Biochemistry-2-1_AA 1 Biochemistry 2.1. Amino acids and peptides Biochemistry-2-1_AA 2 Protein structure & function Proteins serve crucial functions in essentially all biological processes 1 Proteins are built from a repertoire of 21 amino acids 2 Primary structure: amino acids linked by peptide bonds form polypeptide chains 3 Secondary structure: polypeptide chains fold into regular structures such as alpha helix, beta sheet, & turns & loops 4 Tertiary structure: water-soluble proteins fold into compact structures with nonpolar cores 5 Quaternary structure: polypeptide chains can assemble into multisubunit structures 6 The amino acid sequence of a protein determines its three-dimensional structure Biochemistry-2-1_AA 3 Proteins - Key properties - a wide range of functions 1. Proteins are linear polymers built of monomer units called amino acids - spontaneously fold into 3-dimensional structures 2. Proteins contain a wide range of functional groups - alcohols, thiols, thioethers, carboxylic acids, carboxamides, & a variety of basic groups - eg. chemical reactivity essential to function of enzymes 3. Proteins can interact with one another, & with other biological macromolecules to form complex assemblies macromolecular machines 4. Some proteins are quite rigid, whereas others display limited flexibility - structural elements in the cytoskeleton v parts that act as hinges, springs, & levers etc Biochemistry-2-1_AA 4 Amino acids  Proteins are polymers of amino acids, with each amino acid residue joined to its neighbor by a specific type of covalent bond.  L-α-amino acids and their derivatives participate in cellular functions as diverse as nerve transmission and the biosynthesis of porphyrins, purines, pyrimidines, and urea. Proteins: Essential for all organisms  AA  Peptide  Polypeptide  Proteins more 50 AA Biochemistry-2-1_AA 5 Amino acids and Proteins 2.1) Amino acids - Amino acid classes - Modified AA in proteins - AA stereo isomers - Titration of AA - AA reactions 2.2) Peptides 2.3) Protein structure - Protein structure - Fibrous proteins - Globular proteins Biochemistry-2-1_AA 6 AA functions (300 AA)  Primary function (components of proteins)  Chemical messenger: - Neurotransmiters (substance released from one nerve cell - that influence function second nerve cell) - GABA (g-aminobutyric acids), glycin, serotonin (tryptophan) - Hormones (chemical messanger ….produced by one type cell and regulate function of other type of cell) - Thyroxine (tyrosin) - Indole acetic acid (plant) - Precursores for nitrogen containing molecules - Nucleotides, heme, chlorophyl - Metabolic intermediates: arginine, citruline, ornithine – urea cycle Biochemistry-2-1_AA 7 Amino acids  Standard AA ( in proteins 21)  Nonstandard AA (modified after incorporation to polypeptide) General structure L- a- amino acids proline Alpha- imidoaci ds 300 AA 20 AA (proteins, gene code…) Biochemistry-2-1_AA 8 Biochemistry-2-1_AA 9 Protein subunits: aamino acids: L & D isomers Mirror images of each other R group = side chains Amino group Carboxylic acid group Only L amino acids found in proteins. Ca chiral, L & D isomers not symmetrical,except glycine Amino acid stereoisomers enantiomers Biochemistry-2-1_AA 10 Tetrahedral a-carbon atom, Ca chiral center is S configuration. (sinister for left) Counterclockwise arrow indicates chiral center is S configuration Amino group: highest priority substituent (according to atomic #) Only L amino acids in proteins Biochemistry-2-1_AA 11 Amino Acids May Have Positive, Negative, or Zero Net Charge - Titration of Amino acids  -COOH and -NH3 weak acid groups exist in solution in protonic equilibrium: amphoteric zwitterions (neutral, physiological pH) - Full disociation COOH and NH2 groups at physiological pH Biochemistry-2-1_AA 12 Ionization state as a function of pH Physiological pH (measure of [H+]) Biochemistry-2-1_AA 13 pKa express the strengths of weak acids Biochemistry-2-1_AA 14 pKa of ionizable side chains pKa = pH for 50% dissociation, Note range Biochemistry-2-1_AA 15 The 20 Amino Acids Found in Proteins Biochemistry-2-1_AA 16 Classification of AA  Neutral nonpolar  Neutral polar  Acidic  Basic Biochemistry-2-1_AA 17 Neutral nonpolar  Interact poorly with water  Hydrophobic  3D structure of proteins  Aliphatic and aromatic Biochemistry-2-1_AA 18 Neutral polar Amide group is highly polar, affect protein stability…. Biochemistry-2-1_AA 19 Cysteine Similar to Serine with sulfhydryl, or thiol (-SH) group replacing hydroxyl (-OH) group -SH more reactive than -OH. -SH pairs form disulfide bonds (aka bridges), key role stabilizing proteins Biochemistry-2-1_AA 20 Acidic AA  Negative charge at physiological pH Aspartic acid Glutamic acid A A Biochemistry-2-1_AA 21 Basic AA  Positive charge at physiological pH  Ionic bonds with amino acids  Arginine - strong base, no function in acide/base reaction  Lysin – ammonium ion, oxidation of lysines side chain – collagen, linkage  Histidine – weak base, ony partial disociation at ph 7, react as buffer,  Catalytic activity of enzymes B B B Biochemistry-2-1_AA 22 Aromatic side chains Hydrophobic & Hydrophilic properties Aromatic rings have delocalized  electrons, Absorb UV light Biochemistry-2-1_AA 23 Optical properties of AA and proteins TEST Biochemistry-2-1_AA 24 Absorption spectra of Trp & Tyr Beer’s law: A = cl. Used to estimate protein concentration Biochemistry-2-1_AA 25 TEST Biochemistry-2-1_AA 26 Amino acid reactions - peptide bond - disulfide bridge Biochemistry-2-1_AA 27 Primary structure: Peptide bond, between AAs Between a-carboxyl group of one AA & a-amino group of another 2 amino acids Dipeptide Loss of H2O Equilibrium favors hydrolysis, hence, biosynthesis of peptide bonds require free energy input Peptide bonds are stable kinetically Formation of a peptide bond by condensation. The amino group of one amino acid (with R2 group) acts as a nucleophile to displace the hydroxyl group of another amino acid (with R1 group),forming a peptide bond (shaded in yellow). Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily displaced. At physiological pH, the reaction shown does not occur to any appreciable extent. Biochemistry-2-1_AA 28 Formation of a Peptide Biochemistry-2-1_AA 29 Polypeptide bonds are planar Six atoms (Ca, C, O, N, H, Ca) lie in a plane, in a pair of aa Biochemistry-2-1_AA 30 Planarity of Peptide (Amide) Bond Biochemistry-2-1_AA 31 . Biochemistry-2-1_AA 32 Main chain or backbone Constant backbone: regularly repeating part Distinctive side chains (R-groups): variable part AA unit in a polypeptide is called a residue, which contains, a carbonyl group; good hydrogen-bond acceptor, an NH group (except Pro); good hydrogen-bond donor Biochemistry-2-1_AA 33 Polypeptide chain has direction N- C- Biochemistry-2-1_AA 34 Examples of Oligopeptides Biochemistry-2-1_AA 35 Biochemistry-2-1_AA 36 N- and C-Termini May Be Modified in Proteins Biochemistry-2-1_AA 37 Cysteine oxidation -Highly reactive SH- (sulphydryl group of Cysteine) - reversible oxidation – to form disulfide -CYSTINE (disulfide bond) – -DISULFIDE BRIDGE -- singe chain, 2 separate chains, -In proteins: stability Biochemistry-2-1_AA 38 Cross links (disulfide bridges) Prevalent mainly in extracellular proteins Biochemistry-2-1_AA 39  Glutathione –  (g-glutamyl-L-cysteinnylglycine) (g-amide bond, g-carboxy group contributes on peptide bond) Function: - Protein and DNA synthesis, drug and environmental toxin metabolism, amino acid transport - Reducing agent - Protects cells from destructive effects oxidation - GSH/GSSG is high –normally present in cells - Important intracellular reducing agent - Glutathione peroxidase (methemoglobin) TEST Biochemistry-2-1_AA 40 Peptide hormones •Vertebrate hormones: many small peptides exert their effects at very low concentrations - small peptides. •Hypothalamus: Oxytocin (nine amino acid residues), which is secreted by the posterior pituitary and stimulates uterine contractions; Vasopressin bradykinin (nine residues), which inhibits inflammation of tissues; and thyrotropin-releasing factor (three residues), which is formed in the hypothalamus and stimulates the release of another hormone, thyrotropin, from the anterior pituitary gland. Biochemistry-2-1_AA 41 Peptides  Aspartame L-aspartyl-L-phenylalanine methyl ester, the artificial sweetener better known as aspartame or NutraSweet. Biochemistry-2-1_AA 42 Leu L -C-C-CONH2-C-CONH2 -C-COOH -C-C-COOH -H -CH3 -C-OH -C-SH -C-C-S-C PPro -C-C C N N+ -C-C-C-C-NH3 + -C-C- -OH -C- N South line Circular line Aliphatic Amide Acidic Imino, Circular Basic SulfurHydroxy Aromatic -C-C-C-N-C-N N+ = C -C-C-C C -C-C-C C C -C C C C HN C-COOH a-C-C OH Gln QAsn N Asp D Glu EPhe F Arg R Lys K His H Gly G AAAla VVal IIle YTyr Ser S Thr T Met M Cys C Amino Acid Subway Map Trp W Non-polar Polar This is NOT a metabolic pathway TEST Biochemistry-2-1_AA 43 POLAR NON- POLAR Tyr His Gly Acidic Neutral Basic Asp Glu Gln Cys Asn Ser Thr Lys Arg Ala Val Ile Leu Met Phe Trp Pro Classification of Amino Acids by Polarity Polar or non-polar, it is the bases of the amino acid properties. TEST Biochemistry-2-1_AA 44 Primary Structure of Bovine Insulin First protein to be fully sequenced (by Fred Sanger in 1953). For this, he won his first Nobel Prize (his second was for the Sanger dideoxy method of DNA sequencing). Biochemistry-2-1_AA 45 Bovine insulin: AA sequence 1953, Fred Sanger determined aa sequence of insulin, landmark! Showed for 1st time, protein has precisely defined aa sequence Also showed that only L-amino acids were present, linked by peptide bonds Now, aa sequence of > 100,000 proteins are known 1950s-1960s studies showed aa sequence genetically determined Each of 20 aa encoded by one or more specific sequences of 3 nucleotides. Biochemistry-2-1_AA 46 Peptid Some extremely toxic mushroom poisons, such as amanitin, are also small peptides, as are many antibiotics (neomycin, kanamycin). Biochemistry-2-1_AA 47 Biochemistry-2-1_AA 48 Biochemistry-2-1_AA 49 Biochemistry-2-1_AA 50 Evolution and Conservation of Protein Sequences Translation elongation factor Tu/1a Myoglobin Biochemistry-2-1_AA 51 The Genetic Code Biochemistry-2-1_AA 52 DNA RNA Protein Biochemistry-2-1_AA 53 Initiating Amino Acid in Translation N-Formylmethionine in prokaryotes Just methionine in eukaryotes N H H N S CH3 O R OO H H3N H N S CH3 O R O Biochemistry-2-1_AA 54 Charging of tRNAs with Specific Amino Acids Biochemistry-2-1_AA 55 Translation of mRNA into Protein Biochemistry-2-1_AA 56 Ribosomal Peptidyl Transferase Activity Note: the catalytic component of the ribosome’s peptidyl transferase activity is RNA; it’s an example of a catalytic RNA or ribozyme. Biochemistry-2-1_AA 57 Disulfide Bond Formation in Insulin Biochemistry-2-1_AA 58 Methods in Protein Biochemistry Biochemistry-2-1_AA 59 Gel Electrophoresis Biochemistry-2-1_AA 60 Polyampholyte Character of a Tetrapeptide and Isoelectric Points Isoelectric Point (pI), pH at which molecule has net zero charge, determined using computer program for known sequence or empirically (by isoelectric focusing). Group pKa a-NH3 + 9.7 Glu g-COOH 4.2 Lys -NH3 + 10.0 a-COOH 2.2 Biochemistry-2-1_AA 61 Biochemistry-2-1_AA 62 Isoelectric Focusing Electrophoresis through polyacrylamide gel in which there is a pH gradient. Biochemistry-2-1_AA 63 Biochemistry-2-1_AA 64 Two-Dimensional Gel Electrophoresis  Separate proteins based on isolectric point in 1st dimension  Separate proteins based on molecular weight in 2nd dimension Biochemistry-2-1_AA 65 “Salting Out”: Ammonium Sulfate Precipitation in Protein Fractionation Biochemistry-2-1_AA 66 Centrifugation Centrifugation Methods •Differential (Pelletting) – simple method for pelleting large particles using fixedangle rotor (pellet at bottom of tube vs. supernatant solution above) •Zonal ultracentrifugation (e.g., sucrosegradient) – swinging-bucket rotor •Equilibrium-density gradient ultracentrifugation (e.g., CsCl) – swinging-bucket or fixed-angle rotor Low-speed, high-speed, or ultracentrifugation: different spin speeds and g forces Biochemistry-2-1_AA 67 Zonal Centrifugation: SucroseGradient Preparative Ultracentrifugation Separates by sedimentation coefficient (determined by size and shape of solutes) Biochemistry-2-1_AA 68 Sucrose-Gradient Preparative Ultracentrifugation Biochemistry-2-1_AA 69 Equilibrium Density Gradient Ultracentrifugation  Used in Meselsen-Stahl experiment.  Separates based on densities of solutes.  Does not require premade gradient.  Pour dense solution of rapidly diffusing substance in tube (usually CsCl).  Density gradient forms during centrifugation (“self-generating gradient”).  Solutes migrate according to their buoyant density (where density of solute = density of CsCl solution). Biochemistry-2-1_AA 70 Column Chromatography Flow-through Eluate Biochemistry-2-1_AA 71 Different Types of Chromatography  Gel filtration/size exclusion/molecular sieve separates by size (molecular weight) of proteins  Ion exchange (cation exchange and anion exchange) - separates by surface charge on proteins  Cation exchange: separates based on positive charges of solutes/proteins, matrix is negatively charged  Anion exchange: separates based on negative charges of solutes/proteins, matrix is positively charged  Hydrophobic interaction - separates by hydrophobicity of proteins  Affinity - separates by some unique binding characteristic of protein of interest for affinity matrix Biochemistry-2-1_AA 72 Ion-Exchange Chromatography Biochemistry-2-1_AA 73 Gel Filtration Chromatography Biochemistry-2-1_AA 74 Affinity Chromatography Biochemistry-2-1_AA 75 Cleavage of Polypeptides for Analysis  Strong acid (e.g., 6 M HCl) - not sequence specific  Sequence-specific proteolytic enzymes (proteases)  Sequence-specific chemical cleavage (e.g., cyanogen bromide cleavage at methionine residues) Biochemistry-2-1_AA 76 Protease Specificities Biochemistry-2-1_AA 77 Cyanogen Bromide Cleavage at Methionine Residues Biochemistry-2-1_AA 78 Biochemistry-2-1_AA 79 Protein Sequencing: Edman Degradation PTH = phenylthiohydantion F3CCOOH = trifluoroacetic acid PTC = phenylthiocarbamyl Biochemistry-2-1_AA 80 Identification of N-Terminal Residue Note: Identification of C-terminal residue done by hydrazinolysis (reaction with anhydrous hydrazine in presence of mildly acidic ion exchange resin) or with a C-terminus-specific exopeptidase (carboxypeptidase). NO2 Biochemistry-2-1_AA 81 Separation of Amino Acids by HPLC Biochemistry-2-1_AA 82 Protein Identification by Mass Spectrometry Biochemistry-2-1_AA 83 Protein Identification by Mass Spectrometry Two main approaches: 1. Peptide mass fingerprinting: Proteolytic digestion of protein, then determination m/z of peptides by MS (e.g., MALDI-TOF or ESI-TOF), search “fingerprint” against database. Success of ID depends on quality/ completeness of database for specific proteome. 2. Tandem MS (MS/MS – e.g., nanoLC-ESI-MS/MS): Proteolytic digestion of protein, separation and determination of m/z of each (MS-1), then determination of collision-induced dissociation fragment spectrum for each peptide (MS-2). Gives context/sequence-dependent information, so more of a do novo sequencing method. Biochemistry-2-1_AA 84 Locating Disulfide Bonds O O- I iodoacetate Biochemistry-2-1_AA 85 Determing Primary Structure of an Entire Protein Biochemistry-2-1_AA 86 Reactions in Solid-Phase Peptide Synthesis Biochemistry-2-1_AA 87 Biochemistry-2-1_AA 88 Summary 1) Amino acids can be joined covalently through peptide bonds to form peptides and proteins. Cells generally contain thousands of different proteins, each with a different biological activity. 2. Proteins can be very long polypeptide chains of 100 to several thousand amino acid residues. However, some naturally occurring peptides have only a few amino acid residues. Some proteins are composed of several no covalently associated polypeptide chains, called subunits. Simple proteins yield only amino acids on hydrolysis; conjugated proteins contain in addition some other component, such as a metal or organic prosthetic group. 3. The sequence of amino acids in a protein is characteristic of that protein and is called its primary structure. This is one of four generally recognized levels of protein structure.