Study BMB 401H Chapter 9 Flash Cards
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BMB 401H Chapter 9
BMB 401H Chapter 9
| How does GroEL/ES correct proteins? |
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-protein binds inside to hydrophobic patches -Each complex has two large pockets |
| GroEL/ES |
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Chaperonins consist of two family of proteins: Hsp60 (GroEL): two rings of 7 subunits each Hsp10 (GroES): single heptameric ring (toilet lid) -essential for survivial of E. coli under all conditions tested -facilitate corrections of improperly folded proteins |
| Nucleoplasmins |
| -acidic nuclear proteins required for in vivo assembly of nucleosomes |
| Hsp90 |
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-one of the most abundant proteins in eukaryotes -involved in folding of proteins involved in signal transduction |
| Heat shock protein 70: Hsp70 |
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-protein unfolding increased at high temp -expressed constitutively. -bind to newly synthesized peptides coming off ribosomes. -help unfold proteins if they have to go through membranes and subsequently refold them -ATP binding region and a second region that binds hydrophobic regions |
| Molecular Chaperones GroEL/ES |
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-exposure of hydrophobic residues to solvent can cause aggregation -molecular chaperones prevent aggregation by binding with hydrophobic patches -ATPase -use ATP to release proteins that may be more properly folded |
| Proline isomerization |
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-in proline, cis is only around 4 times less stable than trans (usually 1000x) -cis is found at certain prolines at bends -in unfolded state, energy constraints gone and there is equilibrium between cis and trans -catalyze otherwise slow interconversion of cis-trans |
| Protein disulfide isomerase |
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-PDI catalyzes the shuffling of disulfide bonds until native state is reached -a-b-b’-a’ subunits -2nd Cys involved in forming intra disulfide to eliminate the substrate -reactive cys is exposed and in hydrophobic patch -can bind unfolded proteins |
| Folding accessory proteins |
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Protein disulfide isomerase Peptidyl prolyl cis-trans isomerase Molecular chaperones |
| Importance of primary structure |
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-evolved to specify efficient folding pathways in primary structure and also stable native conformations. -mutants fail to renature at 39C but can at 30C -amino acid sequence dictates protein’s native structure by specifying how it folds to its native conformation |
| dithiothreitol |
| used to determine order of disulfide bonds in BPTI |
| Bovine pancreatic trypsin inhibitor BPTI folding |
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-protects pancreas from its own proteases -fully reduced BPTI is inactive. -order of disulfide formation determined by adding oxidized dithiothreitol to reduced BPTI -lower pH to trap -separated on chromatographically -disulfide bonds and structures determined by NMR |
| Free energy funnel |
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-unfolded state is high degree of conformational entropy and high energy. -as folding progresses, narrowing of funnel represents decrease in number of conformational species. -small depressions along side are semistable intermediates that can slow down folding process -reduces to to single native conformation at bottom |
| Final stage |
| involves native interactions, hydrophobic packing, and fixation of surface loops |
| forms tertiary structures |
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-after Molten globule state -slow -can be up to 1 sec -form subdomains that are not fully developed |
| Molten globule state |
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-intermediate -collapse of of flexible disordered polypeptide into partially organized globular state -fast -forms secondary structure -even has correct positions of a-helix and b-sheets -less compact than native protein. -not yet properly packed in interior -ensemble of related structures |
| Phases of protein renaturation |
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1. burst phase 2. Molten globule state 3. forms tertiary structures 4. Final stage |
| burst phase |
| within 5 ms, elements of secondary structure |
| Initial events |
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-within 5 ms, elements of secondary structure are formed -called burst phase -caused by hydrophobic collapse |
| circular dichroism |
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-measure amount of helical structure -proteins absorb in UV region due to aromatic amino acids The extinction coefficient (e) at each wavelength for proteins differs between left and right circular polarized light -variation in wavelength with difference of these quantities constitutes circular dichroism (CD) De = eL - eR -differs for helix, b sheet and random coil (rc) |
| How to denature a protein |
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-heat, chaotropic salts, pH etc. -can monitor denaturation via intrinsic fluorescence of Rnase A or circular dichroism -cooperative: abrupt increase from nature to denatured state |
| Rapid measurement methods |
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-Protein folding can occur in milliseconds -use rapid mixing and detection: stopped-flow -have denatured protein in one syringe and mixing results in refolding |
| Kinetics of refolding |
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-same proteins have same fold -If each peptide group has 3 different conformations and could interconvert in (10-12) sec, it'd take 1048 yr to fold properly -actually 0.1 to 1000 sec |
| protein GB1 |
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-help bacteria by binding to IgG -show that replacement of residues 23-33 (a-helix or 42-52 (b-hairpin) have same fold |
| a hlices and b sheets |
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-not dependent on certain aas being in fold -information specifying helix or sheet can be non-local or context dependent |
| protein structure |
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-no preferred interactions between side chains and found none (e.g. leu always with val). -also, there are proteins with same fold but no similarity in sequence. (alpha beta barrel proteins) |
| Internal Residues important in folding |
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-change Lys on exterior of protein -internal residues direct protein folding -mutations changing surface residues accepted more readily than internal residues. -protein folding directed by hydrophobic forces |
| Determinants of protein folding |
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-hydrophobic interactions are major driving force in protein folding but lack specificity -as protein compaction increases, so does fraction of helices and sheets -important in protein compaction |
| insulin |
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-2 polypeptide chains with 2 disulfides -PDI inactivates it This is because insulin is cleaved after disulfides are formed |
| enzymatic reshuffling after denaturing |
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-letting disulfides form in presence of urea results in 1% active enzyme. -Addition of trace amount of 2-mercaptoethanol results in fully active enzyme after 10 h -takes 2 min if add protein disulfide isomerase (PDI) |
| Mercaptoethanol |
| reduces disulfide bonds |
| Urea |
| chaotropic salt and causes denaturation |
| Refolding of RNAse A |
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-enzyme very stable -unfolded state is inactive -removal of urea and mercaptoethanol resulted in refolding -yields active enzyme -showed was that tertiary structure determined by primary structure |
| Entropy |
| Entropy: denatured state highly disordered, more favorable |
| enthalpy |
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-Enthalpy: energy of non-covalent bonds: hydrophobic interaction, H bonds, ionic bonds -Enthalpy interactions favorable in packed native state |
| Stability of Globular proteins |
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-native to denatured state conversion is easy -total free energy difference between native and denatured is difference of enthalpy and entropy |
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