Sometimes Proteins Don’t Fold ProperlyChaperonesSlide 3Slide 4GroE Helps Proteins FoldPeptide Bonds in Proteins are Almost Always TransCis-Trans Peptide bondsSlide 8Proteins may be posttranslationally modifiedSlide 10Denaturation of ProteinGel ElectrophoresisCharge to Mass RatioSDS-PAGESDS-PAGESlide 16Slide 17Isoelectric FocusingSlide 19pI is the pH at Which a Protein has a Net Charge of ZeropI is the pH at Which a Protein has a Net Charge of ZeroClicker Question2-Dimensional PAGE2 Dimensional Polyacrylamide Gel ElectrophoresisSlide 25Slide 26“ ‘omics “Protein PurificationColumn ChromatographySlide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Importance of Overexpression in Protein PurificationSometimes Proteins Don’t Fold Properly•Not all proteins fold appropriately.•If we think of folding as a pathway leading to the most energetically favorable state, sometimes proteins go “off-pathway” and fold into the wrong conformation.•These wrong conformations can have a favorable enough energy state that the protein cannot refold into the proper state.•These misfolded proteins often have hydrophobic regions that are exposed and they “aggregate”.•Consequences of misfolding proteins–Alzheimer's, Parkinson’s, Mad Cow, Aging?Chaperones•Some proteins will refold into the active state in vitro–Usually small proteins with simple structures•In the cell, folding is assisted by “chaperones” or other proteins– examples:–GroE an ATP-dependent chaperone–Disulfide Bond Isomerases–Peptidylproline IsomerasesChaperones are Important for Folding, Refolding, and Preventing Aggregation•Help in folding newly synthesized proteins •Help in refolding proteins that have become partially unfolded (maybe as a result of some stress like exposure to a high temperature – “heat shock”)•Bind to exposed hydrophobic surfaces preventing aggregation•If refolding fails, misfolded protein is degradedE. Coli Chaperones Assist in the Folding of Newly Synthesized ProteinsDnaJ/DnaK catalyze the folding of about 20% of proteinsTrigger Factor (TF) interacts with nascent proteins as they exit the ribosome. 70% of proteins require no additional help to fold properly.About 10% of proteins require GroEL-GroES system to fold properly.GroE Helps Proteins FoldUnfolded or MisfoldedProteinFolded ProteinGroE ChaperoneATPADPGroELGroESATP hydrolysis causes conformational (and therefore local environment) changes in the interior of the “barrel”Peptide Bonds in Proteins are Almost Always TransA cis peptide bond brings the two C’s closer together.Peptide bonds shown here are trans.Cis-Trans Peptide bonds•Most peptide bonds are trans - the R groups are opposite each other.•With proline the side chain is “tied back” in the proline.•There’s a big energy barrier to rotation around the peptide bond so if a protein wants a cis proline peptide bond, it is formed very slowly.•Cis proline peptide bonds are found in proteins, particularly at turns.•Formation of the cis proline peptide bond can slow down protein folding.PPIs catalyze the interconversion of cis/trans proline peptide bonds and speed up the folding of proteins that contain cis-proline bonds. All organisms contain PPIs. Humans, for example, have 16 PPIs in one structural family + 2 other families.Peptidyl-Prolyl Isomerases (PPIs)Proteins may be posttranslationally modified•Making and folding the polypeptide chain does not necessarily produce the active form of a protein. Many additional modifications may occur.•Addition of a co-factor or prosthetic group, e.g., heme in hemoglobin•Glycosylation - addition of sugar residues•Phosphorylation •Acetylation•Methylation•There are more than 200 types of posttranslational modifications known and more are being discovered.•Posttranslational modifications can have major effects on the properties of proteins. As a result of postranslational modification a single gene can produce a family of proteins with very different properties.Proteins may be posttranslationally modified. Some examples are shown. Many modifications are reversible and can be regulatory.Denaturation of Protein•The structure of a protein is held together by weak non-covalent interactions.•The protein will unfold or “denature” if these interactions are disrupted or overcome.–Temperature–Hydrogen bonding reagents – Urea–pH –Increase in hydrophobicity of solvent – Sodium Dodecyl Sulfate (SDS)–Compounds that disrupt the structure of water - chaotrophs•Upon denaturation, hydrophobic regions are exposed and most proteins aggregate and precipitate – a boiled egg.–This sort of denaturation is generally irreversible.Gel Electrophoresis•Proteins are charged molecules.•Charged molecules will move in an electric field (electrophoresis).•Polyacrylamide gels provide a convenient matrix to “sieve” the protein molecules as they move.–Polyacrylamide Gel Electrophoresis (PAGE)–Movement through gel depends on charge and shape of molecule.•This is “native” or “non-denaturing” PAGE.•In order for movement to be proportional to molecular weight must have a constant charge to mass ratio.Charge to Mass RatioDNA (a regular polymer)++++Proteins (very irregular polymers)SDS-PAGE •Protein charge is dependent on the amino acid sequence.•In order to create a constant charge-to-mass ratio and constant “shape”- proteins can be dissolved in sodium dodecyl sulfate (SDS, a detergent) and treated with a reducing agent to break all disulfide bonds (and prevent any disulfides from forming).•Most proteins bind 1-2 SDS molecules per amino acid. Proteins with bound SDS are unfolded, denatured, “random coil” molecules that are negatively charged. ONa+ -O-S-O OHydrophobicVery HydrophilicSodium dodecyl sulfate (SDS), a detergent, an amphipatic molecule. Also known as sodium lauryl sulfate - look on your shampoo bottle.SDS-PAGE•The protein sample is heated in the presence of SDS and mercaptoethanol.•Mercaptoethanol (HS-CH2-CH2-OH, a reducing agent) is added to reduce -S-S- bonds (if present) to –SH and to prevent them from forming if not originally present.•Multi-subunit proteins are converted to their component peptide chains by this treatment.SDS-PAGEProtein with two subunits, A and B, joined by disulfide bondSingle subunit proteinABCABCABCHeated with SDS and mercaptoethanolPAGE Slab of polyacrylamide gel-+An