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1 Protein folding Primary structure itself results in some folding constraints: See bottom of handout 3-3 2 3 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) 4 5 6 7 8 There’s still plenty of flexibility Secondary structure: the alpha helix 9 Amino acids shown simplified, without side chains and H’s. Almost every N-H and C=O group can participate 10 Alpha helix depictions C = grays N = blue O = red Poly alanine Side chains = -CH3 (lighter gray) H’s not shown 11 Linus Pauling and a model of the alpha helix.1963 Secondary structure: H-bond AA residue beta pleated sheet 12 13 Beta sheet (i.e., beta pleated sheet) antiparallel antiparallel parallel 14 Beta-sheets Anti-parallel Parallel 15 secondary structure (my definition): structure produced by regular repeated interactions between atoms of the backbone. 16 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. 3 alpha helices These “ribbon” depictions do not show the side chains, only the backbone Tertiary structure (overall 3-D) 17 ionic hydrophobic H-bond cys Ion - dipole interaction covalent Van der Waals Examples of bonds determining 3D structure Exist in loop regions and in regions of secondary structure 18 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. 19 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 20 21 Most proteins are organized into 22 Handout 4-2 23 Two different proteins with almost the same 3-D structure ! Handout 4-2 4o, 24 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) 25 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 26 Tetramer Two heavy chains (H), Two light chains (L) Interchain disulfide bonds The 4 weak bond types 27 Sickle cell disease Normal glu glu Sickle cell glu glu val val val val Some small molecules can be bound tightly to a protein. Such associated small molecule are called “prosthetic groups”. Some are even covalently bound to the protein. Pyridoxal phosphate AA side chain = Vitamin B6 Enzyme 28 29 Most prosthetic groups are bound tightly via weak bonds. Tetrahydrofolic acid ~ vitamin B9 Riboflavin ~ vitamin B2 Heme Membrane proteins 30 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 31 Too far 32 Ligand Protein Ligand binding can be equisitely specific: the estrogen reeptor binds estrogen but not testosterone. Testosterone Estrogen 33 34 Protein separation methods Ultracentrifugation Mixture of proteins Ultracentrifuge 35 Causing sedimentation: centrifugal force = m(omega)2r m = mass omega = angular velocity r = distance from the center of rotation Opposing sedimentation = friction = foV. fo = frictional coefficient (shape) V = velocity Constant velocity is soon reached; then, no tnet force So: centrifugal force = frictional force (balanced each other out) And so: m(omega)2r = foV And: V = m(omega)2r/fo, Or: V = [(omega)2r] x [m / fo] V proportional to mass (MW) V inversely proportional to fo V inversely proportional to “non-sphericity” (spherical shape moves fastest) 36 (“native”)37 Glass plates Sample loaded here + + polyacrylamide fibers +++ +++ + +++ + + +++ Winner: Small, +++ High positive charge +++ + +++ Loser: Large, + low positive charge Intermediate: Large, +++ high positive charge Intermediate: Small, + Low positive charge Molecules shown after several hours of electrophoresis 38 Upper resevoir Cut out for contact of buffer with gel 39 Cut out of glass plate for contact of buffer with gel Electrode connection (~ 150 V) Power supply Tracking dyes 40 41 42 SDS PAGE = SDS polyacrylamide gel electrophoresis • sodium dodecyl sulfate, SDS (or SLS): CH3-(CH2)11- SO4-• CH3-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-SO4-- SDS All the polypeptides are denatured and behave as random coils All the polypeptides have the same charge per unit length All are subject to the same electromotive force in the electric field Separation based on the sieving effect of the polyacrylamide gel Separation is by molecular weight only SDS does not break covalent bonds (i.e., disulfides) (but can treat with mercaptoethanol for that) (and perhaps boil for a bit for good measure) 43 Disulfides between 2 cysteines can be cleaved in the laboratory by reduction, i.e., adding 2 Hs (with their electrons) back across the disulfide bond. One adds a reducing agent: mercaptoethanol (HO-CH2-CH2-SH). In the presence of this reagent, one gets exchange among the disulfides and the sulfhydryls: Protein-CH2-S-S-CH2-Protein + 2 HO-CH2CH2-SH ---> Protein-CH2-SH + HS-CH2-Protein + HO-CH2CH2-S-S-CH2CH2-OH The protein's disulfide gets reduced (and the S-S bond cleaved), while the mercaptoethanol gets oxidized, losing electrons and protons and itself forming a disulfide bond. 44 P.A.G.E. e.g., “p53” Molecular weight markers (proteins of known molecular weight) 45 Molecular sieve chromatography (= gel filtration, Sephadex chromatography) Sephadex bead 46 Molecular sieve chromatography Sephadex bead 47 Molecular sieve chromatography Sephadex bead 48 Molecular sieve chromatography Sephadex bead 49 Molecular sieve chromatography Sephadex bead 50 Fancy Plain 4oC (cold room) Larger molecules get to the bottom faster, and …. Non-spherical molecules get to the bottom faster ~infrequent orientation Non-spherical molecules get to the bottom faster 51 52 Handout 4-3: protein separations 53 Winners: Largest and most spherical Lowest MW Largest and least spherical Similar to handout 4-3, but Winners & native PAGE added Winners: Most charged and smallest 54 Enzymes = protein catalysts 55 Flow of glucose in E. coli Macromolecules Polysaccharides Lipids Nucleic Acids Proteins yn th e tic pa t hw ay monomers bi os intermediates glucose Each arrow = an ENZYME Each arrow = a specific chemical reaction 56 Chemical reaction between 2 reactants H 2 + I2 2 HI H 2 + I2 2 HI + energy “Spontaneous” reaction: Energy released Goes to the right H-I is more stable than H-H or I-I here i.e., the H-I bond is stronger, takes more energy to break it That’s why it “goes” to the right, i.e., it will end up with more products than reactants i.e., less tendency to go to the left, since the products are more stable 57 say, 100 kcal/mole say, 103 kcal/mole H2 + I2 2 HI { Change in Energy (Free Energy) 2H + 2I Atom pulled completely apart (a “thought” experiment) -3 kcal/mole Reaction goes spontaneously to the right If energy change is negative: spontaneously to the right = exergonic: energy-releasing If energy change is positive: spontaneously to the left = endergonic: energy-requiring 58 Different ways of writing chemical reactions H 2 + I2 2 HI H 2 + I2 2 HI H 2 + I2 2 HI H 2 + I2 2 HI H 2 + I2 2 HI 59 say, 100 kcal/mole But: it is not necessary to break molecule down to its atoms in order to rearrange them say, 103 kcal/mole H2 + I2 2 HI { Change in Energy (Free Energy) 2H + 2I -3 kcal/mole 60 Reactions proceed through a transition state I I + H H I I + H H I I H H I I H H Transition state (TS) (H2 + I2) I H + I H (2 HI) Products 61 Change in Energy 2H + 2I ~100 kcal/mole H-H | | I-I (TS) Say, ~20 kcal/mole 2 HI { H 2 + I2 -3 kcal/mole Activation energy Allows it to happen Energy needed to bring molecules together to form a TS complex determines speed = VELOCITY = rate of a reaction H 2 + I2 2 HI { Change in Energy (new scale) 62 HHII (TS) Activation energy 3 kcal/mole Net energy change: Which way it will end up. the DIRECTION of the reaction, independent of the rate 2 separate concepts 63 Concerns about the cell’s chemical reactions • Direction – We need it to go in the direction we want • Speed – We need it to go fast enough to have the cell double in one generation 64 Example Biosynthesis of a fatty acid 3 glucose’s 18-carbon fatty acid Free energy change: ~ 300 kcal per mole of glucose used is REQUIRED So: 3 glucose 18-carbon fatty acid So getting a reaction to go in the direction you want is a major problem (to be discussed next time) 65 Concerns about the cell’s chemical reactions • Direction – We need it to go in the direction we want • Speed – We need it to go fast enough to have the cell double in one generation – Catalysts deal with this second problem, which we will now consider 66 The velocity problem is solved by catalysts The catalyzed reaction The catalyst takes part in the reaction, but it itself emerges unchanged 67 Change in Energy HHII (TS) Activation energy without catalyst TS complex with catalyst H 2 + I2 2 HI Activation energy WITH the catalyst 68 Reactants in an enzyme-catalyzed reaction = “substrates” 69 Reactants (substrates) Active site or Not a substrate substrate binding site (not exactly synonymous, could be just part of the active site) 70 Unlike inorganic catalysts, enzymes are specific Substrate Binding 71 Small molecules bind with great specificity to pockets on ENZYME surfaces Too far 72 Unlike inorganic catalysts, enzymes are specific succinic dehydrogenase HOOC-HC=CH-COOH <-------------------------------> HOOC-CH2-CH2-COOH +2H fumaric acid succinic acid NOT a substrate for the enzyme: 1-hydroxy-butenoate: HO-CH=CH-COOH (simple OH instead of one of the carboxyl's) Maleic acid 73 + Enzymes work as catalysts for two reasons: 1. They bind the substrates putting them in close proximity. 2. They participate in the reaction, weakening the covalent bonds of a substrate by its interaction with their amino acid residue side groups (e.g., by stretching).