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| GroEL vs. GroES |
Hsp60 (GroEL): 14 identical 60 kD subunits arranged in two rings of 7 subunits each
Hsp10 (GroES): single heptameric ring of identical 10kD proteins
-both facilitate the folding of improperly folded proteins to their native conformation
-Binding of protein occurs inside to hydrophobic patches
-GroES is like a lid over the barrel
-Each complex has two large pockets formed from two heptameric rings |
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mcs5109 Tue, 21 Oct 2008 22:11:19 GMT |
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| Heat shock protein 70: Hsp70 |
Protein unfolding increased at high temp |
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mcs5109 Tue, 21 Oct 2008 22:11:19 GMT |
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| Molecular Chaperones GroEL/ES |
-Molecular chaperones prevent aggregation by binding with hydrophobic patches
-Many chaperones have ATPase activity to release proteins that may be more properly folded |
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mcs5109 Tue, 21 Oct 2008 22:04:56 GMT |
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| Proline isomerization |
-Peptidyl prolyl cis-trans isomerase catalyzes otherwise slow interconversion of cis-trans
-Cis is 1000 less stable than trans
-In proline, cis is only around 4 times less stable than trans |
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mcs5109 Tue, 21 Oct 2008 22:04:56 GMT |
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| Protein disulfide isomerase |
-PDI is homodimer eukaryotic enzyme (prokaryotes also have this enzyme).
-in reduced form, it catalyzes the shuffling of disulfide bonds until native state is reached
-active site: Cys-Gly-His-Cys
-first Cys participates in catalytic cycle.
-Second Cys forms intra disulfide to eliminate the substrate |
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mcs5109 Tue, 21 Oct 2008 22:04:56 GMT |
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| 3 accessory proteins to help fold |
Protein disulfide isomerase
Peptidyl prolyl cis-trans isomerase
Molecular chaperones |
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mcs5109 Tue, 21 Oct 2008 21:57:07 GMT |
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| Importance of primary structure |
-primary structure evolved to specify specific efficient folding pathways
-mutants fail to renature at 39C but can at 30C.
Once renatured, the mutants have the same Tm as wild type of 88C.
-aa sequence dictates protein’s native structure by specifying how it folds to its native conformation |
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mcs5109 Tue, 21 Oct 2008 21:57:07 GMT |
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| Bovine pancreatic trypsin inhibitor BPTI folding |
-Protects pancreas from its own proteases
-has 3 disulfide bond. Fully reduced BPTI is inactive.
-determined order of disulfide formation by adding oxidized dithiothreitol to reduced BPTI
Intermediates trapped by lowering pH then separated on chromatographically and the disulfide bonds determined
-structures determined by NMR |
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mcs5109 Tue, 21 Oct 2008 21:57:07 GMT |
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| Free energy funnel |
-Unfolded: high entropy and energy
-As folding progresses, decrease in number of conformational species.
-Small depressions= semistable intermediates that can slow down folding process.
At bottom, reduced to single native conformation. |
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mcs5109 Tue, 21 Oct 2008 21:57:07 GMT |
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| molten globular state |
-most 2ary structure of native protein
-correct positions of a-helix and b-sheets
-less compact than native state
-proper interior packing has not yet occurred
-Internal side chains flexible
-Loops/surface components not in correct conformation.
View as ensemble of related structures
Step 2:
-slow (1 sec)
-Native like elements of tertiary structure develop
-form subdomains that are not fully developed
Final stage:
-native interactions
-hydrophobic packing
-fixation of surface loops |
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mcs5109 Tue, 21 Oct 2008 21:49:42 GMT |
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| Denaturing Protein: step 2 |
-after burst phase, collapse of of flexible disordered polypeptide into partially organized globular state
-called molten globule
-fast |
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mcs5109 Tue, 21 Oct 2008 21:47:01 GMT |
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| Denaturing Protein: step 1 |
-2ary structures formed
-burst phase since later steps are slower
-Driving force is hydrophobic collapse |
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mcs5109 Tue, 21 Oct 2008 21:47:01 GMT |
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| Circular Dichroism |
-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 Tue, 21 Oct 2008 21:47:01 GMT |
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| how to denature proteins |
-heat, chaotropic salts, pH etc. denature proteins
-can use Intrinsic fluorescence or circular dichroism (measure amount of helical structure) to measure denatured-ness
Abrupt increase from nature to denatured state suggest that the process is cooperative. |
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mcs5109 Tue, 21 Oct 2008 21:47:01 GMT |
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| kinetics of refolding |
-X-ray structure of several hundred proteins show that specific sequence of polypeptide has the same fold.
Are all conformations sampled in random fashion until lowest energy form is found?
Levinthal showed this can’t be: 150 residues would have 3150 possible conformations and it would take 1048 yr to fold properly. Actually time is 0.1 to 1000 sec
-kinetic pathway exist which prevents sampling irrelevant conformations. |
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mcs5109 Tue, 21 Oct 2008 21:41:04 GMT |
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| Protein structure highly adaptable |
-preferred interactions between side chains?
-no (ex: leu not always with val)
-Thus native fold determines packing but packing does not determine native fold.
-also, proteins families with same fold but no sequence similarity (alpha beta barrel proteins)
-replacement of residues 23-33 in GB1 (a-helix or 42-52 (b-hairpin) have same fold
-shows that information specifying helix or sheet can be non-local or context depended. |
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mcs5109 Tue, 21 Oct 2008 21:41:04 GMT |
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| Internal Residues important in folding |
-With Rnase A, Lys residues changed to polyalanine
no change in activity
-Since Lys is located on exterior of protein, it suggest that internal residues direct protein folding
-folding directed by hydrophobic forces |
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mcs5109 Tue, 21 Oct 2008 21:41:03 GMT |
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| Determinants of protein folding |
-lots of helices and beta sheets.
-Hydrophobic interactions important but lack specificity
-fraction of helices and sheets increases as protein more compact |
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mcs5109 Tue, 21 Oct 2008 21:41:03 GMT |
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| protein disulfide isomerase (PDI) |
-catalyzes the formation and breakage of disulfide bonds between cysteine residues within proteins as they fold. |
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mcs5109 Tue, 21 Oct 2008 21:29:34 GMT |
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| Post-translationally modified proteins |
-disulfide holds proteins in an unstable state.
-Ex: insulin; 2 polypeptide chains with 2 disulfides.
Addition of PDI inactivates it.
This is because insulin is cleaved after disulfides are formed. |
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mcs5109 Tue, 21 Oct 2008 21:29:34 GMT |
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| small amt reducing agent |
-Removing of reducing agent and 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
-active enzyme= energetically most stable |
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mcs5109 Tue, 21 Oct 2008 21:29:34 GMT |
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| Globular proteins: Marginally stable structures |
-Conversion from native to denatured state facile
-covalent bonds don't change from native to denatured state
-Enthalpy interactions more favorable in packed native state rather than unpacked denatured.
Entropy: denatured state highly disordered, more favorable
-Total free energy difference between native and denatured is difference of enthalpy and entropy.
-small difference |
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mcs5109 Tue, 21 Oct 2008 21:29:34 GMT |
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