1. Lecture 13 Protein Structure II Chapter 3

2. PROTEIN FOLDING

3. How does a protein chain achieves its native conformation? As an example, E. coli cells can make a complete, biologically active protein containing 100 amino acids in about 5 sec at 37°C. If we assume that each of the amino acid residues could take up 10 different conformations on average, there will be 10 100 different conformations for this polypeptide. If the protein folds spontaneously by a random process in which it tries all possible conformations before reaching its native state, and each conformation is sampled in the shortest possible time (~10 -13 sec), it would take about 10 77 years to sample all possible conformations.

4. There are two possible models to explain protein folding: In the first model, the folding process is viewed as hierarchical, in which secondary structures form first, followed by longer-range interactions to form stable supersecondary structures. The process continues until complete folding is achieved. In the second model, folding is initiated by a spontaneous collapse of the polypeptide into a compact state, mediated by hydrophobic interactions among non-polar residues. The collapsed state is often referred to as the ‘molten globule’ and it may have a high content of secondary structures. Most proteins fold by a process that incorporates features of both models.

5. Takada, Shoji (1999) Proc. Natl. Acad. Sci. USA 96, 11698-11700 Thermodynamically, protein folding can be viewed as a free-energy funnel - the unfolded states are characterized by a high degree of conformational entropy and relatively high free energy - as folding proceeds, narrowing of the funnel represents a decrease in the number of conformational species present (decrease in entropy) and decreased free energy Schematic pictures of a statistical ensemble of proteins embedded in a folding funnel for a monomeric -repressor domain. At the top, the protein is in its denatured state and thus fluctuating wildly, where both energy and entropy are the largest. In the middle, the transition state forms a minicore made of helices 4 and 5 and a central region of helix 1 drawn in the right half, whereas the rest of protein is still denatured. At the bottom is the native structure with the lowest energy and entropy.

6. A schematic energy landscape for protein folding Nature 426:885

8. Steps in the creation of a functional protein

9. The co-translational folding of a protein

10. The structure of a molten globule – a molten globule form of cytochrome b 562

11. Molecular chaperones help guide the folding of many proteins

12. A current view of protein folding

13. A unified view of some of the structures that can be formed by polypeptide chains Nature 426: 888

14. The hsp70 family of molecular chaperones. These proteins act early, recognizing a small stretch of hydrophobic amino acids on a protein’s surface.

15. The hsp60-like proteins form a large barrel-shaped structure that acts later in a protein’s life, after it has been fully synthesized

17. Nature 379:420-426 (1996)

18. Cellular mechanisms monitor protein quality after protein synthesis Exposed hydrophobic regions provide critical signals for protein quality control

19. The ubiquitin-proteasome pathway Nature 426: 897

20. The proteasome Processive cleavage of proteins

21. Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008) Processive protein digestion by the proteasome

22. Figure 6-91a Molecular Biology of the Cell (© Garland Science 2008) The 19S cap – a hexameric protein unfoldase

23. Figure 6-91b Molecular Biology of the Cell (© Garland Science 2008) Model for the ATP-dependent unfoldase activity of AAA proteins

24. Ubiquitin – a relatively small protein (76 amino acids)

28. Abnormally folded proteins can aggregate to cause destructive human diseases

29. (C)Cross-beta filament, a common type of protease-resistant protein aggregate (D)A model for the conversion of PrP to PrP*, showing the likely change of two α -helices into four β -strands

30. Regulation of protein folding in the ER Nature 426: 886

31. A schematic representation of the general mechanism of aggregation to form amyloid fibrils. Nature 426: 887

32. Proteins can be classified into many families – each family member having an amino acid sequence and a three-dimensional structure that resemble those of the other family members The conformation of two serine proteases

33. A comparison of DNA-binding domains (homeodomains) of the yeast α 2 protein (green) and the Drosophila engrailed protein (red)

34. Sequence homology searches can identify close relatives The first 50 amino acids of the SH2 domain of 100 amino acids compared for the human and Drosophila Src protein

35. Multiple domains and domain shuffling in proteins

36. Domain shuffling An extensive shuffling of blocks of protein sequence (protein domains) has occurred during protein evolution

37. A subset of protein domains, so-called protein modules, are generally somewhat smaller (40-200 amino acids) than an average domain (40-350 amino acids)

38. Each module has a stable core structure formed from strands of β sheet and they can be integrated with ease into other proteins

39. Fibronectin with four fibronectin type 3 modules

40. Figure 3-18 Molecular Biology of the Cell (© Garland Science 2008) Relative frequencies of three protein domains

41. Figure 3-19 Molecular Biology of the Cell (© Garland Science 2008) Domain structure of a group of evolutionarily related proteins that have a similar function – additional domains in more complex organisms

42. Quaternary structure

43. Lambda cro repressor showing “head-to-head” arrangement of identical subunits Larger protein molecules often contain more than one polypeptide chain

44. DNA-binding site for the Cro dimer ATCGCGAT TAGCGCTA

45. The “head-to-tail” arrangement of four identical subunits that form a closed ring in neuraminidase

46. HEMOGLOBIN A symmetric assembly of two different subunits

47. A collection of protein molecules, shown at the same scale

49. Protein Assemblies Some proteins form long helical filaments

50. Helical arrangement of actin molecules in an actin filament

51. Helices occur commonly in biological structures

52. A protein molecule can have an elongated, fibrous shape Collagen

53. Elastin polypeptide chains are cross-linked together to form rubberlike, elastic fibers

54. Disulfide bonds Extracellular proteins are often stabilized by covalent cross-linkages

55. Proteolytic cleavage in insulin assembly