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Lecture 24 Protein Folding and Quality Control Folding Function: making specific functional domains critical for function (occurs following or coincident with synthesis) Sequence dependence: Final structure of protein is dependent on amino acid sequence and properties of amino acids that make up polypeptide being synthesized. Proteins will fold during synthesis to achieve lowest possible energy state. Hydrophobic amino acids will group together forming hydrophobic interactions and a hydrophobic core while hydrophilic amino acids will go outside, interact with water and other water soluble molecules increasing solubility of the amino acid. Refolding: Following denaturation, many proteins can refold when put back into ideal conditions. HOWEVER, there are many proteins that cannot reform and need help along the way. Heat shock proteins: they are molecular chaperones. Problem: at high temperatures, hydrophobic amino acids do not interact properly, don’t form core, interact with other proteins and form aggregates. Solution: HSP aids in regular folding of protein. HSP increase rapidly in presence of heat or other cellular stresses. Method: 1) When in ATP bound state binds to nascent unfolded polypeptide 2)ATP hydrolyzed and HSP form hydrophobic pockets, allows for normal folding of hydrophobic elements. 3+4) When ATP reassociates, goes back to original configuration and folded protein is released. GroEL : acts as bacterial chaperonin (large macromolecular machine devoted to folding) like TriC in humans. Structure: combination of 2 rings, repeated subunits. Barrel structure with interior. 2 different states. QuickTime™ and a Function: 1)In tight conformation, can accept TIFF (Uncompressed) decompressor are needed to see this picture. polypeptides. Allows polypeptide’s hydrophobic residues to interact. Leads to release of properly formed polypeptide. 2) Relaxed conformation allows polypeptide to leave the barrel and do its process. Relaxed by association of GroES and ATP. Disulfide bridges: Function: facilitate appropriate folding and require oxidation of SH groups on cysteines. ER dependence: proteins for processing only present in ER (endoplasmic reticulum) so proteins with S-S bridges must go through ER. Which cells: Proteins to ER are for excretion or membrane. Cells that excrete a lot of QuickTime™ and a TIFF (Uncompressed) decompressor protein have a lot of PDI (ex. Pancreas that secretes insulin) Method: are needed to see this picture. 1) PDI recognizes thiol groups. 2) PDI has reduced active site, so catalyses reaction that oxidizes (removes Hydrogen) from cysteine forming bridge this involves: a) electron transfer from thiol group b) oxidized thiol acting on neighbour forming bridge 3) PDI rearranges bridges if necessary (if protein is not in optimal state after sequential formation of bridge) 4) Ero 1 brings PDI back to oxidized state so that it can be used again (since it was reduced by rxn). IMPORTANT: all proteins with disulfide bridges contain cysteine but NOT ALL CYSTEINE CONTAINING PROTEINS FORM BRIDGES. They MUST be brought to ER to form a bridge. Plaques: Incorrect folding can lead to pathologies involving aggregates of proteins forming plaques: Alzheimers & Huntington’s disease. Alzheimers: Protein aggregates or Amyloid plaques (misfolded amyloid proteins) and tangles present in the diseased brain. Huntington’s: Protein aggregates or plaques arise in neurons within the brain. These are associated with neuronal cell death (necrosis). Majenta Whyte Potter-Mäl 1 of 3 Molecular Biology Lecture 24 Kuru: terrible disease afflicting Fore people. Members of the tribe, particularly women and children, ate parts of the relative. Neuronal tissue was one of first to be consumed. Pathogen was a small protein (prion). Associated with laughing death. People were having ongoing neuronal degradation, and eventually death. Loss of neuronal function. Stopped cannibalism and got rid of disease. Prion disease: related to scrapies, BSE (mad cow disease), Creutzfeldt-Jakob Prion protein (PrP): 2 states: PrPc (conformer state) is non-infectious. Structure: 3 alpha helices. PrPsc (non conformer state) is infectious. Structure: 2 alpha helices + 1 beta sheet. DIFFERENT QuickTime™ and a TIFF (Uncompressed) decompressor STRUCTURE DIFFERENT FUNCTION. are needed to see this picture. Amino acid sequences of both forms are identical. Infection: PrPsc is highly protease resistant thus accumulates and form plaques and results in neuronal cell death. Also, PrPxc can convert PrPc to infectious form! PrPsc thus acts in a dominant manner and causes infection. Quality Control Maturation, export, and pioneering round: involved in translational quality control. a) SR proteins: define exons for proper excision of introns. b) Poly Adenylation. c) Export actors: loaded onto mRNA to bring out of nucleus. All loaded factors removed or you get NMD!!!! NMD (nonsense mediated decay): Wild type situation: proteins knocked off sequentially. Mutant type situation: In frame stop within exon. Ribosome drops off mRNA but still has proteins associated with it. Ex. if had something with only ligand binding domain, could be a dominant negative protein (completes half of function so blocks normal protein from completing function). By keeping SR and other proteins associated to it, tells cell is a bad situation. NMD machinery is set up to chew up mRNA because don’t want to translate that protein. Endoplasmic Reticulum Earmarking: proteins are earmarked for numerous destinations. Info for localization found within polypeptide sequence. Cytosol: contain little localization info so stay in cytoplasm. Nucleus: have a nuclear localization sequence. Mitochondria, chloroplast, peroxisome: other signalling information. ER: secreted proteins come here by recognition of specific sequences called a signal recognition sequence. Rough ER: ribosomes are bound to ER (same as free ribosomes) and introduce growing polypeptides into ER. SRP: recognizes signal sequence and 1) brings down polypeptide 2) inserts it into ER 3) polypeptide modified by proteins in ER (step 3 not related to SRP per se) Ire1 dimerization: important for ER unfolded protein response (UPR) Proteins involved in this are BIP and IRE (ER chaperones that ensure proper folding thus preventing aggregation and binding irreversibly to misfolded proteins). IRE normally in monomeric situation associated with BIP. BIP is a molecular chaperone within the ER. BIP thought to bind to IRE so can’t form dimer when too much protein unfolded. When sequestered away from IRE, can dimerize and increases transcription. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Majenta Whyte Potter-Mäl 2 of 3 Molecular Biology Lecture 24 Alternative explanation: Unfolded protiens forms scaffold that allow to form dimers, through inherent ability to snip transcription factor mRNA, and increases transcription of things that are for folding mrna. Based on structural biology. Perhaps this case with BIP is not the best explanation. IRE is binding to proteins themsleves and that is forming the dimerization. Majenta Whyte Potter-Mäl 3 of 3 Molecular Biology