Molecular Chaperones

Molecular Chaperones

1. A set of diverse, unrelated proteins whose common feature is to interact with protein in non-naive conformations

   • generate native protein structure from unfolded/non native substrates
     • Must stabilise non native confirmations to allow correct folding via use of ATP to overcome energy barrier of trapped proteins in energy well.
     • Can be non specific chaperones or highly specific chaperones
     • Prevent aggregation of misfiled proteins caused by exposed hydrophobic surfaces

2. 6 side examples of Molecular chaperones

   • 1. Protein disulphide isomerase; breaks misfired disulphide bonds to correctly pair them to allow correct burying of phobic residues
   • 2. Peptidyl prolyl cis-trans isomerase; assist switching of proline between cis and trans state, and more stable will remain for correct folding of protein
   • 3. Nucleoplasmin; enzyme that aids assembly of nucleosome
   • 4. Prosequences; Intramolecular chaperones; at ends of protein sequences and ensure correct folding to create WT protein. Also get prepro sequences that stop function before cleaving. LIKE COLLAGEN
   • 5. A-Crystallin; Stabilises eye lens crystalline from degradation to increase life time
   • 6. Heat shock proteins (Hsp); Levels increase in response to increase in temperature. Promote correct folding of proteins
     • e.g Hsp 60 family= Cpn60 in plants that prevent aggregation of denatured protein with its cofactor Cpn10
       • Hsp 60 in mitochondria with cooperating factor Hsp10

3. GroEL-GroES

   • cytoplasm of E.Coli
     • Single mutant experiment of GroEL revealed structure
       • Prise 2 rings apart of GroEL and mutate
         • 2 mutations per ring
           • R13G (arginine to glycine got rid of electrostatic interaction between rings
           • A126V (alanine to valine, valine is bulkier so couldn't sit in its binding pocket on other ring
   • Promote protein folding and required for phage assembly
     • Does not bind native proteins, ONLY NON-NATIVE
   • Split into Large operating factor, EL and small ES
     • GroEL is made of 14 identical subunits of 548 residues
       • these 14 are split into 2x 7 membered rings
         • each ring is 57 kDa
       • both rings of GroEL are needed for protein release, seen with Single ring mutants and GFP protein. Fluoresced so was folded, but couldn't be released.
         • Shows theres cross-talking between rings
           • Inter-ring talking= cross talk between rings, one tells other to remove cap
           • Intra-ring talking= subunits from same ring talk
         • GroEL also has 8 key functional hydrophobic residues
           • Tyr 199, Tyr 203, Phe 204, Leu 234, Leu 237, Leu 259, Val 263, Val 264
             • These key residues allows binding of non-native proteins to GroEL-GroES
             • Mutations/ loss of 1 means no binding. thus essential
       • GroEL has 3 domains in its structure, Equatorial, Intermediate and Apical
         • These are in each subunit, so 14 over the 2 rings
         • GroEL has 2 hinges, in the intermediate and apical domains
           • Intermediate= Pro137 and Gly410 (less flexible due to proline)
           • Apical= Gly192 and Gly 375 (more flexible)
           • A domain is more flexible
     • GroES sits on top of GroEL rings
       • 7 identical subunits of 97 residues
       • has hydrophobic residues of its own that are in arrays of mobile loops
         • These interact with GroEL hydrophobic residues
       • Initiates protein folding as GroES binding to one GroEL ring promotes polypeptide binding to other ring
         • This forms Anfinsens Cage
           • VIA DOMAIN MOVEMENTS
           • Football or bullet shape?
             • Long debate over shape of structure during mechanism
               • Can induce football via mutations to ASP 398 and ASP 52 but only 0.1% ATP hydrolysis activity seen , so ATP binds and sits there
                 • Mutant rotated equatorial ring domain by 7 degrees too, altering allosteric properties, thus why football may have been there
             • structure may be transiently there before release of cap on other ring
           • ANFINSENS CAGE IS PHILIC ENVIRONMENT FOR FOLDING!!!!
         • GroES contains 9 beta strands in each subunit, theres 7 subunits
           • each mobile strand is between strands 2 and 3. theres 7 mobile strands with hydrophobic residues
             • These loops interact with the apical domain/hydrophobic residues of GroEL
     • Provides stabilised environment for hydrophobic exposed surfaces (mis-folded) either by end of rings or in a cavity in both structures= ANFINSENS CAGE
       • Works by timer mechanism, so not always folded into native state under each round
         • Timer mechanism= after a period of time, it releases protein so see if it is folded, if it is not, process is repeated
         • Not all proteins can fit in anfinsens cage, so some proteins fold on edge of GroEL
           • Folding requires nucleotides in form of ADP and ATP
             • around 100 ATP hydrolysis needed per peptide
             • nucleotide binding triggers domain movements in GroEL
               • ATP binding and hydrolysis cause speearte events!
                 • ATP hydrolysis leads to GroES release
                   • Mutations in GroEL showed Asp 398 is required for ATP hydrolysis ( Asp398 to Ala showed loss of negative charge which meant when ATP bound, no hydrolysis occurred.
                     • ASP 398 moves down from intermediate domain to nucleotide binding site through the movements.
                       • Intermediate site creates ATP catalytic site
               • ATP binding causes small apicall domain movements to take place
                 • NOTE WHEN GROES BINDS, LARGE DOMAINS MOVEMENT OCCUR
                   • Equatorial domain moves by 2 degrees
                   • intermediate domain moves by 25 degrees (pro-gly)
                   • Apical moves by 60 degrees away from equatorial and rates 90 degrees its gly-gly hinge
                   • Domain movement is positively coupled, encourage each other.
                   • ANFINSENS CAGE FORMS FROM DOMAIN MOVEMENTS

4. MECHANISM FOR GroEL-GroES= 4 steps

   • 1. In its native state, GroEL is capped by GroES at one end, with ADP bound. ATP comes in and binds to open side which stimulates small apical domain movements in open ring, so its ready to capture protein.
   • 2. Protein now binds to open ring of hydrophobic residues , stretching it out.
   • 3. GroES is on both sides and Mobile loops of GroES interact with GroEL open rings , the apical hydrophobic residues on active side. This triggers large apical domain movements on the gly-gly hinge and the protein is forced into anfinsens cage (56 phobic residues do this) AND THE CAGE THEN SHOWS PHILIC RESIDUES FOR FOLDING . Gross fully caps cage, opposing one comes away and opposing ring ADP also leaves
   • 4. ATP hydrolysis acts as a timer and the cycles may need to be repeated. Step 1-3 will be repeated as if protein is spat out unfolded, its still phobic, so will need to be put into other side and mechanism will be done again