Chapt. 6-7 Amino Acids and ProteinsProteins and amino acidsAmino acidsAmino acidsThe Peptide bondAmino acids side chainsSide chains interact in structure of proteinBasic amino acid Histidine has 3 pKapH affects dissociation of side chains of amino acidsLevels of protein structureProtein structureSecondary structure – alpha helixSecondary structure – b pleated sheetTertiary StructurePost-translational amino acid modificationsTransmembrane proteinsQuaternary structureSpecies conservation - insulinGel electrophoresis separates proteinsDenaturing gel electrophoresisEnzyme isoforms in different tissuesIsoforms in different tissuesProtein familiesStructure-function of myoglobin; hemoglobinOxygen saturation of myoglobin, hemoglobinCooperativity in O2 binding of hemoglobinClinical commentsSlide 28Antibodies are immunoglobulinsChaperoninsPrions are misfolded, infectious proteinsKey conceptsChapt. 6 Review questionsChapt. 7 Review questionChapt. 6-7 Amino Acids and ProteinsChapts. 6-7 Amino Acids, ProteinsStudent Learning Outcomes:• Explain basic structure of amino acids, and classification by side chain• Explain the structure of peptide bond•Describe the different levels of structure of proteins•1o product is polypeptide, from gene sequence• Explain relationship of primary structure of protein to its final function, and modifications of amino acids• Describe effects of amino acids substitutions in the primary sequenceProteins and amino acidsProteins are most diverse macromolecules.Thousands of proteins direct most activities of cell:•Structural components•Transport, storage of small molecules (e.g. O2)•Transmit information between cells (protein hormones), •Defense against infection (antibodies)•EnzymesProteins (polypeptides) are linear polymers formed from 20 different amino acids- translated from mRNA, transcribed from DNAAmino acidsAmino acid structure:• - NH2 end, -COOH end; 2 pKa, (Some side chains 3rd pKa)• Zwitterion at physiological pH: both + and – chargesFig. 6.1Amino acids Fig. 6.2Amino acids can be D and L configuration:•Defined by glyceraldehyde•Asymmetric -carbon•R = side chain•Only L-form in proteins of humans, other organisms•(bacteria have D-aa in cell walls, some antibiotics)The Peptide bondPeptide bond:•Joins 2 amino acids•Condensation reaction•Protein primary sequence: linear order of aa from gene•Note: N- and C- terminus•R = Side chains of the aa •confer functional properties, dictate foldingFig. 6.3Amino acids side chainsSide chains of Amino acids dictate function and reactivities:•pKa for functional groups (Table 1):•-COOH, -NH2,•Side chains•Hydropathic index:•+ = hydrophobic•- = hydrophilicSide chains interact in structure of proteinSide chains of amino acids interact for structure:Hydrophobic, H-bonds, oxidation, electrostaticFigs. 6.5, 6.6Basic amino acid Histidine has 3 pKaBasic amino acid Histidine has 3 pKaFig. 6.8pH affects dissociation of side chains of amino acidspH affects ionized form of side chains of aa:pKa is pH at which half molecules have charged side chainsLevels of protein structure3 levels of structure for a polypeptide:•Primary sequence dictates folding:•Quaternary structure involves multiple polypeptidesFig. 7.1Protein structure1o structure is the linear polypeptidePeptide backbone is rather rigid: Often trans-configuration - alternating side chainsFig. 7.2Secondary structure – alpha helix Secondary structures are localized interactions:•Alpha helix is rigid, stable, compact•Alpha helix from H-bonds of peptide bond •C=O to N-H •Between aa 4 residues awayFigs. 7.3-7.4Top viewSecondary structure –  pleated sheetSecondary structures are localized interactions:-pleated sheet from H bondsC=O to N-H of adjacent strandsFig. 7.5 A. anti-parallel. B.parallel Fig. 7.6 -turn is hairpin;H-bondsTertiary StructureFig. 7.7 -helix and -sheets (arrows); NAD+ is bound upper leftFig. 7.8 G-Actin binds ATP in cleftTertiary structure: Longer range interactions involving 2o structures•Results in domains, basic units•Hydrophobic aa localize in interior•Hydrophilic aa localize on surfacePost-translational amino acid modificationsModified amino acids in proteins•Often post-translational•Target or anchor proteins, •Regulate activity (PO4-2)Fig. 6.13Transmembrane proteinsTransmembrane proteins:•Transport proteins, hormone receptors, ion channels-helical regions: hydrophobic aa expose to lipid bilayer•Hydrophilic in aqueous; often post-translational modified aaFig. 7.9  -adrenergic receptor: heptahelical tube transmembraneQuaternary structureQuaternary structure: interactions between multiple polypeptides to form the active protein•Ex. hemoglobin 2  chains, 2  chains•Myoglobin only 1 chainFig. 7.1Species conservation - insulinSpecies conservation of insulin primary structure:High conservation: porcine and bovine insulin used by diabetics before recombinant human insulinFig. 6.12 Insulinprecursor cleaved to A and B chains;Blue are invariant cys;Red are changes in porcine, bovineGel electrophoresis separates proteinsGel electrophoresis at neutral pH can separate proteins based on overall charge:•Distinguish normal, and person lacking IgG, or myeloma•Distinguish normal and mutant forms of hemoglobin: HbA vs. HbS (Glu6 -> Val); determine genotypes: AA, AS, SSDenaturing gel electrophoresisDenaturing gel electrophoresis (SDS-PAGE) separates proteins based on size (aa length)•Ex. Normal serum proteins, and after depletion of albumin, IgG and transferrin with little columnsGood Biotech Co.Lane 1 serum proteinsLane 1 depleted serumLane 3 eluted proteins(1)IgG; (2) transferrin(3) Albumin (4) IgG H-chain(5) IgG L-chainEnzyme isoforms in different tissuesEnzyme isoforms (isoenzymes) are present in different tissues.• Same function, slightly different size, charge, tissue, developmental stage.•Ex. Creatine Kinase (CK) has B and M forms: BB (brain), MB (heart), MM (skeletal muscle).•After heart attack: diagnosis by increased MB isoform in bloodFig. 6.15Isoforms in different tissues Adenylyl cyclase (3’,5’-cAMP synthesis) • 9 different isoforms, different genes, different tissues (differential response to hormones); •invariant C1 and C2 regions for synthesis of cAMPFig. 6.11M1 and M2 are trans-membrane helicesProtein families Protein families - evolution from ancestral gene•Sequence from proteins, or from DNA of