Transcript CH 2 -CH 2
1 Primary structure itself results in some folding constraints: See bottom of handout 3-3 2 3 4 atoms in one plane 6 atoms in one plane 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. Alpha helix depictions 10 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 12 H-bond AA residue 13 Beta sheet antiparallel antiparallel parallel Peptides can lie either antiparallel and parallel to each other in a beta sheet 14 Beta-sheets Anti-parallel Parallel 15 secondary structure (my definition): structure produced by regular repeated interactions between atoms of the backbone. Tertiary structure: The overall 3-D structure of a polypeptide. Neither Beta-sheets Alpha-helices 16 Tertiary (3o) structure (overall 3-D) ionic hydrophobic H-bond Ionic-H (polar interaction) Van der Waals 17 18 Disulfide bond formation cystine R-CH2-SH cysteine + HS-CH2-R cysteine ½ O2 R-CH2-S-S-CH2-R + HOH An oxidation-reduction reaction: Cysteines are getting oxidized (losing H atoms, with electron, NOT just a proton, as in an acid). 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. Tertiary (3o) structure (overall 3-D) ionic hydrophobic H-bond cys Ionic-H combo Covalent (strong) Van der Waals 19 20 Neither Beta-sheets Alpha-helices Stays intact in the jacuzzi at 37 deg C Usually does not require the strong covalent disulfide bond to maintain its 3-D structure Protein structures are depicted in a variety of ways Backbone only No side chains shown Backbone as a ribbon+line Backbone as a line Space-filing, With surface charge 21 22 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 in the primary structure itself? “Renaturation” of a hard-boiled egg Heat denature Cool, renature? X ovalbumin Too long to sort out Tangle, gel. Probably due to non-productive hydrophobic interactions Cool, entangled 23 24 urea H H H O || N-C-N H chaotropic agent used at very high concentrations (e.g., 7 M) gentler, gradual denaturation, renaturation 25 “Renaturation” of ribonuclease after urea + urea, denature - urea, renature “Native” ribonuclease Active enzyme compact Denatured ribonuclease Inactive enzyme Random coil 26 Reducing the disulfide bonds in ribonuclease to sulfhydryls HO-CH2-CH2-S-S-CH2-CH2-OH + (β-mercaptoethanol) 2 HO-CH2-CH2-SH HS-SH More β-mercaptoethanol Slow denaturation of ribonuclease by urea O || Urea = H2N-C—NH2 27 Ribonuclease in the bag is denatured Now dialyze out the urea Ribonuclease Macromolecules (protein here) cannot permeate bag material Small molecules (H20, urea) can. Urea will move from areas of high concentration to areas of low concentration RENATURES in the absence of any other material 28 Christian Anfinsen: PRIMARY STRUCTURE DETERMINES TERTIARY STRUCTURE. + urea, denatures - urea, renatures 29 Denaturation / renaturation of a protein (titin) using the atomic force microscope. 30 Julio Fernandez and colleagues, Columbia Univ. BUT: Chaperonins 31 (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). 4o, QUATERNARY STRUCTURE 32 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 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 33 34 Tetramer Two heavy chains (H), Two light chains (L) Interchain disulfide bonds The 4 weak bond types 35 Sickle cell disease Normal -glu glu- -glu Sickle cell glu- val val val val 36 Most proteins are organized into 37 Some prosthetic groups 38 Tetrahydrofolic acid ~ vitamin B9 Pyridoxal phosphate ~ vitamin B6 Riboflavin ~ vitamin B2 Heme 39 Membrane proteins Membrane proteins 40 Small molecules bind with great specificity to pockets on protein surfaces 41 Too far Estrogen receptor binding estrogen, a steroid hormone detail estrogen estrogen 42 Protein separation methods Ultracentrifugation Mixture of proteins 43 44 Causing sedimentation: centrifugal force = m(omega)2r m = mass omega = angular velocity r = distance from the center of rotation Opposing sedimentation = friction = foV. Constant velocity is soon reached: centrifugal force = frictional force So: m(omega)2r = foV fo = frictional coefficient (depends on shape) And: V = m(omega)2r/fo, Or: V = [(omega)2r] x [ m / fo ] V proportional to mass (MW) V inversely proportional to fo (shape) V inversely proportional to non-sphericity (Spherical shape moves fastest) Ultracentrifuge 45 Glass plates 46 Large, +++ high positive charge Large, + low positive charge Small, +++ High positive charge Small, + Low positive charge Molecules shown after several hours of electrophoresis 47 Glass plates 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 48 49 Electrode connection Power supply 50 Tracking dyes 51 SDS PAGE = SDS polyacrylamide gel electrophoresis 52 • 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) 53 Molecular weight markers (proteins of known molecular weight) Molecular sieve chromatography (=gel filtration, Sephadex chromatography) Sephadex bead 54 Molecular sieve chromatography Sephadex bead 55 Molecular sieve chromatography Sephadex bead 56 Molecular sieve chromatography Sephadex bead 57 Molecular sieve chromatography Sephadex bead 58 59 Handout 4-3: protein separations 60