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CHAPTER 3- November 1 Proteins HIGHLIGHTS Structure and Chemistry of amino acids Linkage to form a polypeptide monomer to polymer Forces that guide folding Modifications and degradation Functional design Common techniques Function depends on the structure FUNCTIONALLY VERY DIVERSE: Bind ions, nuc acids, other proteins, CHO Catalyze numerous reactions Provide structural rigidity Control flow and conc across plasma membrane Sensors / switches / gene expression Amino acids are the building blocks of Proteins • 20 Different amino acids (a.a.) - Alphabet • unbranched, linear chains of a.a. • correct 3-D structure is essential for function •Monomer= amino acid; polymer=peptide or polypeptide COOH H - C - NH2 R Amino acids (monomeric subunits) R, n=20 – determine its properties - Diversity peptide of 4aa has 204 possible or 160,000 sequences R Chains (Special Properties) • Hydrophilic (surface) - Basic +ve lys(K), arg(R), (His) - Acidic -ve glu(E), asp(D) - polar Ser, Thr, asn(N), gln(Q) • Hydrophobic (core) Ala, Val, Ile, Leu, Met phe(F), tyr(Y), trp(W) • Special Cys, Gly, Pro Polarity is a critical feature for shaping 3D structure Hydrophobic: (aliphatic side chains, hydrocarbons, large bulky aromatic side groups, insoluble or less soluble; non-polar) These line the surface of mem prots within lipid bilayer Alanine ala A CH3-CH(NH2)-COOH Leucine* Isoleucine Methionine** Phenylalanine Tyrosine Tryptophan** Valine leu L ile I met M phe F tyr Y trp W val V (CH3)2-CH-CH2-CH(NH2)-COOH CH3-CH2-CH(CH3)-CH(NH2)-COOH CH3-S-(CH2)2-CH(NH2)-COOH Ph-CH2-CH(NH2)-COOH HO-p-Ph-CH2-CH(NH2)-COOH Ph-NH-CH=C-CH2-CH(NH2)-COOH (CH3)2-CH-CH(NH2)-COOH Glycine Cysteine** gly G cys C NH2-CH2-COOH too flexible, fit tight spaces HS-CH2-CH(NH2)-COOH Sulfhyrdal group (disulfide bond or bridge) Proline pro P NH-(CH2)3-CH-COOH Special: Average mol wt of a.a. is 113 **rare; *most common kink, cyclic ring, rigid Charged amino acids Polar no charge Hydrophobic amino acids Special aa Peptide bond (single chemical linkage for a.a.) From N to C terminus (carboxy gr of the 1st aa and amino gr of the 2nd) Rotation is restricted in pep bond Polyamino acids, peptide, polypeptide Size : mass in daltons (Da) or kilodaltons (kDa) R groups project from the backbone A dalton is 1 atomic mass unit Three types of non-covalent bonds help proteins to fold. Large number of Hydrogen bonds within a polypeptide help to stabilize its three dimensional structure How a protein folds into a compact conformation Elastin molecules are cross-linked together and uncoil upon stretching PROTEIN STRUCTURE (4 distinct levels determine shape) Primary; linear sequence (# and order) Secondary; local spatial organization H bonds (random coil, a-helix spiral, beta-sheet planar and turns 4 residue U shaped seg Tertiary; 3D overall conformation of a polypeptide, hydrophobic interactions, disulfide bonds, folding of domains Quarternary; applies to multimeric protein (2 polypep, noncovalent) The sequence of R-groups along the chain is called the primary structure. Secondary structure refers to the local folding of the polypeptide chain. Tertiary structure is the arrangement of secondary structure elements in 3 dimensions and quaternary structure describes the arrangement of a protein's subunits. Common regular structure; more than 60% of the protein is found to adopt these structures Structures MOTIFS are regular combinations of secondary structures specific combination with a particular topology - helix-loop-helix - zinc finger motif - coiled coil motif DOMIANS (tertiary structures in large proteins): - fibrous / globular - much larger 100-300 a.a. (several alpha-helices and beta sheets) - structural features or functional proline rich; SH3; Kinase domain, DNA binding domain) Alpha Helix • C=O----NH (H – bonded to 4 residues away on C terminal) • 3.6 aa/turn (regular arrangement) • R- outwards (determines hydrophobic/hydrophilic character) differ on each side • proline – rare • functionally important (structural elements) • amphipathic – coiled coils, fibrous proteins Amphipathic Structures a-Helix Hydophobic aa Hydrophilic aa Beta-Pleated Sheet •5-8 a.a. fully extended polypep •Planar structure •H bonds within/different polypep chain •Parallel/anti-parallel •R – project on both faces •Laterally stacked beta strands give beta sheets •Have polarity TURNS : composed of 3 or 4 residues glycine and proline H bonds; located on prot surface beta-beta-alpha zinc finger proteins Helix-loop-helix / split zipper proteins basic zipper proteins •Conformation (Native state) •Key to all higher structures is the a.a. sequence •Function is dependent on its 3D structure •Sequence homology (conserved regions): - function (homologous prots belong to same family - evolutionary relationship •Prosthetic groups - non-covalent / covalent - e.g., zinc for metalloproteinases heme for hemoglobin •Native state (Nascent protein undergoes folding) 8 bond angles are possible; n polypep = 8n most stable conformation (single) native state Modification of Proteins: (almost all prots require this) (alter activity, life span, cellular location) Chemical Modification: Acetylation - N terminal residue CH3CO – most prots - fatty acid acylation – membrane anchored (ras, src) Glycosylation - linear or branched CHO groups - Internal residues - many secreted and cell surface proteins Phosphorylation - phosphate group replaces H on OH group (serine, threonine, tyrosine) Processing: N or C terminal - pre pro insulin -procollagen - pre pro metalloproteinase (important means of keeping activity in check) Denaturation - temp, pH, urea (conformation and activity are lost); disrupt noncov - renature when removed from such condition (regain bioactivity Shows that information for folding is contained within ribonuclease metalloproteinase • Chaperones (proteins found in bacteria and all species) • - facilitate protein folding (molecular chaperones; chaperonins) • large barrel shaped multimeric complex (GroEL/TCiP) Movie Protein degradation: LIFE SPAN IS TIGHTLY CONTROLLED Extracellular -Digestive system (endoproteses or exoproteases) Intracellular -Lysosomes (membrane limited organelles) -Proteososme degrades ubiquitin targeted molecules. prot that contain the sequence (PEST) are degraded by another set of enzymes some degraded within 3 min or as long as 30 hrs (movie) FORM and FUNCTION are inseparable Pores; grooves; barrel-like structure Protein bind other molecules (I.e., ligands for receptors on cell surface) with high degree of specificity or target molecules (substrate for enzymatic activity) Affinity: Strength of binding (Keq; KD) Specificity: preferential binding Both properties depend on structural fit; complementarity Examples: antigen : antibody (Y-shaped molecules immunoglobulins) Complementarity-determining regions at each ends Enzyme : substrate (substrate binding site; active site) Conformational change can be induced by substrate binding Antibodies Made by B-cells of the immune system. Multimeric proteins heavy and light chains linked by disulfide bonds How noncovalent bonds mediate interactions between macromolecules Development of Antibodies for Cell Biology Research Antibodies are secreted by activated B-cells known as plasma cells. Polyclonal all serum from immunized animal contains many different antibodies to different epitopes. Usually made in rabbits, donkeys, goats, sheep, or horse Monoclonal antibodes are produced from one plasma cell so all antibodies are identical against one epitope Usually made in mouse, rat or hamster Making MAb Immunize Mice Test animal for Ab response Remove spleen Harvest B-cells Fuse to hyridoma Screen secreted Ab for reaction to antigen Expand cell line and purify Ab. movie ENZYMES: •Catalysts @ 370C, pH 6.5 – 7.5 and aqueous •Specificity – what they bind and cleavage site •Extracellular/ Intracellular/ Tissue-specific/ House keeping •Active site – 2 important regions – bind substrate - catalytic site •Certain a.a. side chains are important not necessarily adjacent (dependent on specific folding) •Transition state- intermediate state conformation change reduces activation energy movie ENZYME KINETICS: E+S E+P Km = The Michaelis constant Affinity of the enzyme for its substrate Vmax = Maximal velocity at satuarting S concentration Binding E+S catalysis ES release EP E+P Vmax Rate of product formation Km Cons of subs [S] Km affinity [S] Rx. Catalyzed by Lysozyme 1. Enzyme 1st binds the polysaccharide to form enzyme-substrate complex (ES). 2. Catalyzes cleavage of specific colavent bond Forms enzyme-product complex (EP). 3. Release of product allows enzyme to act on another S. Feedback Inhibition A molecule other than the substrate binds to an enzyme at a special regulatory site outside the active site, thereby altering the rate at which the enzymes converts substrate to product. Membrane Proteins (A diverse group) •Integral membrane proteins (intrinsic) embedded or transmembrane •Peripheral (extrinsic) do not interact with hydrophobic core / indirect •Hydrophobic alpha helices in transmembrane prots •Multiple transmembrane a helices •Multiple b strands in membrane spanning barrels •Covalently attached hydrocarbons chains anchor prot to membranes Protein Purification and Detection: 1. 2. 3. 4. 5. 6. 7. Solubilization in detergents Centrifugation (mass or density) Size and charge Electrophoresis (charge, mass) Chromatography (mass, charge, binding affinity) Immunoblotting Mass Spectrometer Detergents Ionic Sodium deoxycolate Sodium dodecylsulfate (SDS) +hydrophilic::hydrophobic Nonionic Triton X-100 Octylglucoside hydrophilic::hydrophobic Micelles Above Critical Micelle Concentration (CGC) detergent phospholipid of cell membrane Mixed Micelles Below CGC, No Micelles Integral proteins dissolve Ionic detergents bind to hydrophobic regions and core of proteins because of charge disrupts ionic and hydrogen bonds. At high conc. Completely denatures proteins. Centrifugation 1st step in purification of a protein Based on differences in Mass and density Mass= weight of sample (grams) Density= ratio of weight to volume (grams/liter) Mass varies greatly Density of protein does not except for lipid or CHO additions Differential centrifugation-separates soluble and insoluble material Rate-Zonal-separates proteins based on their sedimentation rate within a density gradient Rate of sedimentation affected by Mass and Shape Centrifuge too long everything into the pellet too short no separation Electrophoresis Separates proteins based on their Charge:Mass Ratio Under applied electric field proteins move ata speed determined by their charge:mass ratio. Example two proteins of equal mass and shape the one with the greater net charge will move the fastest. SDS-PAGE separates proteins based on chain length, which reflects mass, as the sole determinant of migration rate. Movie Two-Dimensional Electrophoresis 1st dimension separated on charge of protein 2nd dimension separated by SDS-PAGE Charge separation is accomplished by proteins migrating through a pH gradient till the reach their pI, or isoelectric point, the pH at which their net charge is 0. This technique is isoelectric focusing IEF. After IEF strips are treated with SDS and the second dimension is ran. SDS-PAGE 2-D SDS-PAGE Liquid Chromatography Gel-filtration -based on polymer with pore size Ion-exchange -based on resin with either basic or acid charge Affinity -based on protein binding to different matrices -heparin, dyes Antibodies -based on the affinity of Ab for protein. Western Blotting SDS-PAGE Proteins transferred to membrane and antibodies are used to identify protein movie Mass spectrometry Laser to fragment protein and measure peptides produced ESI, MALDI, SELDI, LC-MS