α-amino acids (α means the amine is adjacent to the carboxylate group) 19 out of the 20 are stereoisomers (Glycine does not have a chiral carbon)  Amino acids: Stereoisomers The α-carbon of amino acids is chiral Glycine is the only common amino acid that is not chiral (a chiral molecule is non-superposable to its mirror image due to the presence of an asymmetric carbon atom) https://chem.libretexts.org/Courses/Kenyon_College/Chemistry_231_and_232_-_Kenyon_College_%28Getzler_Hofferberth_and_Hunsen%29/5%3A_Stereoisomers/5.1%3A_Chiral__Molecules Classes of Amino Acids All differences between amino acids depend upon their side-chain R groups Amino acids form classes based on the polarity of their side chains - Nonpolar class has hydrophobic R groups - Polar, neutral have a high affinity for water, but are not ionic at pH - Polar acidic (Negatively charged) have ionized carboxyl groups in their side chains - Polar basic (Positively charged) are basic as the side chain reacts with water to release a hydroxide anion Essential amino acids All amino acids are essential for normal tissue growth and development The term “essential amino acids” is reserved for those amino acids that must be supplied in the diet for proper growth and development. They must be supplied in the diet because either that there are no biochemical pathways available for their synthesis or the available pathway do not provide adequate amounts for proper nutrition. “PVT. TIM HALL” – Phe, Val, Thr, Trp, Ile, Met, His, Arg, Leu, Lys (His and Arg are semi-essential; they not synthesized in sufficient quantities during infancy stage) Amino acids as acids and bases α-carbon is attached to a: Carboxyl group (̶ COOH) Amino group ( ̶ NH2) At physiologic pH the amino acid has: Carboxyl group in –COO- Amino group in –NH3+ Neutral molecule with equal number of + and – charges is called a zwitterion (from the German word “zwitter” which means “hybrid” or “hermaphrodite”) Amino acids as acids and bases Amino acids are white crystalline solids with high melting points and high water solubilities The two charged groups, the basic amino group and the carboxylic acid, at the two ends lead to internal proton transfer, forming zwitterions By changing the pH you can affect the net charge on the zwitterions The pH point at which there is no net charge on the zwitterions is called the isoelectric point (pI) Amino Acids: Isoelectric Point The isoelectric point of an amino acid is the pH at which it bears no net charge. Although they are often drawn in their neutral form, in aqueous solution at pH 7 (physiological pH) their structure is more accurately described as a “zwitterion” (an internal salt) – the product of an acid-base reaction between the carboxylic acid and the amine. In practice, the charges on an amino acid only balance out to zero at one specific pH value, called the isoelectric point pI. At this pH, the amino acid will not migrate in an applied electric field. Amino Acids: Isoelectric Point Amino Acids: Isoelectric Point At pH values below and above the isoelectric point, the molecule will bear a net positive or net negative charge, respectively. It’s possible to test this by applying a sample of the amino acid to specially treated paper or gel and applying an electric field at different pH values – a technique known as electrophoresis. A molecule with a net charge of zero will not migrate in an electric field, whereas one bearing a positive (+) or negative (-) charge will migrate towards the cathode (-) or anode (+), respectively.  Net charge 1+ Net charge 0 Net charge 1- At pH 5.96 (pI of Valine) there will be a range of pH values where the one form dominates  +1 0 -1 Based on the Henderson-Hasselbalch equation, when [HA] = [A-], pH = pKa. Thus, the two points A and B on the sketch above correspond to the pKa values of the amino acid. Sample Problem: The pKa values for glycine are 2.34 (for the carboxylic acid) and 9.60 (for the ammonium). 2.34 9.60 Net charge: (+1) (0) (-1) Sample Problem: pI of amino acids with acidic side chains Sample Problem: pI of amino acids with basic side chains Sample Problem: Amino acids as acids and bases Amino acids as acids and bases 16.3 Draw the structure of alanine at pH=1, 6.02, and 12. pI of Ala is 6.02. Electrophoresis and charged amino acids Electrophoresis is an analytical method for identifying amino acids by observing their migration as a function of pH under an applied electric field gradient. - paper or gel (polyacrylamide) - Saturated with buffer solution - Solution of unknown amino AA is placed at the center of paper - Electrodes are connected to the ends of the paper and electric current is allowed to pass through the sol’n - Ninhydrin is sprayed (reacts with AAs to produce colored products, green/blue) Electrophoresis and charged amino acids The amino acid carries a POSITIVE CHARGE at pHpI and migrates to the POSITIVE ELECTRODE (anode) Follow-up Problem: 1. To which electrode will alanine migrate in electrophoresis at pH=1, 6.02, and 12? 2. An unknown, containing some combination of ALANINE, LYSINE, or ASPARTIC ACID, is subjected to paper electrophoresis at pH=6.01. Ninhydrin treatment shows some amino acid at the negative electrode and some amino acid has not moved from the center. No amino acid is found at the positive electrode. What AA(s) is (are) in the unknown? Ala – 6.01 Lys – 9.74 Asp – 2.77 pH (6.01) > pI (2.77); pH (6.01) = pI (6.01); pH (6.01) < pI (9.74); net negative charge; Zero net charge; Net positive charge; Migrates towards (+) Does not move from migrates towards (-) electrode (absent) the center (present) electrode (present) a. Ion-exchange chromatography b. Size-exclusion chromatography c. affinity chromatography The Peptide Bond Proteins are linear polymers of L-α-amino acids Carboxyl group of one amino acid is linked to the amino group of another amino acid Linkage is an amide bond or peptide linkage This reaction is a dehydration reaction as water is released The Peptide Bond Condensing or dehydrating two amino acids produces a dipeptide Amino acid with a free a-NH3+ group is the amino terminal amino acid, N-terminal for short Amino acid with a free –COO ̶ group is the carboxyl terminal amino acid, C-terminal for short Amino acid structures are written with the N-terminal on the left The Peptide Bond amino acids are polymerized into peptides and proteins; in general, protein is used for molecules composed of over 50 amino acids; peptide is used for molecules of less than 50 amino acids Naming Peptides The far right AA residue retains the name of the amino acid All AAs (except TRYPTOPHAN) → -ine or –ic acid is replaced by –yl Tryptophan becomes tryptophanyl Naming Peptides Writing the Structure of Peptides Writing the Structure of Peptides Writing the Structure of Peptides Some examples of small peptides 1. Aspartame (Asp-Phe) Sold under the trade names Nutrasweet and Equal, aspartame is the artificial sweetener used in almost every diet food on the market today. Its caloric content is the same as sucrose but is ~180 times as sweet. Both AAs present in the dipeptide must be in the L- form for the sweet taste to occur; the L-D, D-L, and D-D forms have a bitter taste. Aspartyl-phenylalanine methyl ester Some examples of small peptides 2. Glutathione (Glu-Cys-Gly) The tripeptide, produced by the body itself, is present in significant concentrations in most cells and is of considerable physiological importance as a regulator of oxidation- reduction reactions. It functions as an antioxidant, protecting cellular contents from oxidizing agents such as peroxides and superoxides, which are highly reactive forms of oxygen often generated within a cell. Glu is bonded to Cys through the side- chain carboxyl group rather than through the α-carbon carboxyl group. Some examples of small peptides 4. Enkephalins (Tyr-Gly-Gly-Phe-Leu & Tyr-Gly-Gly-Phe-Met) Enkephalins and endorphins - natural painkillers produced in the body; bind to receptors in the brain to give relief from pain. This effect appears to be responsible for the runner’s high, for the temporary loss of pain when severe injury occurs, and for the analgesic effects of acupuncture. Β-endorphins and runner’s high Some examples of small peptides 4. Enkephalins (Tyr-Gly-Gly-Phe-Leu & Tyr-Gly-Gly-Phe-Met) Oxytocin stimulates uterine contractions in labor and vasopressin is an antidiuretic hormone that regulates blood pressure by adjusting the amount of water reabsorbed by the kidneys. The sickled cells are unable to pass through the small capillaries of the circulatory system, and circulation is hindered. This results in damage to many organs, especially bone and kidney, and can lead to death at an early age. The Primary Structure of Proteins Primary structure is the amino acid sequence of the polypeptide chain - A result of covalent bonding between the amino acids – the peptide bonds Each protein has a different primary structure with different amino acids in different places along the chain Resonance and rigidity of peptide bonds The peptide bond has a partial double bond character Resonance and rigidity of peptide bonds There is free rotation around only two of the three single bonds of a peptide backbone. https://www.labxchange.org/library/items/lb:LabXchange:b834fc14:html:1 R groups on adjacent amino acids are on opposite sides of the chain because of the rigid peptide bond. R R R R R R R R The Secondary Structure of Proteins When the primary sequence of the polypeptide folds into regularly repeating structures, secondary structure is formed Secondary structure results from hydrogen bonding between the amide hydrogens (N—H) and carbonyl oxygens (C=O) of the peptide bonds Not all regions have a clearly defined secondary structure, some are random or nonregular The Secondary Structure of Proteins: α-Helix Most common type of secondary structure Coiled, helical Important features: - Each amide H and carbonyl O is involved in H bonds locking the helix in place - Carbonyl O links to amide H 4 amino acids away - H bonds are parallel to the long axis of the helix - Helix is right-handed - Repeat distance or pitch is 5.4 angstroms - 3.6 amino acids per turn a-Helix a-Helices in Fibrils Fibrous proteins are arranged in a secondary structure of fibers or sheets with only 1 type of secondary structure Repeated coiling of helices The Secondary Structure of Proteins: β-pleated sheet Second most common secondary structure appears similar to folds of fabric All the carbonyl O and amide H are involved in the H bonds with the chain nearly completely extended The Secondary Structure of Proteins: β-pleated sheet Two possible orientations Parallel if the N-termini are head-to-head Antiparallel if the N-terminus of one chain is aligned with the C-terminus of the other The Tertiary Structure of Proteins The three-dimensional structure, which is distinct from secondary structure is classified as tertiary structure Globular tertiary structure forms spontaneously and is maintained by interactions among the side chains or R groups Tertiary structure defines the biological function of proteins https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/73-translation/protein-structure.html Types of Interactions Maintaining Tertiary Structure Disulfide bridges between two cysteine residues - the only covalent bond, the strongest of the 3o bonds; link chains together and cause chains to twist and bend - a covalent bond between 2 S atoms formed by the oxidation of the –SH groups on two cysteine amino acids - S – S linkage can occur within the same chain (intrachain), or between 2 or more chains (interchain), or both inter- and intra-chain https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/73-translation/protein-structure.html Types of Interactions Maintaining Tertiary Structure Salt bridges (ionic interaction/electrostatic attraction) between ionic side chains –COO– and –NH3+ - some amino acid side chains may contain positively charged groups, others negatively charged groups. If properly positioned these groups may give rise to bonding between different portions of given molecule, or between 2 or more protein chains Hydrogen bonds between polar residue side chains Hydrophobic interactions: two nonpolar groups are attracted by a mutual repulsion of water https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/73-translation/protein-structure.html Interactions Involved in Tertiary Structure https://www.brainkart.com/article/Forces-Involved-in-Tertiary-Structures_27471/ The Quaternary Structure of Proteins The functional form of many proteins is not that of a single polypeptide chain but an aggregate of several globular peptides Quaternary structure: the arrangement of subunits or peptides that form a larger protein Subunit: a polypeptide chain having primary, secondary, and tertiary structural features that is a part of a larger protein Quaternary structure is maintained by the same forces which are active in maintaining tertiary structure https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/73-translation/protein-structure.html https://nationalmaglab.org/about-the-maglab/around-the-lab/what- goes-in-the-magnet/hemoglobin/ Protein Functions Follow Shape Fibrous proteins: Mechanical strength Structural components Movement Globular proteins: Transport Regulatory Enzymes https://www.savemyexams.com/a-level/biology/ocr/17/revision-notes/2-foundations-in-biology/2-2-biological-molecules/2-2-13-fibrous-proteins/  Abnormal hemoglobin in sickle cell anemia https://mysciencesquad.weebly.com/ib-hl-31a1.html Conjugated Proteins a protein to which another chemical group (e.g., carbohydrate) is attached by either covalent bonding or other interactions Class Prosthetic Group Example Nucleoprotein Nucleic acids Viruses Lipoprotein Lipids Serum lipoproteins Glycoprotein Carbohydrates Mucin in saliva Phosphoprotein Phosphate groups Casein in milk Hemoprotein Heme Hemoglobin, cytochormes Metalloprotein Iron, zinc Ferritin, hemoglobin Summary of Protein Structure and Function Types of Protein Structure and Their Interrelationships 1. Primary structure: Amino acid sequence Results from formation of covalent peptide bonds between amino acids 2. Secondary structure: Includes α-helix and β-sheet Hydrogen bonding between amide hydrogens and carbonyl oxygens of the peptide bonds Summary of Protein Structure and Function 3. Tertiary structure: Overall folding of the entire polypeptide chain Interactions between different amino acid side chains 4. Quaternary structure: Concerned with topological, spatial arrangement of two or more polypeptide chains Involves both disulfide bridges and noncovalent interactions  Important Peptides and Protein Hormones Name Origin Action Adrenocorticotropic hormone (ACTH) Pituitary Stimulates production of adrenal Angiotensin II Blood Plasma Cause blood vessels to constrict Follicle-stimulating hormone (FSH) Pituitary Stimulates sperm production and folicle maturation Gastrin Stomach Stimulates stomach to secrete acid Glucagon Pancreas Stimulates glycogen metabolism in liver Human Growth Hormone ( HGH) Pituitary General effect: bone growth Insulin Pancreas Controls metabolism of carbohydrates Oxytocin Pituitary Stimulates contraction of uterus and other smooth muscles Prolactin Pituitary Stimulates lactation Somatostatin Hypothalamus Inhibits production of HGH Vasopressin Pituitary Decreases volume of urine excreted Myoglobin and Hemoglobin Myoglobin and Oxygen Storage Hemoglobin is the oxygen- transport protein of higher animals Myoglobin is the oxygen storage protein of skeletal muscle Oxygen is transferred from hemoglobin to myoglobin as myoglobin has a stronger attraction for oxygen than hemoglobin does Myoglobin and Hemoglobin Heme group is an essential component of the proteins hemoglobin and myoglobin Fe2+ ion in the heme group is the oxygen binding site Each hemoglobin contains a heme group which can hold 1 molecule of oxygen (O2) Protein Denaturation vs Hydrolysis Extremes of temperature and pH have a drastic effect on protein conformation Denaturation is the loss of organized structure of a globular protein; Does not alter primary structure; the disruption of bonds in the secondary, tertiary, and quaternary protein structures Examples: heat and organic compounds that break apart H bonds and disrupt hydrophobic interactions acids and bases that break H bonds between polar R groups and disrupt ionic bonds heavy metal ions that react with S—S bonds to form solids agitation, such as whipping, that stretches peptide chains until bonds break Protein Hydrolysis Protein hydrolysis splits the peptide bonds to give smaller peptides and amino acids occurs in the digestion of proteins; occurs in cells when amino acids are needed to synthesize new proteins and repair tissues In the lab, the hydrolysis of a peptide requires acid or base, water, and heat. In the body, enzymes catalyze the hydrolysis of proteins. Some practical aspects of protein denaturation 1. Heat and UV cooking denatures proteins; e.g., egg white proteins have to be denatured by cooking for them to become utilizable by our system any burn, including sunburn, causes denaturation of protein in the body sterilization uses UV and heat in the form of steam to coagulate the proteins of bacteria Some practical aspects of protein denaturation 2. Salts of heavy metal ions esp. Hg2+, Pb2+, Ag+ used as antiseptics in low concentrations; in higher concentrations they act as poisons. When ingested they precipitate the proteins in cells of body tissues. Effective treatment consists of feeding with egg white, followed by an emetic (the egg white forms complex with the poison and taken out of circulation by emetic) 3. Organic compounds such as soap, detergents, phenol, and aliphatic alcohol the hydrophobic portions of these compounds interact with the hydrophobic core of the protein, while the hydrophilic portion is H- bonded with the aqueous environment. This causes swelling and concomitant unfolding of the protein molecules. https://www.mogenale.pw/products.aspx?cname=curly+to+straight+hair+treatm ent&cid=78 Protein Sequencing The procedure in the determination of amino acid sequence basically involves the following steps: 1. hydrolysis - by acid, alkali, or enzyme 2. identification of the products of hydrolysis 3. fitting the pieces together as you would a jigsaw puzzle Protein Sequencing HYDROLYSIS 1. Acid Hydrolysis involves heating in the presence of 6N HCl in sealed tube at 110oC for 10–100 hrs depending on the nature of peptide or protein to be hydrolyzed the protein is completely hydrolyzed, but Trp, is destroyed completely and Ser, Thr, and Tyr are partially destroyed Protein Sequencing HYDROLYSIS 2. Alkali Hydrolysis heating in the presence of 4N NaOH in sealed tube at 10 – 100 hrs as in acid hydrolysis does not damage Trp, but destroys Arg, Cys, Thr, & Ser; and some amino acids are partly deaminated more disadvantageous but since it does not destroy Trp, it is used in quantitative determination of this amino acid Protein Sequencing HYDROLYSIS 3. Cyanogen bromide cuts peptide bonds on the carboxyl- terminal side of methionine residues. Since this amino acid is relatively infrequent in proteins, this cleavage tends to produce relatively large and relatively few peptides. This reaction is used to reduce the size of polypeptide segments for identification and sequencing. Example A peptide with 12 amino acids has the composition (not sequence) AspCys2Glu2Leu2Ser2Tyr2Val. It also has Ser as the N-terminal residue and Cys as C-terminal. Partial acid hydrolysis gave these peptide sequences: SerLeuTyr TyrCys LeuTyrGlu GluLeuGlu SerValCys CysSerVal GluAspTyr What is the sequence of the original peptide? SerLeuTyrGluLeuGluAspTyrCysSerValCys Protein Sequencing HYDROLYSIS 3. Enzymatic Hydrolysis by proteases/ peptidases a. Exopeptidases – enzymes that cleave external peptide bonds Aminopeptidases sequentially cleaves peptide bonds, beginning at the N-terminal end of the polypeptide; the liberated amino acids are identified one by one Carboxypeptidases sequentially cleaves peptide bonds beginning at the C-terminal end of the polypeptide The exopeptidases may be used in the determination of the amino acid sequence of peptides. By following the increase in amino acid liberated during hydrolysis by the two enzymes it is possible to determine the amino acid sequence in a peptide. EXAMPLE: A time-course analysis of the free amino acid in the hydrolysate of the pentapeptide with aminopeptidase and carboxypeptidase gave the following results: a) with carboxypeptidase [Glu] > [Val] > [Ala] > [Cys] = [Arg] ; cyst and arg appear simultaneously on equal amounts b) with aminopeptidase [Arg] > [Cys] > [Ala] > [Val] = [Glu] ; val and glu appear simultaneously on equal amounts c) what is the primary structure of the pentapeptide? Protein Sequencing HYDROLYSIS 3. Enzymatic Hydrolysis by proteases/ peptidases b. Endopeptidases – enzymes that cleave internal peptide bonds 1. Trypsin cleaves peptide bonds at the carboxyl end of the two strongly basic amino acids: arginine and lysine 2. Chymotrypsin cleaves peptide bonds at the carboxyl end of the three aromatic amino acids: phenylalanine, tyrosine, & trptophan; and Leucine 3. Elastase cleaves on the carboxyl side of Gly and Ala 4. Pepsin cleaves peptide bonds at the amino end of the three aromatic amino acids: phenylalanine, tyrosine, tryptophan; acidic amino acids, Asp and Glu; and Ile 5. Thermolysin cleaves peptide bonds at the amino end of the three aromatic amino acids, Phe, Tyr, Trp; and amino acids with bulky nonpolar R groups, Leu, Ile, and Val Example: Write the peptides generated from chymotrypsin, trypsin, and pepsin digestion of Ala-His-Tyr-Pro-Trp-Arg-Ileu-Phe-Glu-Lys-Cys Chymotrypsin: (carboxyl end) phenylalanine, tyrosine, & trptophan; and Leucine Trypsin: (carboxyl end) arginine and lysine Pepsin: (amino end) phenylalanine, tyrosine, tryptophan; Asp and Glu; and Ileu Other methods for determining the N-terminal end (chemical method) 1. Sanger’s Method. The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4-dinitrobenzene 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. HF Acid hydrolysis cleaves all the peptide bonds leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group. H3O+ 2. Edman Degradation Can be done sequentially one residue at a time on the same sample. Usually, one can determine the first 20 or so amino acids from the N-terminus by this method. The key reagent in the Edman degradation is phenyl isothiocyanate.  Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid. (labeling) phenylthiocarbamoyl (PTC) derivative The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the peptide. * * phenylthiocarbamoyl (PTC) (releasing) HCl derivative * * thiazolone Peptide shortened by 1 residue Under the conditions of its formation, the thiazolone rearranges to a phenylthiohydantoin (PTH) derivative. Thiazolone PTH-derivative The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation. Other methods for determining the C-terminal end (chemical method) 1. Hydrazine Method: hydrazine reacts with all amino acids whose carboxyl group is bound in peptide linkage, creating amino acyl hydrazides. Only the C-terminal amino acid is spared. Example A pentapeptide was found to have the composition Ala, Arg, Gly, Pro, and Trp. Reaction of the pentapeptide with Sanger’s reagent, followed by hydrolysis, gave the DNP derivative of proline. Treatment of the pentapeptide with carboxypeptidase initially produced alanine. Treatment of the pentapeptide with trypsin gave a tetrapeptide which, when treated with chymotrypsin, produced a tripeptide. What is the sequence of the pentapeptide? (note: trypsin cleaves after basic amino acids) Chymotrypsin (C-end) Phe, Tyr, Trp, Leu Trypsin (C-end) Arg, Lys Answer: ProGlyTrpArgAla