Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Chapter 4: Protein Structure and Function• the polypeptide• protein folding• cross-linking• reducing agents and denaturants• prions• post translational modifications• regulation of protein activity• ubiquitylation and proteolytic degradationReferences 4th: p. 121-143, 150-156, 251-251(3rd: p. 119-141, 149-155, 259)Amino acidsDehydration synthesisThe peptide bondThe polypeptidePrimary structureNoncovalent interactions are weak but collectively strong enough to define the folded structure of a proteinNonpolar amino acids• Nonpolar side chains tend to cluster at the interior of a folded polypeptide, away from the aqueous surroundings. • Nonpolar amino acids also form the transmembrane domains of membrane proteins.Polar and charged amino acidsPolar and charged amino acids tend to be near the outside of the protein, the surface exposed to the aqueous surroundings.Hydrophobic interactions and hydrogen bonds between side chains in a folded polypeptideGlobular proteinSecondary structure: the a helixSecondary structure: the b sheet• proteins are synthesized as polypeptides• protein maturation involves:Protein maturation and the regulation of protein function- correct folding- proteolytic cleavage- chemical modifications- formation of quaternary structures- association with co-factors• proteins are synthesized as polypeptides• protein maturation involves:• each step in protein synthesis and maturation can be a target for regulation of protein function• finally: protein degradation is also under tight control Protein maturation and the regulation of protein function- correct folding- proteolytic cleavage- chemical modifications- formation of quaternary structures- association with co-factorsChaperones bind to nascent polypeptides and maintain astable unfolded state.• when synthesis is complete the polypeptide is released and allowed to fold correctlyRole of chaperonesTertiarystructureDisulfide bonds form between adjacent cysteine residues.• they can link two domains of the same polypeptide ordifferent polypeptide chainsCross-linkingCross-linking of elastin polypeptides to form elastic fibersQuaternary structure:multiple polypeptidesThe polypeptideAction of denaturants and reducing agentsDenaturants (such as urea or heat) can unfold (denature) apolypeptide by breaking noncovalent interactionsbetween amino acidsReducing agents (such as Beta-mercaptoethanol) can break disulfide bonds, which areUreaUrea is produced in the liver of mammals as way to excrete ammonia (a toxic metabolic waste product).Urea as a denaturing agentUrea can reversibly break noncovalent interactions between amino acidsBeta-mercaptoethanolOHHSthiolgroupCCOHHCCAction of reducing agentsCollagen foldingFibrillar collagens, themajor structural proteinsof connective tissues, arebuilt of triple helices ofprocollagen polypeptides.Osteogenesis imperfecta- scrapie (sheep)- mad-cow disease (cattle)- Creutzfeldt-Jakob disease (CJD), or Kuru “to tremble with fear,” in humansTSEs are a family of fatal brain diseases characterized by lesions that appear as small cavities (spongy appearance) caused by protein aggregates.Transmissible spongiform encephalopathies• infectious agent: prions (proteinaceous infectious particle)• protein-only hypothesis: diseases are caused by incorrectly folded versions of the prion proteinTransmissible spongiform encephalopathiesDamaged human brain tissue(TSE)PrPcPrPscprion protein cellularprion protein scrapiePrionsPrionsPrPsc is a variation of a normal brain protein PrPc• a PrPsc can direct a PrPc to misfold into PrPsc form• new PrPsc forms can continue the propagation• several strains exist (variations of the prion protein tertiarystructure that are also infectious)Propagation of prionsEvidence for the prion hypothesis1) British cattle were infected because they were fed bonemeal from (presumably infected) cattle carcasses2) Disease is common among the Fore people of Papua NewGuinea, who eat affected brain in a cannibalistic ritual• infectivity of affected brain samples can not be destroyedwith nucleases or radiation (DNA/RNA destroying agents)• if tissue is infected, the conformation of the new PrPsc in thehost will be identical to the strain in the infection source• mutant mice lacking the PrP gene are immune to prions,but:• if normal brain tissue is grafted onto the brain of the mutantsand infected with prions, only the grafted tissue will beaffectedInsulin protein• pancreatic hormone that regulates blood glucose levels• two chain polypeptide linked by disulfide bonds (forming cysteine bridges)• translated as a single polypeptide (preproinsulin)• signal sequence is removed after translocation into the ER• disulfide bonds form (result: proinsulin)• connecting polypeptide is cleaved (result: mature insulin)Polypeptide processing - Proteolytic Cleavage in insulin synthesisInsulin synthesisRegulation of protein activityMay occur at two levels:Examples:1) Regulation of protein localization2) Regulation by ligand binding3) Feedback regulation of enzymes - allosteric regulation – pos and neg4) Phosphorylation5) GTP-binding6) Proteolytic degradation1) Regulation of gene expression: determines the amountof protein produced by the cell by limiting transcription and/or translation.2) Regulation of protein function: the protein is synthesized but its activity is restricted according to needs of the cell.1) Localization - Regulation of nuclear import2) Regulation by binding of specific ligandsThe folding of a polypeptidemay create a specificligand binding siteA ligand binds hereby noncovalent interactions.Binding of cAMP to a specificbinding site on a proteinRegulation of thecAMP-dependent protein kinase APKA: promotes glycogen metabolism• inactive PKA is a tetramer: 2 regulatory subunits bound to 2 catalytic subunits• cAMP activates PKA by binding tothe regulatory subunits, causingthe release of the catalytic subunits• the kinase activity of the releasedcatalytic subunits phosphorylatemultiple effector proteins3) Feedback inhibition= the end product of a biosynthetic pathway