2. Protein Folding, Processing and Degradation

3. 2 Protein Folding Protein in native state is not static Protein in native state is not static –2° structural elements and domains move Function of proteins often dependent on large conformational changes, triggered by ligand binding Function of proteins often dependent on large conformational changes, triggered by ligand binding

4. 3 Energy of Folding Difference in energy (free energy) between folded (native) and unfolded (denatured) state is small, 5-15 kcal/mol. Difference in energy (free energy) between folded (native) and unfolded (denatured) state is small, 5-15 kcal/mol. Two major contributions to energy difference between unfolded and folded state, enthalpy and entropy Two major contributions to energy difference between unfolded and folded state, enthalpy and entropy

5. 4 Free energy of folding Enthalpy (H) is increased upon folding Enthalpy (H) is increased upon folding –Noncovalent interactions are maximized Stronger and more frequent in native state Stronger and more frequent in native state –Enthalpy difference can reach several hundred kcal/mol Entropy is decreased upon folding Entropy is decreased upon folding –Folding causes one main conformation=highly ordered structure –Entropy difference can reach several hundred kcal/mol

6. 5 Free energy of folding Enthalpy and entropy differences balance each other, and  G is a small positive number. Enthalpy and entropy differences balance each other, and  G is a small positive number. Small  G is necessary because too large a free energy change would mean a very stable protein, one that would never change Small  G is necessary because too large a free energy change would mean a very stable protein, one that would never change However, structural flexibility is important to protein function, and proteins need to be degraded However, structural flexibility is important to protein function, and proteins need to be degraded

7. 6 Protein folding For any given protein, there is one conformation that has significantly lower free energy than any other state For any given protein, there is one conformation that has significantly lower free energy than any other state Achieved through kinetic pathway of unstable intermediates (not all intermediates are sampled) Achieved through kinetic pathway of unstable intermediates (not all intermediates are sampled) Assisted by chaperones and protein disulfide isomerases so intermediates are not trapped in a local low energy state Assisted by chaperones and protein disulfide isomerases so intermediates are not trapped in a local low energy state

8. 7 Pathways to folding There are multiple folding pathways for some proteins, but a single main pathway for other proteins There are multiple folding pathways for some proteins, but a single main pathway for other proteins

9. 8 Transition State Model Free energy barrier separates the D (denatured) state from the N (native) state Free energy barrier separates the D (denatured) state from the N (native) state Folding pathway can vary between proteins Folding pathway can vary between proteins –Can be a single energy barrier with one pathway or –A single folding pathway that has sequential transitional states that have limited flexibilities along the pathway or –Can have multiple transition states with similar energy values and a variety of pathways to get to the final folded state

10. 9 Fig. 6-39, p.189

11. 10 Fig. 6-40, p.190

12. 11 Pathways to folded state Molten globular is state in between unfolded and final folded state Molten globular is state in between unfolded and final folded state –Can be multiple molten globular states for each protein Has helices and sheets, which may be positioned correctly Has helices and sheets, which may be positioned correctly Unfolded loops, less compaction, different conformations of surface structures Unfolded loops, less compaction, different conformations of surface structures

13. 12 fast slow DenaturedMolten globularNative

14. 13 Fig. 6-41, p.191

15. 14 Disulfide bond formation Cellular environment is reducing Cellular environment is reducing Cysteines must be oxidized to form S-S bonds Cysteines must be oxidized to form S-S bonds Bacteria use Disulfide bridge-forming protein, Dsb, for oxidation in periplasm Bacteria use Disulfide bridge-forming protein, Dsb, for oxidation in periplasm In eucaryotes, protein disulfide isomerase, PDI, used for oxidation in the endoplasmic reticulum (ER) In eucaryotes, protein disulfide isomerase, PDI, used for oxidation in the endoplasmic reticulum (ER)

16. 15 Dsb Crystal structure shows a thioredoxin-like domain Crystal structure shows a thioredoxin-like domain Mechanism of thioredoxin is based on reversible oxidation of two cysteine thiol groups to a disulfide-- redox-active disulfide bridge is Cys-X-X-Cys (X any amino acid) Mechanism of thioredoxin is based on reversible oxidation of two cysteine thiol groups to a disulfide-- redox-active disulfide bridge is Cys-X-X-Cys (X any amino acid) Oxidized Dsb is less stable than reduced form-- Oxidized Dsb is less stable than reduced form-- –S-S is very reactive, therefore a strong oxidizing agent

17. 16 Isomerization of prolines Most peptide bonds are trans (100x more stable than cis) Most peptide bonds are trans (100x more stable than cis) When second residue is proline, trans form is only 4x more stable When second residue is proline, trans form is only 4x more stable In native proteins, cis-proline peptides are stabilized by tertiary structure but in unfolded state there is an equilibrium between cis and trans isomers In native proteins, cis-proline peptides are stabilized by tertiary structure but in unfolded state there is an equilibrium between cis and trans isomers Cis-trans isomerization of proline peptides is the rate- limiting step in folding for some proteins Cis-trans isomerization of proline peptides is the rate- limiting step in folding for some proteins

18. 17 Protein Folding Proteins are assisted in folding by molecular chaperones Proteins are assisted in folding by molecular chaperones Hsp60 (chaperonins) Hsp70 and Hsp90 are three main classes Hsp60 (chaperonins) Hsp70 and Hsp90 are three main classes Hsp70 recognizes exposed, unfolded regions of new protein chains - especially hydrophobic regions Hsp70 recognizes exposed, unfolded regions of new protein chains - especially hydrophobic regions It binds to these regions, protecting them until productive folding reactions can occur It binds to these regions, protecting them until productive folding reactions can occur

19. 18

20. 19 Fig. 31-2b, p. 1026

21. 20 The GroES-GroEL Complex The principal chaperonin in E. coli The principal chaperonin in E. coli GroEL forms two stacked 7-membered rings of 60 kD subunits; GroES is a dome on the top GroEL forms two stacked 7-membered rings of 60 kD subunits; GroES is a dome on the top Nascent protein apparently binds reversibly many times to the walls of the donut structure, each time driven by ATP hydrolysis, eventually adopting its folded structure, then being released from the GroES-GroEL complex Nascent protein apparently binds reversibly many times to the walls of the donut structure, each time driven by ATP hydrolysis, eventually adopting its folded structure, then being released from the GroES-GroEL complex Rhodanese (as one example) requires hydrolysis of 130 ATP to reach fully folded state Rhodanese (as one example) requires hydrolysis of 130 ATP to reach fully folded state

22. 21 Fig. 31-3a, p. 1027

23. 22 Fig. 31-3b, p. 1027

24. 23 Fig. 31-3c, p. 1027

25. 24 Eucaryotic Hsp90 Chaperones Hsp 90 is 1-2% of total cytosolic protein Hsp 90 is 1-2% of total cytosolic protein Signal transduction molecules such as tyrosine kinase receptors, steroid hormone receptors, non-receptor tyrosine kinases are clients for Hsp90 Signal transduction molecules such as tyrosine kinase receptors, steroid hormone receptors, non-receptor tyrosine kinases are clients for Hsp90

26. 25 Fate of nascent proteins Association of nascent polypeptide with various chaperone systems commits them to folding pathways Association of nascent polypeptide with various chaperone systems commits them to folding pathways If proteins fail to fold, they are recognized and targeted for degradation If proteins fail to fold, they are recognized and targeted for degradation

27. 26 Protein processing Post-translational modifications are covalent modifications that alter function Post-translational modifications are covalent modifications that alter function Hundreds of amino acid variations have been described Hundreds of amino acid variations have been described Methylation, phosphorylation, lipid and carbohydrate addition are a few Methylation, phosphorylation, lipid and carbohydrate addition are a few Human proteins number around 300,000 from 30,000 genes Human proteins number around 300,000 from 30,000 genes

28. 27 Proteolytic cleavage Why remove amino acids? Why remove amino acids? –Create diversity Met-amidopeptidase removes Met from peptide so that not all amino termini are Met Met-amidopeptidase removes Met from peptide so that not all amino termini are Met –Serves as activation mechanism Metabolically active enzymes are pro-proteins or zymogens such as digestive enzymes Metabolically active enzymes are pro-proteins or zymogens such as digestive enzymes –Involves targeting of proteins to proper destinations

29. 28 Protein Translocation Common features in all organisms Proteins are made as preproteins containing domains that act as sorting signals Proteins are made as preproteins containing domains that act as sorting signals Signal recognition particles (SRPs) recognize the sorting signals as they emerge from the ribosome Signal recognition particles (SRPs) recognize the sorting signals as they emerge from the ribosome Membranes involved in protein translocation have specific signal receptors Membranes involved in protein translocation have specific signal receptors Translocons (selectively permeable protein-conducting channels) catalyze the movement of the proteins across the membrane with metabolic energy (ATP, GTP, ion gradients) essential Translocons (selectively permeable protein-conducting channels) catalyze the movement of the proteins across the membrane with metabolic energy (ATP, GTP, ion gradients) essential –Stop-transfer signals allow diffusion of protein into membrane Preproteins bind to chaperones to stay loosely folded Preproteins bind to chaperones to stay loosely folded

30. 29 Prokaryotic Protein Translocation An essential process for membrane proteins and secretory proteins Such proteins are synthesized with a leader peptide, or signal sequence of about 16-26 amino acids Such proteins are synthesized with a leader peptide, or signal sequence of about 16-26 amino acids The signal sequence has a basic N-terminus, a central domain of 7-13 hydrophobic residues, and a nonhelical C-terminus The signal sequence has a basic N-terminus, a central domain of 7-13 hydrophobic residues, and a nonhelical C-terminus The signal sequence directs the newly synthesized protein to its proper destination The signal sequence directs the newly synthesized protein to its proper destination

31. 30 Prokaryotic Protein Transport All non-cytoplasmic proteins must be translocated The leader peptide retards the folding of the protein so that molecular chaperone proteins can interact with it and direct its folding The leader peptide retards the folding of the protein so that molecular chaperone proteins can interact with it and direct its folding The leader peptide also provides recognition signals for the translocation machinery The leader peptide also provides recognition signals for the translocation machinery A leader peptidase removes the leader sequence when folding and targeting are assured A leader peptidase removes the leader sequence when folding and targeting are assured

32. 31 Eukaryotic Protein Sorting Eukaryotic cells contain many membrane-bounded compartments Most (but not all) targeting sequences are N- terminal, cleaveable presequences Most (but not all) targeting sequences are N- terminal, cleaveable presequences Charge distribution, polarity and secondary structure of the signal sequence, rather than a particular sequence, appears to target to particular organelles and membranes Charge distribution, polarity and secondary structure of the signal sequence, rather than a particular sequence, appears to target to particular organelles and membranes Synthesis of secretory and membrane proteins is coupled to translocation across ER membrane Synthesis of secretory and membrane proteins is coupled to translocation across ER membrane

33. 32 Events at the ER Membrane As the signal sequence emerges from the ribosome, a signal recognition particle (SRP) finds it and escorts it to the ER membrane As the signal sequence emerges from the ribosome, a signal recognition particle (SRP) finds it and escorts it to the ER membrane There it docks with a docking protein or signal receptor (SR) - see Figure 31.5 There it docks with a docking protein or signal receptor (SR) - see Figure 31.5 SRP dissociates in a GTP-dependent process SRP dissociates in a GTP-dependent process Protein synthesis resumes and protein passes into ER or into ER membrane; signal is cleaved Protein synthesis resumes and protein passes into ER or into ER membrane; signal is cleaved

39. Ribosome dissociates

41. 40

42. 41 Signal Recognition Particle Complex of six polypepides and one 300- base RNA molecule Complex of six polypepides and one 300- base RNA molecule Signal peptide recognition depends on one protein of 54,000 daltons-SRP54 Signal peptide recognition depends on one protein of 54,000 daltons-SRP54 RNA (7S) is probably structural RNA (7S) is probably structural Removal of RNA disrupts SRP Removal of RNA disrupts SRP

43. 42 Mitochondrial protein import Mitochondria have two membranes, and two spaces in between the membranes Mitochondria have two membranes, and two spaces in between the membranes Signal sequences are N-terminal, positively charged regions of 10-70 aa Signal sequences are N-terminal, positively charged regions of 10-70 aa Form amphipathic  -helices, positive on one side and uncharged, hydrophobic on the other (Fig. 31.6) Form amphipathic  -helices, positive on one side and uncharged, hydrophobic on the other (Fig. 31.6)

44. 43 Mitochondrial protein import (Fig 31.7) Binding of preprotein to TOM (mitochondrial outer membrane translocon) Binding of preprotein to TOM (mitochondrial outer membrane translocon) For outer membrane proteins, transfer to SAM (sorting and assembly complex) For outer membrane proteins, transfer to SAM (sorting and assembly complex) Inner membrane proteins traverse TOM and are taken up by TIM22 (inner mitochondrial membrane translocon) Inner membrane proteins traverse TOM and are taken up by TIM22 (inner mitochondrial membrane translocon) Matrix proteins are taken up by TIM23 and transferred across the inner membrane into the matrix Matrix proteins are taken up by TIM23 and transferred across the inner membrane into the matrix

45. 44 Protein Degradation Some protein degradation pathways are nonspecific - randomly cleaved proteins seem to be rapidly degraded Some protein degradation pathways are nonspecific - randomly cleaved proteins seem to be rapidly degraded However, there is also a selective, ATP- dependent pathway for degradation - the ubiquitin-mediated pathway However, there is also a selective, ATP- dependent pathway for degradation - the ubiquitin-mediated pathway Ubiquitin is a highly-conserved, 76 residue (8.5 kD) protein found widely in eukaryotes Ubiquitin is a highly-conserved, 76 residue (8.5 kD) protein found widely in eukaryotes Proteins are committed to degradation by conjugation with ubiquitin Proteins are committed to degradation by conjugation with ubiquitin

46. 45 Ubiquitin and Degradation Three proteins involved: E 1, E 2 and E 3 E 1 is the ubiquitin-activating enzyme - it forms a thioester bond with C-terminal Gly of ubiquitin E 1 is the ubiquitin-activating enzyme - it forms a thioester bond with C-terminal Gly of ubiquitin Ubiquitin is then transferred to a Cys-thiol of E 2, the ubiquitin-carrier protein Ubiquitin is then transferred to a Cys-thiol of E 2, the ubiquitin-carrier protein Ligase (E 3 ) selects proteins for degradation. the E 2 -S~ubiquitin complex transfers ubiquitin to these selected proteins Ligase (E 3 ) selects proteins for degradation. the E 2 -S~ubiquitin complex transfers ubiquitin to these selected proteins More than one ubiquitin may be attached to a protein target More than one ubiquitin may be attached to a protein target

47. 46 Fig. 31-8, p. 1034

48. 47 Fig. 31-8a, p. 1034

49. 48 Fig. 31-8b, p. 1034

50. 49 Fig. 31-8c, p. 1034

51. 50 Fig. 31-9, p. 1034

52. 51 Fig. 31-10a, p. 1035

53. 52