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| How does GroEL/ES correct proteins? |
-protein binds inside to hydrophobic patches
-Each complex has two large pockets |
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mcs5109 Wed, 12 Nov 2008 06:19:00 GMT |
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| GroEL/ES |
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 |
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mcs5109 Wed, 12 Nov 2008 06:19:00 GMT |
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| Nucleoplasmins |
-acidic nuclear proteins required for in vivo assembly of nucleosomes |
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mcs5109 Wed, 12 Nov 2008 06:09:16 GMT |
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| Hsp90 |
-one of the most abundant proteins in eukaryotes
-involved in folding of proteins involved in signal transduction |
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mcs5109 Wed, 12 Nov 2008 06:09:16 GMT |
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| Heat shock protein 70: Hsp70 |
-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 |
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mcs5109 Wed, 12 Nov 2008 06:09:16 GMT |
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| Molecular Chaperones GroEL/ES |
-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 |
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mcs5109 Wed, 12 Nov 2008 06:09:16 GMT |
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| Proline isomerization |
-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 |
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mcs5109 Wed, 12 Nov 2008 06:01:42 GMT |
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| Protein disulfide isomerase |
-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 |
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mcs5109 Wed, 12 Nov 2008 06:01:42 GMT |
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| Folding accessory proteins |
Protein disulfide isomerase
Peptidyl prolyl cis-trans isomerase
Molecular chaperones |
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mcs5109 Wed, 12 Nov 2008 06:01:41 GMT |
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| Importance of primary structure |
-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 |
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mcs5109 Wed, 12 Nov 2008 06:01:41 GMT |
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| dithiothreitol |
used to determine order of disulfide bonds in BPTI |
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mcs5109 Wed, 12 Nov 2008 05:39:09 GMT |
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| Bovine pancreatic trypsin inhibitor BPTI folding |
-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 |
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mcs5109 Wed, 12 Nov 2008 05:39:09 GMT |
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| Free energy funnel |
-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 |
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mcs5109 Wed, 12 Nov 2008 05:39:09 GMT |
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| Final stage |
involves native interactions, hydrophobic packing, and fixation of surface loops |
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mcs5109 Wed, 12 Nov 2008 05:32:50 GMT |
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| forms tertiary structures |
-after Molten globule state
-slow
-can be up to 1 sec
-form subdomains that are not fully developed |
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mcs5109 Wed, 12 Nov 2008 05:32:50 GMT |
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| Molten globule state |
-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 |
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mcs5109 Wed, 12 Nov 2008 05:32:50 GMT |
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| Phases of protein renaturation |
1. burst phase
2. Molten globule state
3. forms tertiary structures
4. Final stage |
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mcs5109 Wed, 12 Nov 2008 05:32:50 GMT |
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| burst phase |
within 5 ms, elements of secondary structure |
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mcs5109 Wed, 12 Nov 2008 05:32:50 GMT |
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| Initial events |
-within 5 ms, elements of secondary structure are formed
-called burst phase
-caused by hydrophobic collapse |
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mcs5109 Wed, 12 Nov 2008 05:32:50 GMT |
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| circular dichroism |
-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) |
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mcs5109 Wed, 12 Nov 2008 05:17:18 GMT |
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| How to denature a protein |
-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 |
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mcs5109 Wed, 12 Nov 2008 05:11:18 GMT |
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| Rapid measurement methods |
-Protein folding can occur in milliseconds
-use rapid mixing and detection: stopped-flow
-have denatured protein in one syringe and mixing results in refolding |
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mcs5109 Wed, 12 Nov 2008 05:11:18 GMT |
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| Kinetics of refolding |
-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 |
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mcs5109 Wed, 12 Nov 2008 05:05:16 GMT |
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| protein GB1 |
-help bacteria by binding to IgG
-show that replacement of residues 23-33 (a-helix or 42-52 (b-hairpin) have same fold |
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mcs5109 Wed, 12 Nov 2008 05:05:16 GMT |
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| a hlices and b sheets |
-not dependent on certain aas being in fold
-information specifying helix or sheet can be non-local or context dependent |
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mcs5109 Wed, 12 Nov 2008 05:05:16 GMT |
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| protein structure |
-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) |
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mcs5109 Wed, 12 Nov 2008 05:05:16 GMT |
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| Internal Residues important in folding |
-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 |
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mcs5109 Wed, 12 Nov 2008 04:25:38 GMT |
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| Determinants of protein folding |
-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 |
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mcs5109 Wed, 12 Nov 2008 04:25:38 GMT |
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| insulin |
-2 polypeptide chains with 2 disulfides
-PDI inactivates it
This is because insulin is cleaved after disulfides are formed |
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mcs5109 Wed, 12 Nov 2008 04:19:05 GMT |
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| enzymatic reshuffling after denaturing |
-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) |
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mcs5109 Wed, 12 Nov 2008 04:19:05 GMT |
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| Mercaptoethanol |
reduces disulfide bonds |
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mcs5109 Wed, 12 Nov 2008 03:53:10 GMT |
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| Urea |
chaotropic salt and causes denaturation |
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mcs5109 Wed, 12 Nov 2008 03:53:10 GMT |
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| Refolding of RNAse A |
-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 |
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mcs5109 Wed, 12 Nov 2008 03:53:10 GMT |
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| Entropy |
Entropy: denatured state highly disordered, more favorable |
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mcs5109 Wed, 12 Nov 2008 03:53:10 GMT |
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| enthalpy |
-Enthalpy: energy of non-covalent bonds: hydrophobic interaction, H bonds, ionic bonds
-Enthalpy interactions favorable in packed native state |
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mcs5109 Wed, 12 Nov 2008 03:53:09 GMT |
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| Stability of Globular proteins |
-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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mcs5109 Wed, 12 Nov 2008 03:53:09 GMT |
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