2. amino acid

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Information about 2. amino acid

Published on March 6, 2014

Author: invinciblekannde

Source: slideshare.net

AMINO ACIDS • • • Amino acids peptide bond formation peptides as active biomolecules.

Amino acids,

Course Objectives • To know the chemical structure and and be able to identify amino acids of biochemical importance. •To study the physicochemical properties of amino acids •To appreciate the relationship between amino acids and peptides/proteins •To identify specific peptides as active endogenous biomolecules

Amino acids Structure of amino acids •Amino acids are the basic structural units of peptides and proteins. They consist of: • amino group • a carboxyl group • a hydrogen atom • a distinctive R group bonded to a C atom known as the α-carbon • The R group is referred to as the side chain.

Physicochemical properties of amino acids • Amino acids in solution at neutral pH are predominantly dipolar ions (Zwitterions) rather than unionised molecules. • In the dipolar state the amino group is protonated (NH3+) and the carbonyl group is dissociated (-coo- ). • In addition to the ionisable α-substituents several of the amino acids have Other ionisable groups. The pH at which the net ionic charges are balanced is the isoelectric point (IEP).

Amino acids are optically active •The tetrahedral array of 4-different groups about the α-carbon confers optical activity on amino acids • The α-carbon of all amino acids are asymetric so there are two stereoisomers of each except glycine. • The 2 mirror image forms are the: • L-isomer • D-isomer • By convention if the α-NH is projected to the left, the a.acid has an absolute L-configuration. Its optical enantiomer with α-NH3+ projected towards the right has a D-configuration. 3 +

Occurrence of L and D-amino acids in proteins • All amino acids of animal proteins are L-isomers and are sometimes referred to as natural amino acids. •Some D-a.acids occur in plants and bacteria eg. D-alanine and D-glutamic acid occur in bacteria cell wall. • Some antibiotics contain D-amino acids Eg. D-alanine, D-glutamic acid and D-ornithine are present in bacitracin-an active agent against G-ve bacteria. •Polymyxins contain D-phenylalanine

The common amino acids in proteins • 20 kinds of side chains varying in: •Size •Shape •Charge •Hydrogen bonding capacity •Chemical reactivity are commonly found in proteins • All proteins in all species of bacteria & man are constructed from the same set of 20 amino acids. •The remarkable range of functions exhibited by proteins result from the diversity and versatility of the 20 kinds of building blocks-amino acids.

The 20 amino acids and their abbreviations Amino acid 3-letter abbreviation Alanine Arginine Asparagine Aspartic acid Aspartic acid & Asparagine Cysteine Glutamine Glutamic acid Glutamine or Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Ala Arg Asn Asp Asx Cys Gln Glu Glx Gly His Ile Leu Lys Met 1-letter abbreviation A R N D B C Q E Z G H I L K M

Abbreviations cont’d Amino acid Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine 3-letter abbreviation Phe Pro Ser Thr Trp Tyr val 1-letter abbreviation F P S T W Y V

Glycine & Alanine

Glycine • simplest amino acid with H as side chain, not optically active. •Sweet taste •Residues found in almost all proteins particularly abundant in structural proteins •A neurotransmitter in the CNS •Sometimes combined with antacids in the treatment of gastric hyperacidity to reduce gastric irritation due to buffering capacity of the amino group. •Been used in the treatment of myasthenia gravis, benefit is doubtful. Alanine •Has methyl group as its side chain •Occurs in almost all proteins •Its structural isomer β-alanine occurs in tissues in the free form and as a component of coenzyme A and the peptide carnosine & anserine, β-alanine not a component of protein.

Valine, leucine, isoleucine & proline (amino acids with hydrocarbon side chains valine

Proline differs from other amino acids in containing a 20 rather than a 10 amino group. The side chain of proline is bonded to: • both the amino group and the α-carbon to form a cyclic structure. •The cyclic structure results in the distortion of the linear symmetry of the polypepetide chain into which proline is incorporated •Further, when the >NH group is engaged in an amide link, there is no H atom remaining to participate in H-bonding •Therefore, proline is an important determinant of the 3-dimensional shape of peptide chains. •Some hydroxy derivatives of proline are found only in collagen & gelatin •Valine leucine and isoleucine are essential amino acids, thus has to be taken in the diet to support life and growth possibly bcos the body lacks the capacity to synthesize branched aliphatic chains, hydrophobic?

Serine & Threonine: •The –OH group in the side chain of serine can engage in H-bonding and ester formation. •Serine is therefore an important reactive component of proteins. •An antibiotic, a diazoacetyl`ester (azaserine) has anti-tumour and abortifacient activity.

Aromatic amino acids, phenylalanine, tyrosine & tryptophan •Phenylalanine & tyrosine are important constituents of proteins •Are precursors for the synthesis of important regulatory proteins as neurotransmitters and hormones. • Through the formation of another amino acid dihydroxyphenylalanine (DOPA), the catecholamines eg noradrenaline are derived.

Synthesis of catecholamines from phenylalanine and tyrosine •Starting point is the essential aa Phe & Tyr derived from protein in the diet Generally, dietary intake of tyrosine is more than adequate for catecholamine synthesis •Phenylalanine can be converted to tyrosine by the enzyme phenylalanine Hydroxylase. Enzyme absent in the congenital disease phenylketonuria.

Catecholamine synthesis

•L-DOPA used in the treatment of Parkinsons disease. •Note: Rational for L-Dopa/carbidopa combination in Parkinson’s •Methyldopa used to treat high B.P •DOPA involved in melanin formation •Tyrosine gives rise to the thyroid hormones •Tryptophan is the precursor of 5-hydroxytryptamine or serotonin which has several actions on the CVS, GIT, genital tract and the respiratory system. •Side chains of the aas described are uncharged at physiological PH •The ff aas have charged side chains: •Lysine •Arginine •Histidine •Aspartate & glutamate

•Arginine and lysine have basic side chains •Aspartate and glutamate have negatively charged side chains (acidic) •Histidine may have +vely charged or neutral depending on its local environment •Histidine is an important reactive aa in proteins because the pka of the nitrogen in the imidazole ring of histidyl residues in proteins is in the range of pH of the tissues. •Histidine is also an important precursor of the pharmacologically active substance histamine

•Lysine is an important component of proteins due to reactivity of the amino group on the side chain. •Has been used as an adjunct to mercurial diuretics in the treatment of fluid retention •Hydroxylysine, a derivative occurs in collagen. •Arginine involved in the synthesis of urea •Has been given in the treatment of hepatic failure where there is Coma due to high levels of ammonia. •Acts predominantly by stimulating the synthesis of urea. •Large doses stimulate the secretion of insulin & G-hormone

•Aspartate & glutamic acid have carboxyl groups on their side chains which are completely ionised at the pH of the tissues •The acidic groups are masked by being converted to the corresponding amides, asparagine and glutamine

Asparagine and glutamine (Amide derivatives of aspartate & glutamate

•Methionine & cysteine have S in their side chains •Methionine is a component of most proteins acting as a methyl donor in biochemical processes. •The demethylated derivative homocysteine occcurs in tissues •It is a dietary component and may be replaced by D-methionine which is deaminated and then reaminated to the L-form. •Methionine has a lipotropic action and prevents fatty infiltration of the liver. Given as a supplement to patients with liver diseases Unable to take adequate diet.

•Cys plays a role in determining the tertiary structure of proteins •2 mols in adjacent polypeptide chains combine through their thiol groups to form a cystine residue •Cysteine and methylcysteine have been reported to increase the rate of wound healing •Acetylcysteine has been used as a mucolytic •Methionine and acetylcysteine have been used in the treatment of paracetamol overdosage •Acetylcysteine increase GSH formation in the liver •Methionine increases conjugation rxn in the liver •GSH conjugates N-acetyl-p-benzoquinone imine. •Note: Some proteins contain special aas that supplement the 20 basic aas eg. Collagen contains hydroxyproline Such proteins are formed by modification of a common aa after it has been incorporated into a polypeptide chain.

Summary of classification of the amino acids •Nonpolar, aliphatic •Glycine, alanine, proline, valine, leucine & isoleucine •Aromatic •Phenylalanine, tyrosine, tryptophan •Polar, uncharged •Asparagine, glutamine, serine, threonine •Charged •Aspartic, glutamic, arginine, lysine, histidine

The peptide bond •Amino acids are the building blocks of peptides and proteins. • The peptide bond is formed by the interaction of two amino acids with the elimination of a molecule of water between the -NH2 and –COOH

The peptide bond

•Properties & occurrence of peptide bonds in peptides & proteins •The sharing of electrons b/n the –C=O and –C-NH confers rigidity on the peptide bond. •In proteins and polypeptides aas are linked by peptide bonds between the α-amino group of one aa and the carboxyl group of the next. •Peptide bond is therefore an amide linkage •A single polypeptide chain may contain up to several hundreds of aas joined together linearly. •The carbonyl oxygen and the amide hydrogen of the peptide bonds participate in H-bonding interactions leading to the

Structure of Peptide • The peptide bond is an amide bond. • Amides are very stable and neutral. => Chapter 24 30

Oligopeptides, polypeptides and proteins •Amino acids may be linked by peptide bonds to form: •Oligopeptides (several aa residues) •Polypeptides (many residues) •Proteins consist of one to several peptides

Protein terminals •N-terminal or the amino terminal is the terminal bearing the free amino group. •C-terminal or the carboxyl terminal is the one bearing the free carboxyl group. •The amino acid residues are numbered starting at the Nterminal, the direction in which aas are incorporated during protein synthesis.

Small peptides •Small peptides may break the rule governing peptides •They may contain D-amino acids (some antibiotics) •The amino acids may be linked by bonds other than the standard peptide bond (the glutamyl residues of glutathione)

Dipeptides: •2 dipeptides carnosine & anserine occur particularly in muscle. •Carnosine in man and other animals and anserine in birds

Tripeptides •Glutathione is a tripeptide of considerable biochemical importance •Amino acid residues in glutathione are: •glutamic acid •Cysteine •Glycine •GSH acts as a coenzyme for several enzymatic reactions •2 mols may be linked through their –SH group under Oxidising conditions to form oxidised glutathione GSSH

Polypeptides •Several members of biochemical & Pharmacological interest. •Include the following nonapeptides of the Post Pituitary hormones: •Oxytocin •Vassopressin •Antidiuretic hormone (ADH) •Longer chain polypeptide hormones of the pancreas •Insulin 51 •Glucagon 29

•Hormones of the anterior Pituitary •ACTH OR Corticotrophin 39 •Melanotrophins (13-22) •Substances having intense activity on smooth muscle: •Kinins •Bradykinins •Kallidin •Substances regulating secretions in the GIT: •Gastrin •Secretin •A substance regulating circulation •Angiotensin II

Liver Angiotensinogen Renin Angiotensin I ACE Vasoconstriction Raised BP Angiotensin II Role of Angiotensin in BP control

Have we achieved our objectives? To know the chemical structure and be able to identify amino acids of biochemical importance. To study the physicochemical properties of amino acids To appreciate the relationship between amino acids and peptides/proteins To identify specific peptides as active endogenous biomolecules

CLASSIFICATION OF PROTEINS •May be classified as follows: •Composition •Simple (contain only amino acids •Conjugated (contain additional substances) •Molecular weight •Low (5000-20,000) •Medium (20,000-50,000) •High (50,000-several millions) •Molecular shape •Fibrous (long in proportion to their diameter) •Globular (less asymmetric)

•Function •Enzymes •Structural •Antibodies •Source •Tissue protein •Plant •Bacteria •Viral •Physicochemical properties •Solubility soluble insoluble •Thermal stability stable unstable

Generally however, proteins are classified as: •SIMPLE •Albumins Water soluble, and soluble in dilute salt solutions Precipitated with full saturation in ammonium sulphate Eg. Plasma albumin •Globulins Soluble in dilute salt solutions Insoluble in water and strong salt solutions •Scleroproteins Insoluble in aqueous solutions eg. Keratin, collagen, fibrin •Protamines Contain High proportion of arginine Of low MW, not coagulated by heat, soluble in water to give appreciably alkali solution

•Histones •Soluble in water to give weakly alkali solution, conjugated as nucleoprotein •CONJUGATED PROTEINS: •Nucleoproteins-nucleic acids eg chromosomes •Glycoproteins or mucoproteins carbohydrate derivatives-blood group substances •Lipoproteins-plasma lipoproteins, components of cell Membranes and subcellular organells •Phosphoproteins-phosphoesters with serine or threonine residues eg. casein

•Flavoproteins-flavine-adenine dinucleotide, various reduction and oxidative enzymes •Haemoproteins-Iron-porphyrin (haem) groups eg. Hb, Myoglobin, cytochrome c •Metalloproteins-containing metal groups eg carbonic anhydrase

•Proteins •Greek – proteios – of 1° importance •Polymers of amino acids linked by peptide bonds. •Proteins are the most important of all biological compounds.

•Components of Proteins •A copolymer is a polymer made from more than one type of monomer molecule. •Twenty different α-amino acids can link to form polypeptides.

•Distribution Proteins of Body 20% 20% 10% 10% 50% 50% 20% 20% Muscle Muscle Bone Bone Skin Skin Other Other

•Protein for Energy •Prefer to use fat, CHO for energy •CHO and fat are protein sparing •EXCEPTIONS –During prolonged strenuous exercise, about 15% of the muscles need met with protein (break down own tissue) –If protein intake is inadequate, body protein → energy e.g. starvation

•Blood Levels •Total plasma proteins – 6.0-8.4 g/dL –Albumin – 3.5-5.0 g/dL –Globulin – 2.3-3.5 g/dL

•Proteins - Properties & Functions •Size •Proteins are extremely large natural polymers with molecular weights reaching several million. •Compare a typical organic molecule -benzoic acid (C6H5COOH MW = 132). •The small protein haemoglobin has the formula C2952H4664O832N812S8Fe4. •Its molecular weight = 65,000.

•Size contd. •Proteins are too large to pass through cell membranes and remain trapped in the cells where they are made. •In disease or trauma, cells are damaged and proteins can escape. •Detection of proteins in urine indicates kidney damage. Heart attack releases specific heart cell proteins into the blood.

•Size of Some Important Proteins Protein Insulin Cytochrome c Hemoglobin Gamma globulin Myosin Molecular wt 6,000 16,000 65,000 176,000 800,000 No. of aa residues 51 104 574 1320 6100

•Properties contd. •Proteins are linear polymers built of monomer units called amino acids •Proteins contain a wide range of functional groups. •Proteins can interact with one another and with other biological macromolecules to form complex assemblies •Some proteins are quite rigid, whereas others display limited flexibility

•Linear Polymer •Function of a protein is directly dependent on its three-dimensional structure •Proteins spontaneously fold up into three-dimensional structures that are determined by the sequence of amino acids in the protein polymer.

•Functional Groups •alcohols, thiols, thioethers, carboxylic acids, carboxamides, and a variety of basic groups. •combined in various sequences, this array of functional groups accounts for the broad spectrum of protein function.

•Interaction + macromolecules •assemblies include –macro-molecular machines that carry out the accurate replication of DNA, – the transmission of signals within cells, and –many other essential processes.

•Rigidity & Flexibility •Rigid units can function as structural elements in the cytoskeleton (the internal scaffolding within cells) or in connective tissue. •may act as hinges, springs, and levers that are crucial to protein function, to the assembly of proteins with one another and with other molecules into complex units, and to the transmission of information within and between cells

•Other properties of proteins •Sedimentation -a protein containing solution centrifuged at sufficiently high speed will have its molecules settled at a constant rate when the centrifugal force exceeds the dispersant forces on the molecules •pH -The pH determines the properties of the protein such as solubility, viscousity and enzymatic activity. •Immunofluorescent histochemistry -the precise location of an antigenic substance can be determinedby an antibody that reacts specifically with it. •Electrophoresis -At any pH other than the IEP, a protein will migrate in an electric field. The differential rates of migration can be used to separate proteins.

•Hydrolysis Hydrolysis (enzymatic or heat) of the amides regenerates the amino acids: H O N C R R H N C H O O N C R R H N C H N O •The amide linkage is split as indicated.

•Regeneration of component amino acids The very large protein is broken down into smaller, water soluble components: O H2N R COH H2N O R H2N COH O R COH H2N R COH O •These small molecules may move through the organism to be reassembled elsewhere.

•General functions of proteins •Most versatile macromolecules in living systems •serve crucial functions in essentially all biological processes –catalysts, –transport and store other molecules such as oxygen, – provide mechanical support and immune protection, –generate movement, – transmit nerve impulses, and – control growth and differentiation

•Functional Roles of Proteins •Dynamic Functions Transport, metabolic control, contraction, and catalysis of chemical transformations. •Structural Functions provide the matrix for bone and connective tissue give structure and form to the human organism.

•Dynamic Functions I •Enzymatic Catalysis •Enzymes-dynamic proteins. almost all biological reactions are enzyme catalyzed . Allows the reaction to occur at a rate compatible with life. •Transport •Haemoglobin and myoglobin •transport oxygen in blood and in muscle respectively •Transferrin •transports iron in blood. •Albumin •many drugs and xenobiotics compounds are transported bound to albumin. •Others transport hormones in blood from their site of synthesis to their site of action

•Dynamic Functions II Protective Role –Immunoglobulins and interferons act against bacterial or viral infection. –Fibrin formed where required to stop the loss of blood on injury to the vascular system. Metabolic Control –Many hormones are proteins. –Protein hormones include insulin, thyrotropin, somatotropin (growth hormone), luteinizing hormone, and follicle stimulating hormone. Important peptide hormones include adrenocorticotropin, antidiuretic hormone, glucagon, and calcitonin.

•Dynamic Functions III •Contractile Mechanisms –Myosin and actin function in muscle contraction. •Control And Regulation Of Gene Transcription And Translation –histone proteins closely associated with DNA, the repressor and enhancer proteins that control gene expression, and the proteins that form a part of the ribosomes.

•Structural Functions •brick-and-mortar" roles –collagen and elastin, form the matrix for bone, ligaments, connective tissue and skin Provide structural strength and elasticity to organs α-keratin – Keratin is the 1º component of human hair, nails, skin, and tooth enamel – fibrous sulfur-containing protein.

Protein Structure

•Levels of protein structure Primary structure –The amino acid sequence in a polypeptide chain Secondary structure –Consists of local regions of polypeptide chains formed into structures that are usually stabilized by hydrogen bonds Tertiary structure –Involves folding of the secondary elements into an overall three-dimensional conformation Quaternary structure –Combination of 2 or more subunits each composed of a polypeptide chain

Protein Organization Four levels of organization –Primary structure –Secondary structure α- helix –Tertiary structure –Quaternary structure Myoglobin Hemoglobin

•Primary Structure 1˚ structure = specification of the sequence of amino acids i.e. the order in which amino acid residues are linked together in a protein. Note: since every polypeptide begins with free amino group, this is called the N-terminus. The opposite end of the polypeptide has a free carboxyl group, called the C-terminus.

N and C t er m inal of polypept ides R H N C H C H N R R R H C C N C C N O C C OH H Amino or N terminus O H O H O H Carboxyl or C terminus

•Amino Acid Sequences Have Direction •Leu-enkephalin - an opioid peptide, modulates the perception of pain. • reverse pentapeptide, Leu-Phe-Gly-Gly-Tyr (LFGGY), is a different molecule and shows no such effects

Polypeptide chains consists of a regularly repeating part, called the main chain or backbone and a variable part, comprising the distinctive side chains

•Why know the sequence of amino acids in a polypeptide chain? • Elucidating its mechanism of action (e.g., the catalytic mechanism of an enzyme) – proteins with novel properties can be generated by varying the sequence of known proteins. • Second, amino acid sequences determine the three-dimensional structures of proteins. – sequence is the link between the genetic message in DNA and the three-dimensional structure that performs a protein's biological function.

•Oxytocin & Vasopressin •ADH and oxytocin each have nine (9) amino acids. •Each has cysteine residues at amino acid positions 1 and 6. These cysteine residues form a disulfide bond with one another to create a cyclic six amino acid ring with 3 amino acid residues hanging off. •ADH and oxytocin share 7 amino acids in common and differ only at amino acid positions 3 and 8. •Oxytoxin is Isoleucine-3, Leucine-8 while ADH is Phenylalanine-3, Arginine-8.

•Functions of Oxytocin & ADH Oxytocin stimulates contraction of uterine smooth muscle. It is secreted during labor to effect delivery of the fetus. Oxytocin also stimulates contraction of smooth muscle in the mammary glands (myoepithelial cells). ADH in low doses controls the resorption of water by the distal tubules of the kidneys and regulates the osmotic content of blood. At high doses, ADH causes contraction of arterioles and capillaries, especially those of the coronary vessels, to produce localized increases in blood pressure Receptors, V1 – bood vessels, V2- kidney

1 0 structure of Insulins Used in the Treatment of DM Species Human A8 Thr A9 Ser A10 Ile B30 Thr Cow Ala Ser Val Ala Pig Thr Ser Ile Ala Sheep Ala Gly Val Ala Horse Thr Gly Ile Ala Dog Chicken* Thr His Ser Asn Ile Thr V2kidney Ala Ala Duck* Glu Asn Pro Thr *Positions 1 and 2 of B chain are both Ala in chicken and duck; whereas in the other species in the table, position 1 is Phe and position 2 is Val in B chain.

•Recombinant DNA Technology

•Restriction Enzymes Produced by various kinds of bacteria restriction enzymes recognize specific sequences of DNA and cut the double strand where the sequence occurs. Treating the DNA of two different organisms with the same restriction enzyme produces complementary fragments, or fragments with ends that fit together These can be combined in a hybrid DNA molecule that, if part of a living cell, expresses traits of both parents.

•Recombinant DNA

•Recombinant DNA Technology In genetic engineering, scientists use restriction enzymes to isolate a segment of DNA that contains a gene of interest—for example, the gene regulating insulin production. 2. A plasmid extracted from its bacteria and treated with the same restriction enzyme can hybridize with this fragment’s “sticky” ends of complementary DNA. 3. The hybrid plasmid is reincorporated into the bacterial cell, where it replicates as part of the cell’s DNA. 4. A large number of daughter cells can be cultured and their gene products extracted for human use. 1.

•Insulin Lispro •Short-acting insulin analogs are designed to overcome the limitations of conventional regular human insulin. •Insulin lispro (Humalog), formerly called LYSPRO from the chemical nomenclature [LYS(B28), PRO(B29) •Advantages •faster subcutaneous absorption, •an earlier and greater insulin peak, and •a shorter duration of action

•Insulin lispro In insulin lispro, reversal of the proline at B-28 and the lysine at B-29 results in more rapid dissolution of this insulin to a dimer and then to a monomer that is absorbed more rapidly after subcutaneous injection


•Secondary Structure Polypeptides fold in a series of stages. The first level of folding is called the secondary (2˚) structure. One of the most common 2˚ folding patterns is called the alpha-helix , discovered by Pauling and Corey. –Alpha helix: Hydrogen bonds can form readily between C=O groups in the backbone and N-H groups four amino acid residues further along the chain. –This regular pairing pulls the polypeptide into a helical shape that resembles a coiled ribbon.

20 structure contd •Another common folding pattern is called beta pleated sheet . •Some protein regions remain in random coil, no regular pattern of secondary structure. •Different proteins have different degrees of alpha helix, beta sheet, and random coil . •Silk is a protein stabilized entirely by pleated sheet; keratin (in hair) is stabilized entirely by alpha helix. Most proteins have some of both.

Alpha helix

•Hydrogen-Bonding Scheme For an α helix the CO group of residue n forms a hydrogen bond with the NH group of residue n+ 4.

Structure of an α-helix The polypeptide backbone is folded into a spiral that is held in place by hydrogen bonds (black dots) between backbone oxygen atoms and hydrogen atoms.  Note that all the hydrogen bonds have the same polarity. The outer surface of the helix is covered by the sidechain R groups.

Beta sheet

•A simple two-stranded β sheet with antiparallel β strands. • A sheet is stabilized by hydrogen bonds (black dots) between the β strands. •The planarity of the peptide bond forces a β sheet to be pleated; hence, this structure is also called a β pleated sheet, or simply a pleated sheet.

view of a β sheet showing how the R groups protrude above and below the plane of the sheet. Side

SECONDARY STRUCTURE Secondary structure is not just hydrogen bonds. Helix: Right-handed helix with 3.6 amino acid residues per turn. Hydrogen bonds are formed parallel to the helix axis. Sheet: A parallel or antiparallel arrangement of the polypeptide chain. Hydrogen bonds are formed between the two (or more) polypeptide strands. Turn: A structure in which the polypeptide backbone folds back on itself. Turns are useful for connecting helices and sheets.

•Secondary structure exists to provide a way to form hydrogen bonds in the interior of a protein. •These structures (helix, sheet, turn) provide ways to form regular hydrogen bonds. •These hydrogen bonds are just replacing those originally made with water. • As a protein folds, many hydrogen bonds to water must be broken. •If these broken hydrogen bonds are replaced by hydrogen bonds within the protein, there is no net change in the number of hydrogen bonds •Hydrogen bonding does contribute somewhat to the overall stability of a protein; however, the hydrophobic interaction usually dominates the overall stability

Fibrous Proteins Highly elongated protein molecules whose shapes are dominated by a single type of secondary structure. Example Characteristics 1. Coiled Coil Keratin durable, insoluble, unreactive 2. β Sheet Silk soft, flexible 3. Triple Helix Collagen strong, high tensile strength Type

Keratin • principal component of hair, nails, wool, horns, hooves, scales, feathers, shells ∀ α keratin - in mammals ∀ β keratin - in birds and reptiles The α-keratin chain is an α-helix. Pairs of α-helix chains are interwound to form a two-chain coiled coil. The chains wind in a left-handed sense. Each α-keratin chain consists of ~310 residues having a 7-residue repeat: a-b-c-d-e-f-g where residues a and d are nonpolar

Silk - a β sheet • consists of antiparallel β sheets • 6-residue repeat (-Gly-Ser-Gly-Ala-Gly-Ala-)n • The β sheets stack to form a microcrystalline array.

Collagen - a triple helix • Single collagen molecule contains 3 polypeptide chains. • Each chain is a left-handed helix (3 residues/turn). • 3 helical chains are twisted together in a right-handed manner to form a superhelical structure. • Many varieties - eg., Type I has two α 1 and one α 2 chains

Collagen - distinctive amino acid composition 30% Gly and 15-30% Pro or Hyp (hydroxyproline) (-Gly-X-Pro-) repeats or (-Gly-X-Hyp-) repeats Pro Hyp (4-hydroxyproline) O O C N CH H2C CH2 C H2 prolyl hydroxylase (requires ascorbic acid) C CH N H2C CH2 C HO H

Collagen Diseases • Scurvy (vitamin C deficiency) - improper fibers, skin lesions, fragile blood vessels, poor wound healing, due to decreased Hyp formation • Osteogenesis imperfecta (brittle bone disease) (OI) a group of heritable disorders with an incidence of about 1 in 10, 000- abnormal bone formation in infants, varies from mild to lethal. • Defect due to mutation in the genes for procollagen Type I, single base change in the codon for glycine resulting in the disruption of the triple helical structure. • Ehler-Danlos syndrome - hyperextensibility of joints and skin (“loose” skin), mutations: Gly replaced with Ser or Cys

Schematic Views of α-Helices • A ball-and-stick model. • A ribbon depiction. • A cylindrical depiction.

Ferritin • Ferritin, an iron-storage protein, is built from a bundle of a helices.

Major Histocompatibility Complex • • • Model of binding site in class I MHC (major histocompatibility complex) molecules, which are involved in graft rejection. A sheet comprising eight antiparallel β strands (green) forms the bottom of the binding cleft, which is lined by a pair of α helices (blue). A disulfide bond is shown as two connected yellow spheres. The MHC binding cleft is large enough to bind a peptide 8 10 residues long.

Tertiary Structure Polypeptides continue folding beyond the formation of secondary structure. It is only with the complete, compact folding into tertiary (3°) structure that they attain their "native conformation" and become active proteins (as a result of the creation of active sites).

Forces that contribute to tertiary folding include: –hydrogen bonds –hydrophobic bonds –ionic bonds –sulfhydryl bonds (-S-S- bonds). These are especially important, because they are covalent bonds and quite strong compared to H-bonds.

Tertiary Structure

Protein Folding Protein synthesis generates a linear sequence that has to be folded with hydrophilic groups on the outside and hydrophobic groups buried (if it is water soluble). The primary structure determines the folding pattern. Given the number of possible structures it is not possible that the protein tests every one of them to find the lowest energy state.

Protein Folding It is thought that secondary structures, called ‘molten globules’, facilitate the folding process. Another problem is that as proteins are synthesised hydrophobic regions must not be exposed to an aqueous environment or they will associate to form aggregates. This is achieved by chaperones that bind to hydrophobic regions and subsequently detach to allow correct folding.

Protein Folding contd This process allows the correct folding of even large proteins since these fold sequentially as they are synthesised. Some proteins require chaperonins that enclose the protein to be folded in a cavity away from the rest of the cell. Chaperones and chaperonins do not direct protein folding but simply provide conditions where it can occur properly. In cells exposed to a near lethal temperature rise heat shock proteins are synthesised. These allow existing proteins to refold correctly. Examples include Hsp 70 and Hsp 60

Prion diseases and protein folding Novel pathogens composed entirely of proteins A number of neurological degenerative diseases are known to be caused by prions –These include Creutzfeldt–Jacob disease (CJD) and kuru in humans and scrapie (Bovine spongiform encephalopathy,BSE) in sheep. –Mad cow disease is also caused by a prion. Although they are infectious no nucleic acid has been identified and it is now thought that a protein infectious agent or prion is responsible. In scrapie there is a normal brain protein (PrPc) which becomes converted to the scrapie form (PrPsc). These have the same primary structure but different secondary and tertiary structures.

Prion Diseases It is suggested that the prion form converts the normal form to the prion form, i.e. the process is autocatalytic. There are two possible mechanisms for this –The association of the normal form with the prion form may be sufficient to cause the change –There may be an involvement of a chaperone and ATP in the unfolding and refolding Mutations in the normal gene for PrP may make the formation of PrPsc more likely.

Assignment ! Alzheimer’s Disease ? Pathophysiology? Which protein?

Domains A long protein sequence frequently folds into a series of compact, semi-independent regions called domains. Each domain has a hydrophobic core and a hydrophilic exterior and generally are 100-150 amino acids in length. Domains by a of a single protein are usually connected stretch of polypeptide chain lacking a usual secondary structure (random coil) or a cleft or less dense region of tertiary structure. Sometimes a binding site is found in a cleft between domains. 

Domains contd Domains are frequently associated with a specific function of the protein. For example: binding sites for two different substrates or a substrate and effector could be in two different domains. Example: Glyceraldehyde-3-phosphate dehydrogenase..one domain binds NAD+ and the second domain binds glyceraldehyde-3phosphate.

The cell-surface protein, CD4 •Cell surface protein found on some cells of the immune system. •Has an extracellular and cytoplasmic portions. •(HIV) attaches itself to the extracellular portion, which comprises of four similar domains of approximately 100 amino acids each

Quaternary Structure Some proteins are made of multiple polypeptide subunits, which must be assembled together after each individual polypeptide has reached its 3° structure. Examples: –Hemoglobin (blood protein involved in oxygen transport) has four subunits . –Pyruvate dehydrogenase (mitochondrial protein involved in energy metabolism) has 72 subunits.

Immunoglobulins (Igs) Consist of 2 heavy and 2 light chains.  A disulfide bond joins a L chain to a H chain and the two L-H chain pairs are bound together by two disulfide bonds between the H chains. The variable regions of an L and H chain come together to form the antigen binding site of the immunoglobulin.

Structure of Antibodies

Structure of antibodies The heavy and light chains come together to form Fab domains, which have the antigen-binding sites at the ends. The two heavy chains form the Fc domain. The Fab domains are linked to the Fc domain by flexible linkers

Myoglobin and Hemoglobin Both proteins are involved in oxygen transport. myoglobin = intracellular protein in muscle hemoglobin = intracellular protein in red blood cells Why study them? vital proteins in human health valuable model in studying protein structure, binding, function

Myoglobin 153 a.a. residues MW 16,700 X-ray structure, 1959 eight α-helices contains a heme group –iron atom –porphyrin ring system

Heme group Fe(II) coordinated to N atoms in porphyrin ring Fe(II) binds O2 –with O2 = scarlet –no O2 = dark purple Fe(II) can be oxidized to Fe(III) dark brown, does not bind O2

Myoglobin Function Major physiological role is to facilitate oxygen transport in muscle. Essentially solutions. increases oxygen solubility in aqueous In aquatic mammals, myoglobin also functions to store oxygen (10-fold more in seals and whales) Reversible binding of O2 to myoglobin (Mb) Mb + O2 MbO2

Hemoglobin intracellular protein in red blood cells physiological function is to transport oxygen binds oxygen in lungs and releases oxygen into tissues quaternary structure –tetrameric protein –two α-subunits and two β subunits - α2β2 –each subunit contains a heme group –Fe(II) binds O2 with no O2 = scarlet O2 = dark purple

Haemoglobinopathies •Over 300 variations of amino acid sequences of normal adult haemoglobin (HbA) have been reported. •Differ by: -insertion of incorrect amino acid into either β or α-chain during protein synthesis •Haemoglobin variants may function normally or abnormally depending on the nature and position of the substitution

Haemoglobin variants Name Hammersmith Bristol Bibba Savannah Philly Mutation Phe CD1(42)β → Ser Val E11(67) β → Asp Leu H19(136) α → Pro Gly B6(24) β → Val Tyr C1(35) α → Phe Boston Milwaukee Iwate Yakima His E7(58) α → Tyr Val E11(67) β → Glu His F8(87) α → Tyr Asp G1(99) β → His Kansas Asn G4(102) β → Thr Sickle-cell anemia Effect Weakens heme binding Weakens heme binding Disrupts the H helix Disrupts the B-E helix interface Disrupts hydrogen bonding at the α1-β1 interface Promotes methemoglobin formation Promotes methemoglobin formation Promotes methemoglobin formation Disrupts a hydrogen bond that stabilizes the T conformation Disrupts a hydrogen bond that stabilizes the R conformation Glu A6(6) β → Val Deoxyhemoglobin S forms insoluble filaments that deform erythrocytes. (hemoglobin S) Mutant Val on one β subunit interacts in hydrophobic pocket of another β subunit , forming linear polymers.

Haemoglobin variants

Sickle Cell Disease Most common hereditary blood disorder Most common of the conditions is sickle cell anaemia (SCA) affecting mainly the black population. In SCA, the Haemoglobin called HbS contains normal α-chains but its β-chain contain valine instead of glutamate at residue 6, ie, a hydrophobic amino acid replaces an acidic one. The hydrophobic valine is able to interact with the β85-Phe and β88-leu of an adjacent deoxy HbS.

Consequences of the alteration: •Modification of the Hb conformation, stacking of 280 million Hb molecules within each erythrocyte altered by the production of fibrous aggregates. •Change in shape of erythrocytes from a biconcave disc to a crescent or sickle shape on deoxygenation In homozygotes the erythrocytes interact to form clumps, occlusion of capilaries and consequent reduction in blood flow. Organ damage! • SCA is characterized by episodes of pain, chronic hemolytic anemia and severe infections, usually beginning in early childhood

Sickle cell anaemia Under certain conditions such as low O2 levels, RBCs with HbS distort into sickle cells The sickled cells can block small vessels producing microvascular occlusions which may cause necrosis of the tissue

Sickle Cell Anaemia Detection –gel electrophoresis. Because sickle hemoglobin lacks a glutamate, it is less acidic than HbA. Hemoglobin HbS, therefore, does not migrate as rapidly towards the anode as does HbA. –It is also possible to diagnose sickle-cell anemia by recombinant DNA techniques.

SCA – Management a combination of fluids, analgesics, antibiotics and transfusions are used to symptoms and complications. treat – –Hydroxyurea, an antitumor drug, has been shown to be effective in preventing painful crises. –Hydroxyurea induces the formation of fetal Hb (HbF) - a Hb normally found in the fetus or newborn - which, when present in individuals with SCA, prevents sickling.

Degradation of Proteins Cells have both extracellular and intracellular pathways for degrading proteins. The major extracellular pathway is the system of digestive proteases, which break down ingested proteins to polypeptides in the intestinal tract.  endoproteases such as trypsin and chymotrypsin, which cleave the protein backbone adjacent to basic and aromatic residues exopeptidases, which sequentially remove residues from the N-terminus (aminopeptidases) or C-terminus (carboxypeptidases) of proteins; and  peptidases, which split oligopeptides into di- and tripeptides and individual amino acids. These small molecules then are transported across the intestinal lining into the bloodstream

Protein Degradation: intracellular Pathways The life span of intracellular proteins varies from as short as a few minutes for mitotic cyclins, which help regulate passage through mitosis, to as long as the age of an organism for proteins in the lens of the eye. Cells have several intracellular proteolytic pathways for degrading misfolded or denatured proteins, normal proteins whose concentration must be decreased, and foreign proteins taken up by the cell. One major intracellular pathway involves degradation by enzymes within lysosomes, membrane-limited organelles whose interior is acidic.

Protein Degradation Distinct from the lysosomal pathway are cytosolic mechanisms for degrading proteins. The best-understood pathway, the ubiquitinmediated pathway, involves two steps: •addition of a chain of ubiquitin molecules to an internal lysine side chain of a target protein •proteolysis of the ubiquitinated protein by a proteasome, a large, cylindrical multisubunit complex

Ubiquitin The pathway contd numerous proteasomes present in the cell cytosol proteolytically cleave ubiquitin-tagged proteins in an ATP-dependent process that yields peptides and intact ubiquitin molecules

The Ubiquitin-mediated Pathway To be targeted for degradation by the ubiquitinmediated pathway, a protein must contain a structure that is recognized by a ubiquitinating enzyme complex. Different conjugating enzymes recognize different degradation signals in target proteins. – For example, the internal sequence Arg-X-X-Leu-Gly-X-Ile- Gly-Asx in mitotic cyclin is recognized by the ubiquitinconjugating enzyme E1. –Internal sequences enriched in proline, glutamic acid, serine, and threonine are recognized by other enzymes.

The Ubiquitin-mediated Pathway contd The life span of many cytosolic proteins is correlated with the identity of the N-terminal residue, suggesting that certain residues at the Nterminus favor rapid ubiquitination. – –For example, short-lived proteins that are degraded within 3 minutes in vivo commonly have Arg, Lys, Phe, Leu, or Trp at their N-terminus. –In contrast, a stabilizing amino acid such as Cys, Ala, Ser, Thr, Gly, Val, or Met is present at the N-terminus in long-lived proteins that resist proteolytic attack for more than 30 hours.

The Ubiquitin-mediated Pathway contd all newly synthesized proteins have methionine, a stabilizing amino acid, at the N-terminus. subsequent enzymatic alteration that generates one of the destabilizing amino acids at the N-terminus is necessary to target a protein for degradation

Denaturation Denaturation is the breaking of the noncovalent bonds which determine the structure of a protein. Complete disruption of tertiary structure is achieved by reduction of the disulfide bonds in a protein. Generally, the denatured protein will lose its activity, antigenicity, and become insoluble. 

Denaturation Denaturation occurs when: –hydrogen bonds are disrupted –disulfides are reduced –soaps separate the hydrophobic sections –acids or bases neutralise the salt bridges –metals complex with functional groups to form insoluble salts.

Denaturation Any chemical or physical agent that destroys and changes protein conformations causes denaturation. Heat Urea Reducing agents Surfactants Acids Bases Heavy metals UV Alcohols Amines Free radicals

Mechanisms of Denaturation Heat: Disrupts low energy van der Waals forces in proteins. Extremes of pH: Lead to changes in the charge of the protein’s amino acid side chains and results in the disruption of electrostatic and hydrogen bonds. Detergents like Triton X-100 (nonionic, uncharged) and sodium dodecyl sulfate (SDS, anionic, charged) disrupt the hydrophobic forces which fold proteins. Charged detergents like SDS also disrupt electrostatic interactions.

Mechanisms of denaturation contd Urea and guanidine hydrochloride disrupt hydrogen bonding by forming hydrogen bonds with the protein’s amino acid side chains that are stronger than those in the undenatured protein. – In addition, these two reagents can disrupt hydrophobic effects much like detergents. β-mercaptoethanol (βME) and dithiothreitol (DTT) reduce disulfide bonds.

In vitro denaturation and renaturation of proteins Treatment with an 8 M urea solution containing mercaptoethanol (HSCH2CH2OH) completely denatures most proteins. The urea breaks intramolecular hydrogen and hydrophobic bonds, and the mercaptoethanol reduces each disulfide bridge (-S-S-) to two sulfhydryl (-SH) groups.  When these chemicals are removed by dialysis, the SH groups on the unfolded chain oxidize spontaneously to re-form disulfide bridges, and the polypeptide chain simultaneously refolds into its native conformation

Mechanisms of denaturation contd Agents such as urea or guanidinium chloride effectively disrupt the noncovalent bonds β-mercaptoethanol - In the presence of a large excess of β-mercaptoethanol, a protein is produced in which the disulfides (cystines) are fully converted into sulfhydryls (cysteines). 

Role of β -Mercaptoethanol in Reducing Disulfide Bonds Disulfides are reduced, and the βmercaptoethanol is oxidized and forms dimers.

Denaturation of Ribonuclease Most polypeptide chains devoid of cross-links assume a random-coil conformation in 8 M urea or 6 M guanidinium chloride When ribonuclease was treated with β-mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity.

Reestablishing Correct Disulfide Pairing Native ribonuclease can be reformed from scrambled ribonuclease in the presence of a trace of b-mercaptoethanol

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