Protein metabolism

1. Protein metabolism

2. 5’-GATGCCCCTCGAATAA-3’ 3’-CTACGGGGAGCTTATT-5’ DNA mRNA 5’-GAUGCCCCAGCAAUAA-3’ PROTEIN (PEPTIDE) M—P—Q—Q--STOP Birth of a protein Predicted genes or genes of unknown function are typically called open reading frames (ORF’s)

3. Complexity of protein synthesis

4. Aminoacyl-tRNA synthetases charge tRNAs with amino acids • Specificity is shown Here  Note two non-standard amino acids have been shown to be incorporated into proteins in this way selenocysteine, and pyrrolysine

5. Initiation requires several auxiliary factors • Bacterial translation is regulated by specific incorporation of fMet Note three sites for tRNA binding two encompass both subunits

6. Structural analysis reveals RNA binding sites on 50S subunit

7. Structures have been solved for both 50S and 30S ribosomal subunits

9. RNA as a catalyst in peptide bond formation

10. Translation elongation

11. The ribosome is a molecular machine

12. Ribosomes are the site for various antibiotic action

13. A Protein’s Adolescence

14. Fate of proteins in cells Prokaryote vs. Eukaryote

15. Additional “codes” are intrinsic to a protein’s primary sequence Acquire secondary structure either coming off ribosome (a), or by interactions with chaperones (b), however,some proteins fold into tertiary structures autonomously Post-translational processing and modifications Insulin and proteases Inteins Glycosylation, etc.

16. Protein targeting utilizes signal peptides Various protein activities are regulated by processing to yield a “mature” enzyme

17. The PCC functions in protein translocation

18. Introns versus Inteins IntronIntein DNA DNA RNA RNA RNA Protein Protein Protein Splicing event

19. Formation of disulfide bonds; addition of prosthetic groups

20. Glycosylation provides a ligand and targeting to proteins

21. “Buttering” proteins

22. Some amino acids are modified post-translationally

23. Some nascent polypeptides can fold spontaneously

24. …while others need help

25. DnaKJ and GrpE are chaperones

26. GroESL is a chaperonin

27. Other important enzymes in protein folding… • Protein disulfide isomerase (PDI) – assists protein in forming proper disulfide bridges • Peptide prolyl cis-trans isomerase (PPI) – interconverts cis-trans isomers of proline

28. The state of proteins are monitored by the cell ~20% of new polypeptides are immediately degraded because of abnormalities, incomplete assembly; others inactivated by heat or chemical stress, while some are unstable Quality control for proteins Signal for quality control is surface-exposed hydrophobic regions

29. Why do some proteins need help folding, while others do not? • What are the molecular mechanisms of protein folding? • How important is protein folding? • Why should I care?

30. Two-state folding of small proteins • Modeled after Anfinsen experiments • A large number of proteins fewer than 150 amino acids can efficiently refold upon dilution • During refolding, only observe two distinct states – folded and unfolded on a time scale of seconds or less

31. A non-random process • The unfolded protein undergoes specific kinetically preferred steps on way to the native state

32. Long-range interactions lead to non-randomness • In non-native proteins residual structure appears as hydrophobic clusters, in which tryptophan or histidine residues are surrounded by hydrophobic residues • Do these act as nucleation sites for protein folding?

33. Lysozyme folding

34. H-exchange and lysozyme folding • Refolding experiments indicated both domains achieve native structure in folding intermediates prior to tertiary interactions spanning the two domains • Amide hydrogens become protected in the alpha domain much faster than beta

35. Hydrogen exchange measures solvent accessibility of amide hydrogens

36. Trp-63 is an exception • The amide hydrogen of Trp-63 becomes protected as rapidly as the alpha domain residues, despite it’s location in the beta domain • This result suggests Trp-63 may be involved in alpha domain folding

37. Other Trp’s are important too • Replacement of Trp-62 and Trp-108 with Tyr lead to increased rate of refolding • Chemical modification of Trp-62 leads to increased misfolding • Trp-62 is also important for correct formation of disulfide binds in peptide fragments

38. Folding interactions • Native state Trp62 is solvent accessible and side chain disordered in crystal structure • Denatured state, this Trp and others are inaccessible • Trp62 and 63 (in a non-native b domain state) associate with native hydrophobic cluster in a (Trp108-111) • These non-native interactions stabilize a native core (W62G destabilizes core and causes misfolding)

39. Take home • Although a residue such as tryptophan may be exposed in the native state for functional reasons, it could be buried in early stages of folding to reduce the tendency of transiently populated species to aggregate • Protein sequence codes for structural characteristics other than those of the native fold!

40. Larger proteins have a more difficult time becoming native

41. Kinetic barriers • Folding intermediates can become trapped in energy minima • Some may be necessary intermediates, which can accumulate to significant populations • This may lead to aggregation

42. Tryptophanase folding • 8 M urea  Dilution  aggregates and native protein • 3 M urea  Dilution  only aggregates • Suggests a folding intermediate whose population is favored under this [urea] • Occurs in the presence of other denatured proteins generating a homogenous population

43. A competition • Kinetics of aggregate formation indicates a competition between unimolecular, intrachain reaction and multimolecular, interchain reaction

44. Competition evident in domain swapping between monomers • Three domains in diptheria toxin • In dimer, one domain loses contact with other two (rotates 180 degrees and translates 65 angstroms) and forms similar contacts with other chain. • Observed in several proteins

46. Chaperonin function • Open rings of chaperonin provide a hydrophobic binding surface to bind hydrophobic clusters of non-native structures (compete with multimer formation, no access in cavity) • GroES and ATP binding cause conformational changes in GroEL  leading to alternation of surface from hydrophobic to hydrophilic (encourages burial of hydrophobic surfaces of folding protein) • Oligomeric structures form in bulk solution

47. GroESL cycle

48. Why should I care? • Amyloid – Protein deposit in b-pleated sheets • Associated with numerous disease states • Alzheimer’s  Ab peptides • Creutzfeldt-Jacob  Prion • Elderly cardiac  Transthyretin

49. The amyloid state • Various proteins share NO primary sequence identity • Amyloid state must be accessible to any protein as a very stable alternative state (may be more thermodynamically stable than native) • Expect sequences that readily form amyloids to be selected against (very few form in vivo)

50. Amyloid formation • Often results from destabilization of already folded native protein • Is structural disruption thermodynamic or kinetic? • Thermodynamic  free energy difference between native and monomeric intermediate leading to amyloid formation • Kinetic  refers to free energy barrier between native state and transition state for amyloid, reflected in rate constant for conversion