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1 Determining the sequence One way: use an enzyme: Carboxypeptidase: hydrolyzes the peptide bond (an old method, but useful for teaching) , identify e.g., …. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu-asp-ser …. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu-asp + …. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu + ser asp asp ser 2 METHODS . . . AA mixture (ala, glu, lys Anode (-) (+) Cathode Note: The cathode is negative in an electrophoresis apparatus even though it is positive in a battery (voltaic cell). 3 A paper electrophoresis apparatus 2000 to 4000 volts DC, dangerous 4 Handout 3-4 Side view AAs applied at lower end Isopropanol 5 “front” =1.00 “Rf” 0.82 After stopping the paper chromatography and staining for the amino acids: 0.69 0.45 0.27 Most hydrophobic = furthest Most hydrophilic = least far 0.11 6 Paper chromatography apparatus (felt tip black marker ink demonstration) 7 Ordering the sub-peptides within the polypeptifde: • • • Treatment of a polypeptide with trypsin Trypsin is a proteolytic enzyme. It catalyzes cleavage (hydrolysis) after lysine and arginine residues Polypeptide chain “Sub-peptides” Determine sequence of each subpeptide using the carboxypeptidase technique 8 The order of the subpeptides is unknown. The sequence is reconstructed by noting the overlap between differently produced subpeptides Trypsin (lys, arg) (1) Chymotrypsin (trp, tyr, phe) (2) N C Sequence overlap Done! 9 Quick way to compare two proteins without sequencing: Fingerprinting a protein: analysis of the sub-peptides themselves. (Without sequencing, i.e., without breaking them down to their constituent amino acids) Application to sickle cell disease (Vernon Ingram, 1960’s) Hemoglobin protein trypsin Sub-peptides No further digestion to amino acids; left as sub-peptides 10 Oligopeptides behave as a composite of their constituent amino acids + - - Net charge = -2+1= -1: moves toward the anode in paper electrophoreses Fairly hydrophobic (~5/6): expected to move moderately well in paper chromatography Nomenclature: ala-tyr-glu-pro-val-trp or AYEPVW or alanyl-tyrosyl-glutamyl-prolyl-valyl-tryptophan 11 Hb protein In fingerprinting, these spots contain peptides, not amino acids trypsin The mixture of all sub-peptides formed Less negatively charged, more hydrophobic Negatively charged Sequence just this peptide: ------valine-----(sickle) Positively charged Single AA substitution ---glutamate--(normal) More hydrophobic negatively charged positively charged More hydrophilic 12 Every different polypeptide has a different primary structure (sequence). By definiton. The migration behavior of each sub-peptide depends on its composite properties. The properties are suffiently complex such that most subpeptides in a given polypeptide will behave differently. Every polypeptide will have different arrangement of spots after fingerprinting. Four polypeptide fingerprints 13 3-dimensional structure of proteins One given purified polypeptide • Molecule #1: N-met-leu-ala-asp-val-val-lys-.... • Molecule #2: N-met-leu-ala-asp-val-val-lys-... • Molecule #3: N-met-leu-ala-asp-val-val-lys-... • Molecule #4: N-met-leu-ala-asp-val-val-lys-... etc. clothesline . . . 14 Information for proper exact folding (How does a polypeptide fold correctly?) Predicting protein 3-dimensional structure Determining protein 3-dimensional structure Where is the information for choosing the correct folded structure? Is it being provided by another source (e.g., a template) or does it reside in the primary structure itself? “Renaturation” of a hard-boiled egg Denature by heat Cool, renature? X ovalbumin Too long Cool, entangled to sort out Tangle, gel. Probably due to non-productive hydrophobic interactions 15 16 urea H H H O || N-C-N H chaotropic agent used at very high concentrations (e.g., 7 M) gentler, gradual denaturation, renaturation 17 “Renaturation” of ribonuclease after urea + urea, denature -urea, renature “native” ribonuclease active enzyme compact ?? denatured ribonuclease inactive enzyme random coil 18 Slow denaturation of ribonuclease by urea O || Urea = H2N-C—NH2 Ribonuclease in the bag is denatured Now dialyze out the urea Macromolecules (protein here) cannot permeate bag material Small molecules (H20, urea) can permeate. Ribonuclease RENATURES in the absence of any other material Urea will move from areas of high concentration to areas of low concentration. Christian Anfinsen: PRIMARY STRUCTURE DETERMINES TERTIARY STRUCTURE. + urea, denatures - urea, renatures “The Anfinsen Experiment” 19 Julio Fernandez lab, CU: a modern version of the Anfinson experiment Force needed to pull Pull Length 20 21 Denaturation/renaturation of domains ofa protein (titin) using the atomic force microscope. 22 BUT: Chaperonins (made of proteins themselves) • Help fold proteins during synthesis • Perhaps by preventing illegitimate interactions, like intermolecular contacts via exposed hydrophobic groups of partially folded proteins • Also help re-fold proteins that have denatured after passing through a membrane’s P-lipid bilayer, e.g., during transport into a mitochondrion (organelle). 23 Zsolt Török, Laszlo Vigh and Pierre Goloubinoff, 1996 The Journal of Biological Chemistry, 271, 16180-16186. 24 Protein folding Primary structure itself results in some folding constraints: See bottom of handout 3-3 25 26 And these 4 atoms are in one plane (N central) These 4 redatoms are ininone so 6 atoms oneplane plane (C of C=O central) 27 28 29 30 31 There’s still plenty of flexibility 32 This graphic intentionally left blank. Secondary structure: the alpha helix 33 Amino acids shown simplified, without side chains and H’s. Almost every N-H and C=O group can participate 34 Alpha helix depictions C = grays N = blue O = red Poly alanine Side chains = -CH3 (lighter gray) H’s not shown 35 Linus Pauling and a model of the alpha helix.1963 Secondary structure: H-bond AA residue beta pleated sheet 36 37 Beta sheet (i.e., beta pleated sheet) antiparallel antiparallel parallel 38 Beta-sheets Anti-parallel Parallel 39 secondary structure (my definition): structure produced by regular repeated interactions between atoms of the backbone. 40 Tertiary structure: The overall 3-D structure of a polypeptide. Neither This is a popular “ribbon” model of protein structure. Get familiar with it. The ribbons are stretches of single polypeptide chains. A single ribbon is NOT a sheet. A beta sheet 3 alpha helices These “ribbon” depictions do not show the side chains, only the backbone Tertiary structure (overall 3-D) 41 ionic hydrophobic H-bond cys Ion - dipole interaction covalent Van der Waals In loop regions and in regions of secondary structure 42 Disulfide bond formation Disulfide bond (covalent, strong) Sulfhydryl group R-CH2-SH cysteine + HS-CH2-R cysteine ½ O2 R-CH2-S-S-CH2-R + HOH cystine Two sulfhydryls have been oxidized (lost H’s) Oxygen has been reduced (gained H’s). Oxygen was the oxidizing agent (acceptor of the H’s). An oxidation-reduction reaction: Cysteines are getting oxidized (losing H atoms, with electron; NOT losing a proton, not like acids.) Oxygen is getting reduced, gaining H-atoms and electrons Actually it’s the loss and gain of the electrons that constitutes oxidation and reduction, respectively. No catalyst is usually needed here. 43 Overall 3-D structure of a polypeptide is tertiary structure Stays intact in the jacuzzi at 37 deg C Usually does not require the strong covalent disulfide bond to maintain its 3-D structure [Tuber mode]l Protein structures are depicted in a variety of ways Backbone only Ribbon Small molecule bound Drawing attention to a few side groups Continuous lines, ribbons= backbone (not sheets) Space-filing, with surface charge blue = + red = - Space-filling 44 45 Most proteins are organized into 46 4o, 47 QUATERNARY STRUCTURE Monomeric protein (no quaternary structure) Dimeric protein (a homodimer) The usual weak bonds Dimeric protein (a heterodimer) Also called: multimeric proteins A heterotetramer A heteropolymeric protein (large one) 48 Hemoglobin $ One protein $ Four polypeptide chains, 2 identical alphas and 2 identical betas Four “subunits” Molecular weight $ 16,000 Subunit molecular weight 16,000 Subunit molecular weight 64,000 Protein molecular weight $ $ 64,000, even though the 4 chains are not covalently bonded to each other 49 Tetramer Two heavy chains (H), Two light chains (L) Interchain disulfide bonds The 4 weak bond types 50 Sickle cell disease Normal glu glu Sickle cell glu glu val val val val Some small molecules can by added to a protein via covalent bonds. One form of a “prosthetic group”. Pyridoxal phosphate = Vitamin B6 51 52 Most prosthetic groups are bound tightly via weak bonds. Tetrahydrofolic acid ~ vitamin B9 Riboflavin ~ vitamin B2 Heme 53 Membrane proteins 54 Membrane proteins Hydrophobic side chains on the protein exterior for the portion in contact with the interior of the phospholipid bilayer. Anions are negatively charged. Cations are positively charged Small molecules bind with great specificity to pockets on protein surfaces 55 Too far 56 Estrogen receptor binding estrogen, a steroid hormone detail estrogen estrogen Estrogen receptor is specific, does not bind testosterone 57 58 Protein binding can be very specific Testosterone Estrogen