Protein folding in the cell (I)

1. 3-1 Protein folding in the cell (I) Basics - cell compartments, molecular crowding: cytosol, ER, etc. Folding on the ribosome - co-translational protein folding Molecular chaperones - concepts, introduction - intramolecular chaperones - chemical chaperones - protein chaperones

2. 3-2 • eukaryotes - cytosol ..................................protein synthesis, folding/assembly - extracellular .........................proteins are exported in folded form - mitochondria ........................limited protein synthesis; energy production - chloroplasts ..........................limited protein synthesis; light harvesting - endoplasmic reticulum.......... import of unfolded proteins; protein processing - peroxisome ........................... import of folded proteins; anab./catab. pathways - nucleus ................................. import of folded proteins - lysosome................................ import of unfolded proteins; degradation Cell compartments and folding • bacteria - cytosol ..................................protein synthesis, etc. - periplasm .............................import and folding of periplasmic proteins - extracellular .........................proteins are exported • archaea - cytosol ..................................protein synthesis, etc. - extracellular .........................proteins are exported

3. 3-3 Folding in vitro vs.in vivo in vitro in vivo protein denatured in a chaotrope Differences: 1. One has all of the information immediately available for folding; the other process is gradual 2. the cellular environment is very different (much more crowded) folding by dilution in buffer folding folded protein folded protein

4. 3-4 Co-translational protein folding Fact: - first ~30 amino acids of the polypeptide chain present within the ribosome is constrained (the N-terminus emerges first) Assumption: as soon as the nascent chain is extruded, it will start to fold co-translationally (i.e., acquire secondary structures, super-secondary structures, domains) until the complete polypeptide is produced and extruded folding assembly

5. 3-5 Sindbis Virus Capsid Protein (SCP) • SCP is the capsid protein of the Sindbis virus • 26S Sindbis RNA encodes a polyprotein • SCP is auto-proteotically cleaved from the rest of the polyprotein • other cellular proteases cleave E1-E3 from the polyprotein to generate the mature proteins; E1, the envelope protein, is 9 kDa • SCP is a 33 kDa serine protease • WT SCP self-cleaves; Ser215 => Ala215 mutant doesn’t N C SCP E1 E2 E3 catalytic triad & C-terminus of SCP

6. 3-6 SCP folds co-translationally Experiment: 1. make and translate different SCP construct RNAs in vitro in the presence of 35S-methionine for 2 min 2. Prevent re-initiation of translation with aurintricarboxylic acid (ATCA): ‘synchronizing’ 3. at set timepoints, add SDS buffer and perform SDS-PAGE 4. observe by autoradiography 2 3 4 5 6 7 8 10 12 min Result: * Mut SCP-E1 42 kDa N C 33 kDa SCP E1 9 kDa 2 3 4 5 6 7 8 10 12 min 42 kDa N C WT SCP 33 kDa SCP 9 kDa 2 3 4 5 6 7 8 10 12 min WT SCP- E1 42 kDa N C 33 kDa SCP E1 9 kDa

7. 3-7 in vitro E. coli cytosol Macromolecular crowding ~340 mg/ml <0.1 mg/ml ribosome Ellis and Hartl (1996) FASEB J. 10:20-26 proteins other macromolecules chaperonin nucleic acids When doing experiments in vitro, we should all be thinking about this:proteins in isolated (pure) systems may not behave as they do in the cell- binding partner(s) might be missing - cell conditions (pH, salts, etc.- post-translational modifications might be missing may be dramatically different

8. 3-8 Effects of crowding Definition: Molecular crowding is a generic term for the condition where a significant volume of a solution, or cytoplasm for example, is occupied with things other than water Fact: - association constants (ka) increase significantly - dissociation constants (kd) decrease significantly (kd=1/ka) - increased on-rates for protein-protein interactions (see for example Rohwer et al. (2000) J. Biol. Chem.275, 34909) Assumption: - non-native polypeptides will have greater tendency to associate intermolecularly, enhancing the propensity of aggregation

9. 3-9 Effects of crowding: example denatured lysozyme, reduced or oxidized oxidized lysozyme dilution in buffer with different crowding agents loss of activity due to protein aggregation reduced lysozyme measure lysozyme activity crowding agents: ficoll 70*, dextran 70, protein (BSA, ovalbumin) *roughly spherical polysaccharide van den Berg et al. (1999) EMBO J. 18, 6927.

10. 3-10 Problem: non-native proteins • non-native proteins expose hydrophobic residues that are normally buried within the ‘core’ of the protein • these hydrophobic amino acids have a strong tendency to interact with other hydrophobic (apolar) residues - especially under crowding conditions exposed hydrophobic residues X X incorrect molecular interactions & loss of activity X X intramolecular intermolecular X X X X X X misfolding aggregation

11. 3-11 Solution: molecular chaperones • in the late 1970’s, the term molecular chaperone was coined to describe the properties of nucleoplasmin: Nucleoplasmin prevents incorrect interactions between histones and DNA Laskey, RA, Honda, BM, Mills, AD, and Finch, JT (1978). Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA. Nature275, 416-420. Dictionary definition: 1: a person (as a matron) who for propriety accompanies one or more young unmarried women in public or in mixed company 2: an older person who accompanies young people at a social gathering to ensure proper behavior; broadly: one delegated to ensure proper behavior • in the late 1980’s, the term molecular chaperone was used more broadly by John Ellis to describe the roles of various cellular proteins in protein folding and assembly

12. 3-12 Molecular chaperones:general concepts Requirements for a protein to be considered a chaperone: (1) interacts with and stabilizes non-native forms of protein(s)- technically also: folded forms that adopt different protein conformations (2) not part of the final assembly of protein(s) Functions of a chaperone: “classical” - assist folding and assembly more recent - modulation of conformation - transport - disaggregation of protein aggregates - unfolding of proteins self-assembly refers to the folding of the polypeptide, as well as to its assembly into functional homo- or hetero-oligomeric structures assisted self-assembly (as opposed to spontaneous self-assembly) prevention of assembly assisted disassembly

13. 3-13 Molecular chaperones:common functional assays Type of assay Rationale Binary complex formation If chaperone has high enough affinity for an unfolded polypeptide, it will form a complex detectable by: • co-migration by SEC; • co-migration by native gel electrophoresis • co-immunoprecipitation Prevention of aggregation Binding of chaperones to non-native proteins often reduces or eliminates their tendency to aggregate. Assay may detect weaker interactions than is possible with SEC Refolding Chaperones stabilize non-native proteins; some can assist the refolding of the proteins to their native state. Usually, chaperones that assist refolding are ATP-dependent Assembly Some chaperones assist protein complex assembly Activity Some chaperones modulate the conformation/activity of proteins (Miscellaneous) A number of chaperones have specialized functions

14. 3-14 Intramolecular chaperones Concept: - portions of a polypeptide may assist the biogenesis of the mature protein without being part of the final folded structure - these regions are chaperones by definition, although “classical” molecular chaperones act inter-molecularly, not intra-molecularly.

15. 3-15 acid-unfolded; with 77aa propeptide Intramolecular chaperone: example Subtilisin E - non-specific protease - mature protein cannot fold properly if propeptide is removed Gdn-HCl unfolded; with propeptide Gdn-HCl unfolded; without 77aa propeptide propeptide (77 aa) precursor (352 aa) mature protein (275 aa) Shinde et al. (1993) PNAS 90, 6924.

16. 3-16 Subtilisin E propeptide - unstructured alone in solution - alpha-helical when complexed with subtilisin? propeptide is ~ 20% of preprotein; CD suggests combination mature subtilisin + propeptide mostly helical Intramolecular chaperone: continued propeptide with subtilisin propeptide in TFE Interpretation of CD data alpha-helical: minima @ 208, 222 nm maximum @ 192 nm - more pronounced minimum at 208 nm compared to 222 nm suggests less helical Structure beta-sheet: minimum @ 220 nm Maximum @ 193 nm random coil: maximum ~220 nm propeptide alpha ellipticity beta coil subtilisin nm Note:CD traces are additive • Propeptide must interact with subtilisin

17. 3-17 Intramolecular cleavage or intermolecular? released propeptide Result: Fact: unfolded His10-preprotein can refold alone in solution Experiment: 1. prepare subtilisin pre-protein containing an N-terminal polyhistidine tag (His10) 2. unfold in denaturant 3. bind different concentrations of the protein to Ni2+-NTA resin 4. assay for folding by measuring propeptide release full-length protein Q: what do the results mean? Q: why bind the protein to a resin? Q: why use different concentrations of proteins? Li et al. (1996) J. Mol. Biol.262, 591.

18. 3-18 Chemical chaperones Concept: - small molecules could enhance the stability and assist the folding or assembly of proteins - under conditions of cellular stress, such as a heat-shock, small molecules may help proteins from misfolding and aggregating - one easy way to test is to see how they can prevent loss of activity, or, prevent the aggregation of a protein- protein aggregation can be conveniently monitoredspectrophotometrically at 360 nm, where light scatteringfrom the aggregates is detected

19. 3-19 A Chemical chaperones: example in vitro studies B protein aggregation Singer and Lindquist (1998) Mol. Cell1, 639.

20. 3-20 bacterial luciferase expressed in yeast; subjected to heat shock conditions Chemical chaperones: example B in vivo studies C 40ºC heat shock 40ºC heat shock Note:tps1 yeast cells have a deletion in the trehalose synthase

21. 3-21 without Different chemical chaperones with protein aggregation protein aggregation glycerol is often used to stabilize proteins in vitro

22. 3-22 trans-acting protein molecular chaperones - cis-acting (intramolecular) chaperones are relatively rare - chemical chaperones may play an important role in protecting proteins in the cell, but their extent of action is likely to be limited - organisms have evolved large families of protein molecular chaperones that have either general functions in the cell, or have highly specific functions - the expression of many of the chaperones is induced under cellular stress conditions--giving rise to the name “Heat-shock proteins”, or Hsps, followed by their Molecular Weight (MW) BUT: - not all chaperones are Hsps - not all Hsps are chaperones Best characterized: small Hsps (12-42 kDa), Hsp40, Hsp60 (chaperonins), Hsp70, Hsp90, Hsp100/Clp/AAA ATPases

23. 3-23 Functional proteins from a random-sequence library Anthony D. Keefe & Jack W. Szostak Nature410, 715-718 (2001) The PDF file of this manuscript is available on the MBB443 web site There will be one question on the first exam relating to this paper