Published on February 5, 2014
Amino Acids, Peptides and Proteins Learning objectives • Amino acids share a common structure • R groups provide different chemical properties • Amino acids can ionize in aqueous solutions • Proteins can be purified and studied in a variety of ways • Protein structure has four levels of organization • Sequence homology generally translates to shared function
Proteins • Proteins serve many functions: – 1.Structure: collagen and keratin are the chief constituents of skin, bone, hair, and nails. – 2. Catalysts: virtually all reactions in living systems are catalyzed by proteins called enzymes. – 3. Movement: muscles are made up of proteins called myosin and actin. – 4. Transport: hemoglobin transports oxygen from the lungs to cells; other proteins transport molecules across cell membranes. – 5. Hormones: many hormones are proteins, among them insulin, oxytocin, and human growth hormone.
Proteins – 6. Protection: blood clotting involves the protein fibrinogen; the body used proteins called antibodies to fight disease. – 7. Storage: casein in milk and ovalbumin in eggs store nutrients for newborn infants and birds; ferritin, a protein in the liver, stores iron. – 8. Regulation: certain proteins not only control the expression of genes, but also control when gene expression takes place.
Amino Acids •Have an alpha- carbon attached to: • an amino group • carboxyl group • a hydrogen • an R group
Chirality of Amino Acids • With the exception of glycine, all protein-derived amino acids have at least one stereocenter (the carbon) and are chiral. – The vast majority of protein-derived amino acids have the L-configuration
Each R group determines the properties of the amino acid R groups can be polar, nonpolar, acidic, basic Hundreds of modified amino acids
Each R group determines the properties of an amino acid R groups can be polar, nonpolar, acidic, basic
Proteins are made of 20 amino acids
amino acids can act as acids and bases • Amino acids exist in solution as dipolar ions (Zwitterions) • Like buffers, AA’s can act as proton donors or acceptors – “Amphoteric” compounds or “amphoteric electrolytes”
titration of amino acids Ex. Glycine Deprotonation • Two distinct plateaus, each correspond to deprotonation of glycine • Titration curves can be used to predict AA charge at a given pH • The isoelectric point (pI) is the pH at 0 charge
formation of peptide bonds Peptides and proteins are polymers of amino acids • Two amino acids are covalently joined in condensation reaction N-terminal C-terminal
Peptides: how aa are linked • proteins are long chains of amino acids joined by amide bonds. peptide bond: – amino acids become linked together to form peptide bonds with the elimination of water – The reaction takes place between the -COOH of one amino acid and the -NH2
α-carbons separated by 3 covalent bonds Partial sharing of e- • A small electric dipole results from the partial negative charge on oxygen and the partial positive charge on nitrogen • The shared electrons result in some double bond character and the lack of rotation
planar nature of peptide bonds • The N-Cα and Cα-C bonds can rotate
Primary Structure • Just how important is the exact amino acid sequence? – Human insulin consists of two polypeptide chains having a total of 51 amino acids. – In the table are differences between four types of insulin. A Chain positions 8-9-10 B Chain position 30 Human Cow -Thr-Ser-Ile-Ala-Ser-Val- -Thr -Ala Hog Sheep -Thr-Ser-Ile-Ala-Gly-Val- -Ala -Ala
Primary Structure – Vasopressin and oxytocin are both nonapeptides but have quite different biological functions. – Vasopressin is an antidiuretic hormone. – Oxytocin affects contractions of the uterus in childbirth and the muscles of the breast that aid in the secretion of milk.
proteins range in size
proteins contain prosthetic groups Non-amino acid part of proteins
protein purification (chromatography) Ion-Exchange Size-Exclusion Affinity Uses protein characteristics, such as charge, size and binding affinity to separate the protein
electrophoresis (SDS-PAGE) Purification steps Stain and blot the gel Migration of charged proteins in an electric field Gel slows migration in proportion to mass
2D gel electrophoresis 1. Isoelectric focusing 2. SDS PAGE
Mass Spectrometry 1. First treat isolated protein with a protease 2. Mixture is vaporized and peptides separated 3. One peptide is selected and further fragmented 4. MS measures m/z ratios for all the fragments
Mass Spectrum of Ethanol
Levels of Structure • Primary structure: the sequence of amino acids • Secondary structure: conformations of amino acids in localized regions of a polypeptide chain; examples are -helix, -pleated sheet, and random coil. • Tertiary structure: the complete three-dimensional arrangement of atoms of a polypeptide chain. • Quaternary structure: the spatial relationship and interactions between subunits in a protein that has more than one polypeptide chain.
4 levels of protein structure • Primary – sequence of amino acids • Secondary – interactions between adjacent amino acids • Tertiary – 3D folding of the polypeptide • Quaternary – arrangements of multiple polypeptides
Secondary Structure • conformations of amino acids in localized regions of a polypeptide chain. – The most common types of secondary structure are helix and -pleated sheet. -Helix: a type of secondary structure in which a section of polypeptide chain coils into a spiral, most commonly a right-handed spiral. -Pleated sheet: a type of secondary structure in which two polypeptide chains or sections of the same polypeptide chain align parallel to each other
-Helix • The -helix structure: held together by hydrogen bonds
-Helix • In a section of -helix; – The C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino acid units away from it. – All R- groups point outward from the helix.
secondary structure • Note the position of the purple R groups relative to the backbone of the polypeptide
all α helices are right handed • But some supramolecular complexes are left handed (keratin, collage n) right-handed = clockwise
β sheet secondary structure • More extended • H-bonds may occur between amino acids some distance from one another • Adjacent chains can run parallel or anti-parallel to each other
β sheets require β turns • One third of amino acids are in turns or loops • Gly and Pro are frequently found in turns
-Pleated Sheet • In a section of -pleated sheet; – The C=O and N-H groups of peptide bonds from adjacent chains point toward each other so that hydrogen bonding is possible between them. – All R- groups on any one chain alternate, first above, then below the plane of the sheet, etc.
Pleated Sheet Structure of Proteins
secondary structure and function
Tertiary Structure • the overall conformation of an entire polypeptide chain. • Tertiary structure is stabilized in several ways: – Covalent bonds, as for example, the formation of disulfide bonds between cysteine side chains. – Hydrogen bonding between polar groups of side chains, as for example between the -OH groups of serine and threonine. – Electrostatic interaction or Salt bridges, as for example, the attraction of the -NH3+ group of lysine and the -COO- group of aspartic acid. – Hydrophobic interactions, as for example, between the nonpolar side chains of phenylalanine and isoleucine.
Cysteine • The -SH (sulfhydryl) group of cysteine is easily oxidized to an -S-S- (disulfide).
the permanent wave that isn’t Heat + New S-S bonds
Tertiary Structure • Forces that stabilize 3° structure of proteins
Tertiary Structures of Proteins • the three dimensional shape of proteins that results from further crosslinking, folding and interaction between R groups
protein structure ribbon mesh surface contour Sperm Whale Myoglobin ribbon + side chains space-filling model
relative compactness of proteins • Hypothetical chain length of a protein if it were to appear either as an α helix or β sheet
Common Motifs stable folding patterns in globular proteins
Common Motifs β microglobulin
Complex domain from simple motifs immunoglobulin
Quaternary Structure • the arrangement of polypeptide chains into a noncovalently bonded aggregation. – The individual chains are held together by hydrogen bonds, electrostatic interactions, and hydrophobic interactions. • Hemoglobin – Adult hemoglobin: two chains of 141 amino acids each, and two chains of 146 amino acids each. – Each chain surrounds an iron-containing heme unit.
Hemoglobin • The 4° structure of hemoglobin: made up of 4 subunits
Fibrous proteins: α keratin • Evolved for strength (hair, wool, nails, claws, quills…) • Right handed α helix • Coiled-coil provides added strength (like a twisted rope)
Fibrous proteins: collagen • Like keratin, collagen also evolved to provide strength • Left-handed a chain (not an α helix) • Right handed coiled coils – 3-stranded coil
Fibrous protein: silk • Fibroin, the silk protein is in the β conformation • Rich in Ala and Gly (for close packing) • More extended than α helix conformation
Denaturation • the process of destroying the native conformation of a protein by chemical or physical means. – Some denaturations are reversible, while others permanently damage the protein.
protein folding and misfolding • Molecular chaperones assist in protein folding for many • Interact with partially folded or improperly folded polypeptides • Misfolded proteins can be lethal Vacuoles associated with spongiform encephalopathies
Protein Function • Protein function often includes reversible binding interactions with other molecules. • Complementary interactions between proteins and ligands are the basis of self vs non-self recognition by the immune system. • Specific protein interactions modulated by chemical energy are the basis of muscle movement.
oxygen-binding proteins have a heme prosthetic group
oxygen-binding proteins have a heme prosthetic group hemoglobin http://www.youtube.com/watch?v=5LjLFrmKTSA&feature=related
protein-ligand interactions can be measured association equilibrium: Ka = [PL] / [P] [L] dissociation equilibrium: Kd = [P] [L] / [PL] O2 binding to myoglobin θ = fraction of ligand-binding sites occupied Which protein (X or Y) has greater affinity for ligand A?
Hemoglobin Binds O2 is a cooperative process. Binding affinity of Hb for O2 is increased by the O2 saturation of the molecule with the first O2 bound influencing the shape of the binding sites (conformation change) for the next O2
hemoglobin-O2 binding is influenced by pH Hb, binds H+ and CO2 as well as O2, but all at different sites. Binding of H+ and CO2 is inversely related to binding of O2. Low pH = high [H+] = lower O2 binding.
hemoglobin-O2 binding allosterically modulated by 2,3-bisphosphoglycerate BPG reduces the affinity of Hb for O2. BPG binds at a site distant from the O2-binding site and regulates the affinity of Hb for O2.
immune responses are mediated by protein interactions that distinguish self and non-self Cellular immune response - T cells destroy host cells infected by viruses Humoral immune response – B cells produce antibodies or immunoglobulins against bacteria, viruses and foreign molecules
muscle contraction is also based on protein interactions and conformational changes Muscle contraction occurs by the sliding of the thick (myosin) and thin (actin) filaments past each other Conformational changes in the myosin head are coupled to ATP hydrolysis http://www.sci.sdsu.edu/movies/actin_myosin _gif.html
1. What 2 functional groups are present in all amino acids? 2. Name the simplest amino acid. Is it a chiral molecule? 3. Approximately how many amino acids are needed to make the proteins found in the body?
5. What is meant by the primary, secondary and tertiary structures of proteins? 6. What type of bonds are responsible for the helix structure of some proteins? 5. Linus Pauling and Robert Corey found that the C—N bond in the peptide link is intermediate in length between a single and double bond. They also found that the peptide bond is planar. a) What does the length of the bond tell us about the strength and bond order? b) What does the observations tell us about the ease of rotation about the C—N peptide bond?
9. What is the effect of the following changes on the O2 affinity of hemoglobin? a) Drop in pH of blood plasma a) A decrease of partial pressure of CO2 in the lungs a) Increase in BPG levels a) Increase in CO
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