Chaperone proteins

Question 1

What do chaperone proteins do?

Answer 1

Prevent inappropriate interactions between proteins which haven’t folded correctly, but play no part in their final folded state.

Question 2

What are the main classes of chaperone proteins?

Answer 2

• Hsp70s
• Hsp90s
• Hsp100s

Question 3

What are the characteristics of Hsp70s?

Answer 3

They are approximately 70 kDa and many examples are up-regulated after heat shock (hence Heat Shock Proteins). They bind to short hydrophobic stretches of extended or helical protein, approximately 7 residues, with high aliphatic content. It is estimated that there are hsp70 binding sites every 35-40 residues in most proteins. This prevents hydrophobic interactions. Hsp70s often work with other chaperones to complete their function, e.g. hsp60, hsp90 and hsp104. The independent action of the two co-chaperones is also exploited to allow translocation in mitochondrial import.

Question 4

How is Hsp70 affinity for unfolded proteins modulated?

Answer 4

by ATP hydrolysis, which in turn is regulated by Hsp40 co-chaperones (stimulate the ATPase) and nucleotide exchange factors (NEFs). This regulation allows targeting of protein release. In Eubacteria (E. coli), this protein is called DnaK (Hsp70) and the co-chaperones DnaJ (Hsp40) and GrpE (NEF). The rate determining step in folding is the ATP-dependent dissociation of the polypeptide. Note that the chaperone does not direct the folding pathway in any way, except to prevent side reactions that make kinetic traps such as aggregates.

Question 5

What helps protein fold when the folding rate is slow?

Answer 5

In eubacteria, and mitochondria, this is provided by Hsp60s. GroEL is the bacterial Hsp60. It is a dimer of heptamers; i.e. it has 14 subunits of 60 kDa arranged in to 2 rings of 7, making an 840kDa complex. In common with Hsp70, its affinity for unfolded protein is modulated by nucleotide binding, and its full activity requires a co-chaperone, Hsp10, called GroES in bacteria. GroES is a ring of 7 subunits, and binds to one of the rings of GroEL in the presence of nucleotide, forming a cap. The cavity produced when GroES binds has hydrophilic walls, allowing an encapsulated protein to fold without the possibility of aggregation.

Question 6

What are the characteristics of Hsp90s?

Answer 6

Up-regulated upon heat shock, and has its binding affinity regulated by ATP. Its role in eukaryotic cytosol is understood best, where it is the most abundant cytosolic protein. It is a homodimer of 90kDa subunits. There are isoforms in ER, chloroplasts and eubacteria. In contrast to the general chaperones considered so far, a defined sub-set of eukaryotic proteins are substrates, or clients.

Question 7

How are Hsp90s regulated?

Answer 7

In addition to ATP hydrolysis, the action of Hsp90s is heavily regulated by a large number of other co-chaperones. The role of all of these is not well understood. They interact with Hsp90 in a stepwise manner. One of these is kinase specific, cdc37. Co-chaperones include PPIases, protein phosphatases and factors that modulate the Hsp90 ATPase and protein binding capacity, including p23, Cns1, Aha1.

Question 8

What is an example of Hsp90 use?

Answer 8

Oestrogen receptor, which cannot fold in the absence of hormone, and so forms a stable complex with hsp90 until hormone binds. Hormone binding also induces nuclear transport of the hsp90:OR complex. Once in the nucleus the receptor acts as a transcription factor, eliciting the response to hormone.

Question 9

What are the characteristics of Hsp100s?

Answer 9

Hsp100s are members of the AAA+ ATPase family. One of the most well characterised examples is Hsp104 from yeast, because of its role in the suppression of the yeast prion Psi. It forms a hexameric ring structure, with each monomer containing 2 nucleotide binding domains, which form two rings. In collaboration with Hsp70 and Hsp40, it can utilise ATP hydrolysis to resolubilize insoluble protein aggregates. Substrate polypeptides are forced through the central channel of the ring, which unfolds the polypeptide or aggregate.

Question 10

What are the functions of Hsp100s?

Answer 10

This function is often combined with proteolytic functions, and is important in the breakdown of proteins that have been damaged and will no longer fold. In bacteria, the homologue, ClpB interacts with a similarly hexameric protease called ClpXP. In eukaryotes, components of the 19S proteosome regulatory particle are members of the same family, and another homologue, p97, is involved in the transport of protein requiring breakdown from the ER.

Question 11

What are the two main families of folding catalysts?

Answer 11

the cyclophilins and the FK binding proteins (FKBP).

Question 12

What drug targets each folding catalyst family?

Answer 12

cyclophilins being the target of the immunosuppressant cyclosporin, and FKBPs being the target of ascomycin.

Question 13

What is the structure of the two main folding catalyst families?

Answer 13

Structurally different but catalytically similar, dehydration of peptide bond lowers the energy barrier between forms. Good enzymes, ~300-fold rate enhancement at cellular concentrations.

Question 14

What assists in the rearrangement of disulphide bonds in eubacterial periplasm and eukaryotic ER?

Answer 14

Protein disulphide isomerases. Different systems exist in the two locations. The principles can be understood from the Dsb system of bacteria. PDIs cause the oxidation, reduction or disulphide exchange via mixed disulphides with the substrate protein. PDIs contain a CXXC motif in the centre of a hydrophobic surface, one Cys having an unusually low pKa (6.7) that starts the reaction.

Question 15

How are the enzymes (DsbA and thioredoxin) redoxed?

Answer 15

Require redox power from an external source. The bacterial DsbA is oxidised in a series of steps. DsbA is oxidised by DsbB, which is oxidised by ubiquinone, which is oxidised by cytochrome oxidase and oxygen. DsbA introduces a disulphide bridge into proteins at random. DsbC is reduced by DsbD, which is reduced by NADPH via thioredoxin. This rearranges disulphide bonds through mixed disulphides with two invariant cysteines.

Question 16

What is the bacterial Hsp60?

Answer 16

GroEL is the bacterial Hsp60. This class of chaperones was discovered independently in three kingdoms of life, by genetic means: Hsp60 was found to be an essential gene in chloroplast biogenesis, linked to the synthesis of Ribulose bisphosphate carboxylase (Rubisco). Hsp60 was found to be required for the import of mitochondrial proteins. GroEL was found to be required for phage assembly by E. coli.

Question 17

What is the regulation and structure of GroEL?

Answer 17

In addition, GroEL was found to be massively up-regulated upon heat shock, under the control of the heat shock promoter. It becomes 11 % of the cell protein when the growth temperature rises from 37° to 46° C. This gives rise to the term heat shock protein.
GroEL forms a dimer of heptameric rings. The subunits are composed of three domains, whose orientations change upon binding different nucleotides. GroES binds in the presence of these forming a lid over the protein binding cavity.

Question 18

How do we study chaperone protiens?

Answer 18

In order to study chaperones, we first need a folding assay. In our cuvette is a mixture of NADH (0.2 mM) and pyruvate (10mM), which are both in excess of their respective KM (20mM and 1mM). We also have a concentrated solutions of Lactate dehydrogenase (LDH). Dilution (1:2000) of folded LDH to our assay mix. We also have a 6M solution of Guanidine hydrochloride (GuHCl), a powerful denaturant. We mix our LDH solution with an equal volume of GuHCl, to give a solution of LDH unfolded in 3M GuHCl. Dilution of this into our assay mix leaves a very small residual concentration of GuHCl, allowing the protein to refold. The refolding rate is significantly slower than the catalytic rate (>1000x), giving a lag time while LDH folds.

Question 19

How is the refolding experiment repeated?

Answer 19

In the presence a 5-fold excess of GroEL. Because it binds hydrophobic regions, and therefore unfolded protein, the folding is retarded and is halted. Addition of ATP decreases the affinity for protein, and so repeating the experiment in the presence of GroEL and ATP means the protein is released once every ATP turnover, allowing the protein to fold at the rate of hydrolysis. The yield of folded protein increases.

Question 20

How does GroES bind?

Answer 20

GroES binds in the presence of nucleotide, displacing the bound protein into the cavity in the centre of the GroEL ring. Therefore, repeating the experiment in the presence of GroEL, GroES and ATP allows folding to proceed faster. In the case of LDH, it does not increase the yield compared with GroEL alone.

Question 21

What happens when we do a refolding experiment with a different enzyme?

Answer 21

We can do this experiment with a different enzyme, like mitochondrial malate dehydrogenase (mMDH). This aggregates very quickly. GroEL plus ATP; we get some refolding, but reduced yield. In the case of a fast aggregating protein, GroEL:ATP alone is not enough. Chaperone plus ATP and GroES; because folding can take place within the GroEL cavity, in isolation from other proteins, get 100% yield and increase in rate of folding.

Question 22

What are the main characteristics of the refolding model?

Answer 22

The model only includes the effects of preventing aggregation (ATP alone); and creating a cavity where folding can take place (ATP plus GroES). No acceleration of the underlying folding process is assumed. The hydrophobic interaction between unfolded protein and GroEL prevents other processes dominated by this effect, like folding or aggregation. In the absence of ATP, get no folding OR aggregation. ATP switches GroEL to a weaker binding form, releasing the protein and allowing some folding. Therefore, increased yields of spontaneously refolding proteins can be provided by the action of GroEL and ATP Even greater yields of refolding can be provided by the inclusion of GroES. This creates a cavity which allows folding but not aggregation.

Question 23

What cooperativity is shown through GroEL?

Answer 23

The release of protein into the central cavity requires the coordinated action of the GroEL subunits of one ring: this is positive cooperativity. ATP binding and hydrolysis by GroEL has a sigmoidal concentration dependence indicating a cooperativity in ATP binding. Full loading of ATP in one ring strongly inhibits ATP binding to the other ring, and this is enhanced by GroES binding: this is negative cooperativity. This results in a system where ATP hydrolysis occurs on alternating rings.

Question 24

What does GroEL simulation show?

Answer 24

Assistance of protein folding does not REQUIRE any active unfolding. One class of chaperones can solubilise stably aggregated protein including amyloid forms.

Question 25

What is the best example of GroEL simulation?

Answer 25

Best example of this is action of Hsp104 on yeast prion aggregates. Overexpression of Hsp104 prevents susceptibility of Sac. cerevisiae to yeast prion. In combination with Hsp70 and Hsp40, the amyloid form of the prion protein (Sup5) is solubilised, resulting in resistance. Prokaryotes have an equivalent system called Clp. In addition to solubilising function (ClpB); a co-protein protease, ClpX, can digest the solubilised protein.

Question 26

What is the structure of Hsp104?

Answer 26

Hsp104 is a hexamer arranged as a ring. The Nucleotide binding domains (NBDs) are AAA+ ATPases, similar to helicases, dynein, and numerous other proteins. Use the hydrolysis of ATP to generate force. The protein binding site either face the central channel or are buried in subunit interfaces. Suggests that protein is passed through central channel from NBD1 to NBD2.

Question 27

What do mutations of Walker A motifs show?

Answer 27

Mutation of the Walker A motifs in the two NBDs shows that there is stronger ATPase activity in NBD1, but it requires binding of ATP in NBD2. The ATPase activity is stimulated by presence of protein. Role of NBD2 ATPase less well characterised, important for protein translocation. One site at a time ATPase, which induces rotation out of ring by one domain at a time (ATP-bound). This motion of NBD1 moves peptide binding site from exposed at ring entrance when ATP is bound, to buried in subunit interface when it is hydrolysed. Domains in turn bind protein, and then move it into channel, to be bound by peptide sites in NBD2.

Question 28

What can ClpXP induce?

Answer 28

ClpXP can induce unfolding and proteolysis of protein domains and the denaturation rates correlates with the protein stability. This is only true up to a limit, which is required so that stably folded proteins do not get processed in this way. The number of ATPs used to do this correlates with denaturation time and hence with protein stability.